1P Plenary Lectures PL1 PL2

Plenary Lectures

Where applicable, the authors confirm that the experiments PL1 described here conform with The Physiological Society ethical requirements. Pacemakers and/or dynamic plasticity of rhythm generation in the in vivo respiratory network - The Physiological Society Paton Lecture D.W. Richter PL2 Department of Neuro- and Sensory Physiology, University of The Beginnings of Optogenetics – The Physiological Society’s Göttingen, Göttingen, Germany Bayliss-Starling Prize Lecture Eupneic breathing of mammals is not simply an alternation G. Miesenboeck between inspiratory lung inflation and expiratory lung defla- tion as a passive relapse of the lungs. Normal quiet breathing Department of Physiology, Anatomy and Genetics, University of in vivo involves a precisely co-ordinated interplay between inspi- Oxford, Oxford, UK ratory and post-inspiratory muscle contractions, variable but rare expiratory muscle contractions, and a concomitant adjust- Light-sensitive proteins encoded in DNA can serve as selective ment of sympathetic cardiovascular and vagal cardiac activi- optical interfaces for observing and controlling genetically tar- ties, all together being organized by a common cardio-respi- geted neurons in functioning circuits, in vitro and in vivo. Light- ratory control system. As we do not only breathe quietly but emitting sensors of neuronal activity (reporting calcium possibly also want to dine, sing and dance, for example, such increase, neurotransmitter release, or membrane depolariza- in vivo control cannot just rely upon autonomous pacemaker tion) have begun to reveal how information is represented by cells rigidly driving a ventilator pump, but requires in a highly neuronal assemblies, and how these representations are trans- dynamic plasticity of neural network control. formed during the computations that inform behaviour. Light- To increase the options for such varying behavioural pheno- driven actuators control the electrical activity of central neu- types, mammals have developed quite a complex network rons in freely moving animals and establish causal connections involving diverse assemblies of distinct classes of respiratory between the activities of specific neurons and the expression neurones localized in the ventral region of the brainstem that of particular behaviours. The combination of finely resolved extends in a rostro-caudal direction from the pons to the cer- optical field sensing and finely resolved optical field actuation vical spinal cord. A commanding “kernel”, however, is localized is opening new dimensions for the analysis of the connectivity, in the pre-Bötzinger Complex (pre-BötC) just caudal to the com- dynamics, and plasticity of neuronal circuits, and perhaps even pact division of the nucleus ambiguus. Its vital role was demon- for replacing lost - or designing novel - functionalities. strated by the finding that in vivo rhythmic respiratory output Where applicable, the authors confirm that the experiments from the brainstem disappears when the pre-BötC is lesioned. described here conform with The Physiological Society ethical Such findings allowed the isolatation of a spontaneously active requirements. respiratory network in “reduced” preparations, such as the “en bloc” brainstem-spinal cord preparation or the ‘rhythmic’ brainstem slice of neonatal rat or mouse containing the pre-BötC. These preparations endogenously generate a “respiratory-like” PL3 rhythmic activity. The activity pattern is, however, quite different to the normal (eupneic) pattern as seen in more intact My genes made me eat that! - The Physiological Society preparations like the in situ network in the arterially perfused Public Lecture working heart-brainstem preparation of rat and mouse or the fully intact in vivo preparations of anaesthetized mini-pig, cat, S. O’Rahilly rat or mouse. A clear distinction between the “quality” of the Institute of Metabolic Science, University of Cambridge Metabolic various preparations can be drawn by the capacity of the net- Research Laboratories, Cambridge, UK work in the preparation to alter inspiratory and post-inspiratory activity patterns and oscillatory frequency to changes in The recent and rapid increase in the prevalence of obesity in behaviour or metabolic demands as well as their ability to most developed and developing countries has correctly focused demonstrate dynamic plasticity in response to modulating attention on environmental determinants of that secular trend. influences. However, a fuller understanding of the factors determining any The lecture will start with the cellular biophysics and continue individual person’s adiposity requires appropriate considera- with various aspects of synaptic interaction and integration to tion of inheritance. Studies of twins, adoptees and adopted explain how under in vivo conditions potential pacemakers are twins provide incontrovertible evidence that heritable factors kept under harsh control. It will then address the repertoire of play a major, perhaps even the major factor, in determining a metabotropic receptors acting through separate and conver- person’s fatness. Until recently, the precise mechanisms gent signal pathways onto ionotropic receptors and describe whereby such genes might influence fatness were obscure. the molecular and cellular processes underlying a vital network However, in the past decade we have witnessed an explosion plasticity that includes unexpected changes in network con- of information regarding the molecular mechanisms underly- figuration to maintain rhythmicity. The final aspect will deal ing the control of mammalian energy balance. That informa- with translational approaches to pharmacotherapies treating tion, much of it originating from animal models, is beginning life-threatening disturbances of network functions. to demonstrate its clear relevance to human energy balance.

1P Plenary Lectures

Thus, defects in several individual genes have now been demon- Bridget Marie Flaherty Professor of Neurology, Neurobiology strated to result in human obesity. One of these (MC4R defi- and Pharmacology ciency) is not uncommon, being responsible for up to 5% of Chairman, Dept. of Neurology severe obesity in childhood, and one rare form (congenital leptin deficiency) is amenable to life-saving treatment. Impor- Director, Center for Neuroscience & Regeneration tantly, the vast majority of single gene /Neurorehabilitation Research defects causing human obesity do so through the impairment of satiety. Yale University School of Medicine & VA Connecticut Evidence is accumulating that more subtle genetic variants affecting these pathways underlie susceptibility and resistance Where applicable, the authors confirm that the experiments to common forms of obesity in the general population. Far from described here conform with The Physiological Society ethical encouraging a mood of deterministic nihilism, the more pre- requirements. cise knowledge of biological pathways that genetic information provides should assist the prevention and treatment of human obesity by allowing the design of better therapies and PL5 improving the focus and precision of behavioural strategies for treatment and prevention. Neurotransmitter and neuromodulatory mechanisms at Where applicable, the authors confirm that the experiments peripheral arterial chemoreceptors – The Physiological described here conform with The Physiological Society ethical Society Michael de Burgh Daly Lecture requirements. C.A. Nurse Department of Biology, McMaster University, Hamilton, ON, Canada In mammals, peripheral arterial chemoreceptors located pri- PL4 marily in the carotid body (CB) play a key role in the maintenance of blood PO2, PCO2, and pH homeostasis, via the reflex Fire, Fantoms and Fugu: Sodium Channels from Squid to control of ventilation. The polymodal nature of this sensory Clinic - The Physiological Society Annual Review Prize Lecture organ is further emphasized by more recent evidence sug- S.G. Waxman gesting a role in the sensing of blood osmolarity, temperature, and glucose. The CB contains clusters of chemoreceptor (type Department of Neurology, Yale University School of Medicine & 1) cells which receive afferent sensory innervation from the pet- VA Connecticut, New Haven, CT, USA rosal ganglion via the carotid sinus nerve (CSN), a branch of the glossopharyngeal (GPN) nerve. The afferent CSN discharge in Hodgkin and Huxley’s seminal studies on the squid giant axon turn provides the peripheral input to the central pattern gen- - carried out before the advent of patch clamp or modern molec- erator in the brainstem, leading to the control of respiration. ular biology - clearly established the pivotal role of sodium chan- Since the pioneering studies of Fernando De Castro and nels in action potential conduction. Since then, it has become Corneille Heymans in the 1920’s and 1930’s on the structure clear that there are ten isoforms of sodium channels, sharing a and function of the CB, there has been considerable interest in common overall motif but with different amino acid sequences the transduction and neurotransmitter mechanisms that oper- and different physiological properties. This review will exam- ate during chemoexcitation. In this lecture, I will begin with a ine recent progress on the roles of these different sodium chan- brief historical overview of some of the key studies that led to nels in mammalian neurons, with emphasis on their multiple our current understanding of the role of CB neurotransmitters roles in the human nervous system, both healthy and diseased, and neuromodulators in shaping sensory output and CSN dis- using multiple sclerosis and neuropathic pain as model diseases. charge. While it is now generally accepted that the neuro- We will discuss the role of specific sodium channels in driving transmitter ATP and P2X purinergic receptors play important reverse sodium-calcium exchange which triggers axonal degen- roles in chemoexcitation, other CB transmitters and modula- eration and irreversible worsening, and the role of other sodium tors including ACh, dopamine (DA), 5-HT, adenosine, and GABA channels in restoring conduction along demyelinated axons, are also thought to contribute to the afferent response. I will which permits remission (recovery of function) in multiple scle- also review studies from my laboratory based on a unique co- rosis. We will also discuss changes in sodium channel expres- culture model containing rat type 1 cell clusters and petrosal sion following axonal injury which contribute to DRG neuron neurons, that contributed to current ideas and working mod- hyperexcitability that underlies neuropathic pain. Finally, we els on sensory processing in the CB. will describe loss-of-function and gain-of-function mutations In addition to the ‘afferent excitatory’ pathway discussed above, of the Nav1.7 sodium channel which produce insensitivity to the CB also receives an ‘efferent inhibitory’ innervation via a pain, and the “man on fire” syndrome, erythromelalgia, in plexus of neuronal nitric oxide synthase (nNOS) -positive fibers humans, and which both establish the role of this channel in originating from autonomic neurons embedded within the GPN human nociception and suggest new therapeutic strategies. nerve (i.e. GPN neurons). Much less is known about the mech- These examples underscore the continuing relevance of the anisms underlying activation of this pathway, leading to release original studies on sodium channels, carried out in a model sys- of nitric oxide (NO) and inhibition of CB receptors. I will review tem, the squid. some of our more recent studies, based on a different co-cul- Stephen G. Waxman, M.D., Ph.D. ture model of type 1 cell clusters and isolated GPN neurons,

2P Plenary Lectures suggesting an additional involvement of ATP and P2X receptors in the generation of the NO signal and negative feedback inhibition of CB receptors. The reasons for this complex pattern of regulation of CB function by multiple neuroactive agents are presently unclear. Conceivably, they may contribute to the mechanisms underlying CB plasticity during patterned stimulation, e.g. chronic sustained hypoxia and chronic intermittent hypoxia, as experienced during cardiorespiratory disorders such as chronic obstructive pulmonary disease and sleep apnea. Studies in the author’s laboratory were supported by grants from the Canadian Institutes of Health Research. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

3P Demonstration

Analysis of the RyR-Channel Stochastic Dynamics in the Electron-Conformational Model A.M. Ryvkin1, A.S. Moskvin2 and O.E. Solovyova1,2 1Laboratory of Mathematical Physiology, Institute of Immunology & Physiology RAS, Ekaterinburg, Russia and 2Ural State University, Ekaterinburg, Russia Methods: We have proposed a simple, biophysically reason- able electron-conformational theory for the ryanodine receptor channel (RyR) to be a main element governing the calcium dynamics in a cardiac cell [1,2], starting with the well-known model theory of the photo-induced structural phase transitions [3]. The main feature of our model is that, in addition to a fast electronic (dichotomic) degree of freedom, the RyR channel is characterized by a slow classical conformational coordinate, Q, that obeys the Langevin dynamics. Two minima of a conformational potential (CP) are related with a closed and open RyR state, respectively. We took into account the calcium induced direct electronic Franck-Condon transitions between the CP branches and different relaxation mechanisms. Results: We have performed a series of computer simulations of a single RyR stochastic gating under steady-state conditions and made a Hurst analysis of the channel’s conformational coordinate Q(t). Dynamics of our system appears to be strongly correlated (H ≈ 1.0) for relatively short times compared with the characteristic times of conformational relaxation to the metastable minimum of CP, and weakly correlated (H ≈ 0.5) for larger time intervals. A.S. Moskvin et al, Doklady Biochemistry and Biophysics, 400 : 32- 37, 2005. A.S. Moskvin et al, Progress in Biophysics and Molecular Biology, 90: 88-103, 2006. K. Koshino, T. Ogawa, Journal of Luminescence, 87-89: 642, 2000.

Supported by RFBR Grants No 07-04-96126. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

4P Oral Communications

Rook WHR, Glen KE, Marshall JM, Coney AM. Changes in the regulation C1 of vascular tone in adult male rats exposed to chronic hypoxia in utero. Proc Physiol Soc. 2008;11: C85. Chronic hypoxia in utero (CHU) increases superoxide Cosentino F, Luscher TF. Tetrahydrobiopterin and endothelial nitric oxide production in adult rat skeletal muscle vasculature synthase activity. Cardiovasc Res. 1999;43: 274-8.

W.H. Rook, J.M. Marshall and A.M. Coney Funded by BHF

School of Clinical and Experimental Medicine (Physiology), Where applicable, the authors confirm that the experiments University Of Birmingham, Birmingham, UK described here conform with The Physiological Society ethical requirements. We have shown that chronic hypoxia in-utero (pregnant dams breathing 12% O2 during the second half of pregnancy) may lead to a reduction in nitric oxide (NO) bioavailability in the vas- 1 - culature of the adult offspring . Superoxide anions (O2 )are implicated in reducing NO bioavailability by directly reacting C2 with NO to produce peroxynitrite. Superoxide dismutase (SOD) - catalyses the dismuting of O2 into H2O2 and O2,thuspro- The respiratory profiles of Dexmedetomidine in paediatric tecting NO bioavailability. In light of this, we have now inves- patients following cardiac surgery tigated the role of SOD in skeletal muscle vasculature in CHU 1 2 2 3 offspring. N.J. Nordin , S. Kadiman , F. Jaffar and S. Hamil -1 -1 Under anaesthesia (Alfaxan; 3-6ml hr kg i.v.), we recorded 1Physiology, Science Islam University of Malaysia, Kuala Lumpur, arterial blood pressure (ABP) and femoral blood flow (FBF) Wilayah Persekutuan, Malaysia, 2Anaesthesiology, National Heart in 9-10 week old normal (N, n=12) rats and in age-matched Institute, Malaysia, Kuala Lumpur, Wilayah Persekutuan, Malaysia CHU (CHU, n=8) offspring. Femoral vascular conductance and 3Physiology, National University Malaysia, Kuala Lumpur, (FVC) was calculated (FBF/ABP) and integrated FVC (IntFVC) Wilayah Persekutuan, Malaysia used to give an indication of vascular tone. Variables were recorded during air breathing and during 10min periods of Dexmedetomidine is a potent sedative, analgesic and sym- acute systemic hypoxia (breathing 8% O2) before and dur- patholytic agent. The sympatholytic effects of Dexmedito- ing infusion of the cell permeant SOD inhibitor midine are associated with decrease in endogenous concen- Diethyldithiocarbamate trihydrate (DETC; 5mg-1.kg-1.min- trations of noradrenaline resulting in dose-dependant 1 i.v.) decreases in arterial blood pressure and heart rate that lead Baseline ABP was similar in both groups (N:136±3, to perioperative hemodynamic stability with a decrease risk CHU:143±5mmHg) as was baseline IntFVC (N:7.9±1.1, of adverse perioperative cardiac events(1,2). Series of case CHU:6.3±1.1 CU) resulting in similar FBF (N:5.4±0.6, reports by Tobias et. al enhanced the focus on the usage of CHU:4.6±0.9ml-1min-1kg-1). As previously shown, responses Dexmedetomidine among pediatric patients(3). The objective to acute hypoxia were similar in both N and CHU with both of this study was to assess the respiratory profiles including showing a fall in ABP, an increase in IntFVC which maintained respiratory rate, SPO2 and PaO2/ FIO2 ratio of pediatric FBF at control levels. DETC caused a small increase in ABP, patients on Dexmedetomidine. It is hoped that we may eval- although not statistically significant (N:137±3, CHU:149±3 uate the potential benefits of Dexmeditomidine in pediatric mmHg). DETC also caused a significant reduction in IntFVC cardiac patients. The study was randomized controlled dou- (N:0.6±0.7, CHU:1.8±0.6) in CHU, but not N rats, which was ble blinded comparing Dexmedetomidine sedation to insti- mirrored by changes in FBF (N:-0.2±0.4, CHU:-1.1±0.3). tution’s standard for postsurgical sedation. The study was Inducing acute hypoxia during DETC infusion caused ABP to approved by the institution’s ethical committee. Patients were fall. However, it remained at a level higher than under con- randomized in a 1:1 fashion into two study arms, Group A trol conditions in both N and CHU (N:79±5, CHU:77±3). received infusion of dexmedetomidine between 0.1 to 0.7 There was no difference in the magnitude of muscle vasodi- mcg/kg/hr (n= 25) and Group B, as control group received latation induced by hypoxia as indicated by a similar increase midazolam between 0.5 to 2 mcg/kg/min (n: 25). The drug in IntFVC. infusion was performed under strict protocol by the anesthe- - SOD normally dismutes O2 into the vasodilator H2O2, there- siologist involved. The study drugs were started after patients - fore, SOD antagonism results in the build up of (O2 and induces arrived in ICU with stable haemodynamic. Results showed no vasoconstriction. The reduction in FVC in air breathing CHU rats significant differences in respiratory rate; O2 saturations; arte- during DETC administration suggests increased basal produc- rial pH and PaCO2 between both groups compared (p>0.05). - tion of O2 anions that is not apparent in N rats. It is possible Although the arterial partial O2 tension (PaO2): fractional - that this O2 production is through uncoupling of nitric oxide inspire O2 (FiO2) ratios were slightly lower in dexmeditomi- synthase2. However, in keeping with our previous findings with dine group, they was not significantly different from the con- shorter periods of hypoxia1, the acute response to hypoxia and trol group (p>0.05). Our observation tends to show that is similar in both N and CHU rats. Further, there is no apparent dexmeditomidine does not have clinically important adverse - difference during DETC infusion suggesting the increased O2 impacts on respiration in the postsurgical paediatric patients production in CHU rats is only evident during air breathing. Pre- who requires intensive care. Hence it appears that dexmedit- liminary findings in the carotid artery suggest there may also omidineissafeforpostoperationsedationinselectedpaedi- be differences in other vascular beds, warranting further work. atric heart surgery(4).

5P Oral Communications

affected by dietary restriction. This work evaluated the alterations in MAP and heart rate (HR) of Fisher rats fed for 35 days after weaning with regular (15%) or low (6%) protein diets, before and after systemic administration of the angiotensin converting enzyme inhibitor (iACE) or AT1 receptor antagonist. Under ketamine (80 mg/kg) plus xylazine (7 mg/kg) anesthesia, polyethylene cannulas were inserted into the femoral artery to record arterial pressure and into the vein for drug injections one day before the experiments. All procedures and experimental protocols were conducted in accordance with the Brazil- ian Society for Neuroscience and Behavior instructions for the use of animals in research. MAP levels in malnourished rats were greatly dependent on RAS because the inhibition of angiotensin converting enzyme with enalapril or the blockade of AT1 receptors with losartan produced greater fall in the blood pressure of malnourished rats compared to control rats (-37±4 mmHg vs. -3±2 mmHg, n=8 and -41±5 mmHg vs. -3±1 mmHg, n=8, respectively). To further investigate the relative contributions of angiotensin II acting directly on smooth muscle cells versus through the sympathetic nervous system to maintain blood pressure in malnourished rats, experiments were carried out with peripheral α1 adrenergic and AT1 receptors blockade. The blockade of α1 receptors with prazosin after losartan further decreased the MAP in both groups (-29±7 mmHg in control, n=8 and -17±7 mmHg in malnourished, n=8). Prazosin was less effective than losartan to reduce MAP in malnourished rats when compared to control rats that underwent the same treat- Mukhtar,A.M.,Obayah,E.M.,Hassona,A.M.2006.TheuseofDexmedeto- ment judged by the changes due to AT1 blockade versus AT1 midine in Pediatric Cardiac Surgery. Anesth Analg. 103: 52-56. plus α1 blockade (-8±3 mmHg and -29±4 mmHg respectively Venn, R.M., Grounds, R.M. 2001. Comparison between dexmedeto- in control, n=8 vs. -44±5 mmHg and -17±7 mmHg respectively midine and propofol for sedation in the intensive care unit: patient and in malnourished, n=8). When prazosin was given first, mal- clinical perceptions. British Journal of Anaesthesia. 87(5): 684-690. nourished rats also presented greater fall in MAP compared to Tobias, J.D. 2004. Sedation during mechanical ventilation in infants and control (-38±3 mmHg vs -8±2 mmHg respectively). The sub- children: dexmedetomidine versus midazolam. South Med J. 97(5): sequent administration of losartan produced further and sim- 451-455. ilar fall in MAP for both groups (-25±3 mmHg in control and - Koroglu, A., Demirbilek, S., Teksan, H., Sagir, O., But, A.K., Ersoy, M.O. 22±4 mmHg in malnourished). The present results suggest that 2005. Sedative, haemodynamic and respiratory effects of dexmedeto- ongoing production of angiotensin II and its action on AT1 midine in children undergoing magnetic resonance imaging examination: preliminary results. Br J Anaesth. 94: 821-824. receptors are critical factors supporting the blood pressure in malnourished rats and that α1 receptors activation could be ThisstudywassupportedbytheNationalHeartInstitute,Malaysia. under strong influence of angiotensin II. Where applicable, the authors confirm that the experiments FAPEMIG and CNPq. described here conform with The Physiological Society ethical requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C3

Blood pressure in low protein diet rats is highly dependent C4 on renin-angiotensin system Gender differences in the renal sympathetic nerve response J.M. Gomide1, L.G. Fernandes1, F.C. Silva1, M.E. Silva2, to myocardial infarction L.M. Cardoso1, A.R. Massensini3, M.F. Moraes3 and D. Chianca1 1Biological Sciences, Universidade Federal de Ouro Preto, Ouro M. Pinkham, C. Barrett, S. Guild and S. Malpas Preto, Minas Gerais, Brazil, 2Department of Foods, Universidade Physiology, University of Auckland, Auckland, New Zealand Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil and Elevated renal sympathetic nerve activity (RSNA) in heart fail- 3 Department of Physiology and Biophysics, Universidade Federal ure is correlated with poor outcome, but little is known about de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil what triggers the increase in sympathetic tone or indeed at Previous data from our laboratory showed that 344 Fisher rats what stage the increased occurs. While males are more likely fed with a protein deficient diet presented a slight increase in to experience myocardial infarction (MI) than females, females mean arterial blood pressure (MAP). It has been suggested in often have a worse prognosis. Interestingly females are also the literature that the renin-angiotensin system (RAS) could be less likely to be prescribed beta-blockers.

6P Oral Communications

The aim of this project was to determine if there are gender dif- the major integrative site of stress response. Under ferences in the RSNA response immediately following a myocar- xylazine/ketamine anesthesia (0.4ml 10% ketamine IP, 0.1ml dial infarct (MI). 2% xylazine IP per animal) TA-11-PA C40 DSI implants were The University of Auckland Animal Ethics Committee approved introduced in aorta followed by metamizol treatment (200mg, allexperiments.MaleandfemaleWistarratswereanaesthetised IP). After ten days rats were exposed to stereotaxic surgery with urethane (1000-1500 mg/kg I.P) and α-chloralose (80-120 (anaesthetics as above): glass micropipettes containing 1:1 mg/kgI.P).Ratswereintubatedandventilated.Theleftrenalartery mixture of the replication-deficient adenoviral vectors wasexposedandtherenalnervesdissectedforrecordingofRSNA Ad.CMV.eGFP (titer, 1.57x1010 pfu/ml) plus Ad.CMV.V2 (2.5 alongwitharterialpressure(femoralartery)andECG.Aleftinter- x1010pfu/ml) or Ad.CMV.eGFP (control), were injected in PVN costalthoracotomywasperformedtoexposetheheart,theperi- (Qiu et al. (2007)). Seven days elapsed before full expression of cardiumwasremoved.Afteraonehourperiodofbaselinerecord- transfected genes and beginning of the 10 minute air-jet stress ing the left coronary artery was ligated using a 6-0 suture. In the protocol. Only animals in which eGFP expression was observed shamgroupthechestwasopened,pericardiumremoved,asuture within the PVN were subjects of cardiovascular signal analysis inserted around the coronary artery but not tied. and one-way ANOVA statistical significance evaluation. All Inmalerats(n=7)coronaryligationresultedinabruptincrease(32 experiments were performed in accordance with Directive ±12%,mean+sem)inRSNAbeforesubsidingtolowersteadystate 86/609/ECC. Systolic (SBP), diastolic blood pressure (DBP) and within10minutesoftheinfarctforthenext2hours(13±4%above heart rate (HR) were derived from the arterial blood pressure thebaselinelevel,P<0.05,ANOVA).Incontrastinfemalerats(n=7) digitalized at 1000Hz, as maximum, minimum and inverse of no significant changes in RSNA were observed in response to interbeat interval, respectively. Evaluation of the spontaneous myocardial infarction. No change in RSNA was observed in sham BRR was performed by using the method of sequences and cal- operated rats (n=10). Arterial pressure responses were also dif- culation of baroreceptor reflex sensitivity (BRS) and effective- ferent between the male and female rats; two hours post-infarct ness index (BEI). Time spectra were created using fast Fourier arterial pressure was 10 ± 5mmHg below that seen in the sham transform algorithm on 15 overlapping 2048 point time series group (P < 0.05), whereas arterial pressure in the male rats was involving 410-s registration period. Spectra were analyzed in not significantly different to the shams. Baseline baroreflex con- very-low-frequency (VLF:0.0195-0.195Hz), low-frequency trolofRSNAdifferedbetweenthesexeswiththefemalesdisplaying (LF:0.195-0.8Hz) and high-frequency (HF:0.8-3Hz) range. Basal a significantly reduced maximum RSNA response to decreases in values of cardiovascular parameters did not differ between mean arterial pressure compared to males (64±7% vs. 89±6%, groups. Exposure of rats to air-jet stress increased P>0.05).Inboththemalesandfemales,neithershamsurgerynor SBP(137.81±5.36mmHg, p<0.05, and 144.23±5.14mmHg, myocardial infarction had a significant effect on baroreflex con- p<0.05), DBP(107.94±3.36 mmHg, p<0.01 and troloverheartrateorRSNAat60minafteroftheintervention.Ani- 107.92±5.85mmHg, p<0.01), HR(447.92±16.59Hz p<0.01 and malswerehumanelykilledwitheuthatalattheendofthesestud- 465.57 Hz p<0.01), LF SBP(3.97±0.56mmHg2/Hz, p<0.05 and ies. Post-mortem analysis showed the coronary ligation resulted 4.58±0.7256mmHg2/Hz, p<0.05) and HF SBP(1.79±0.36 in a non-perfused area of left ventricle of 35-50%. mmHg2/Hz, p<0.05 and 3.20±1.75 mmHg2/Hz, p<0.001)in These results indicate that gender exerts a major influence on both groups of rats. In HR spectra, only transfected rats exhib- the resulting sympathoactivation occurring after MI. We sug- ited significant increase of LH HR(81.59±47.12 mmHg2/Hz, gest that different treatment strategies may justified based p<0.05) and HF HR(119.5±63.2 mmHg2/Hz, p<0.05) and on gender after MI. reduced LF/HF HR ratio. In control rats exposure to air-jet stress This work was supported by the University of Auckland and the reduced BRS, whereas in transfected rats BRS remained close Health Research Council of New Zealand to baseline values. The results indicate that overexpression of V2 receptors in PVN of rats increases vagal control of the heart Where applicable, the authors confirm that the experiments and preserves the baroreceptor reflex functioning during stress. described here conform with The Physiological Society ethical Qiu J et al.(2007). J Neurosci 27(9):2196-2203. requirements. Stojicic S et al. (2008). Neuropharmacology 54(5):824-36. Where applicable, the authors confirm that the experiments C5 described here conform with The Physiological Society ethical requirements. Upregulation of vasopressin V2 receptors in paraventricular nucleusofratsmodulatescardiovascularresponsetoacutepanic M. Lozic1, O. Sarenac1, J.F.R. Paton2,D.Murphy2 and C6 N. Japundzic-Zigon1 Central drives to cardiac ganglion neurons of rat: an 1 Institute of Pharmacology, Clinical Pharmacology and Toxicology, intracellular analysis in situ School of Medicine, Belgrade, Serbia and 2Bristol University, 1 1 2 3 Bristol, UK A.E. Pickering , J.F.R. Paton , A.A. Harper and R.M. McAllen 1 2 In a paper by Stojicic et al (2008) we found that central V2 vaso- University of Bristol, Bristol, UK, University of Dundee, Dundee, 3 pressin antagonist modulates cardiovascular homeostasis. To UK and Howard Florey Institute, Melbourne, VIC, Australia further asses the role of central vasopressin V2 receptors we Heart rate variability is largely dependent on cardiac vagal activ- performed experiments in adult male Wistar rats overex- ity and its loss is an independent risk factor for arrhythmias and pressing V2 receptors in hypothalamic paraventricular nucleus, cardiac mortality. Thus, it is important to elucidate the factors

7P Oral Communications that modulate cardiac vagal ganglionic transmission in health h) activity patterns are observed and are strongly influenced and in cardiovascular disease states. Although cardiac vagal by environmental stimuli (1). Evidence to date suggests that ganglionic transmission has been studied in a number of in vitro horses housed in stabled conditions display diurnal (daytime) preparations (Edwards et al 1995) there have been no intra- activity rhythms (2). However, this contrasts with observations cellular recordings from cardiac ganglion neurones in a func- of feral horse populations where ultradian activity bouts tionally intact setting to permit the detailed analysis of synap- throughout the 24-hr cycle have been suggested (3). The true tic integration and modulation at this site. endogenous nature of a circadian rhythm can only be confirmed Using the working heart-brainstem preparation (Paton, 1996) under constant conditions in the absence of time cues. This from Wistar rats (aged 4-5 weeks, initially anaesthetised with study determined the activity patterns of horses in their natu- Halothane), we have obtained the first intracellular recordings ral environment (Pasture) and under both a light/dark (LD) and from functionally connected cardiac vagal ganglion cells in situ. constant dark (DD) stabled environment. Six mares of light- The atria were dissected from the ventricles, pinned flat and weight breed were fitted with Actiwatch-L (Respironics, Bend, stabilized with a nylon mesh foot. Intracellular recordings of 33 OR) monitors (for measurement of activity and light intensity) cardiac ganglion cells were made with sharp microelectrodes and were maintained for successive 48-h periods at pasture, (0.5M KCl; 80-120 MOhm). Stable recordings were obtained for in individual stalls within a lightproof barn under LD, and finally periods of over 30 minutes allowing examination of intrinsic in DD. Actiwatch data were used to create ClockLab (Actimet- properties, spontaneous firing pattern and responses to vagus rics, Evanston, IL) compatible files. ClockLab’s batch analysis nerve (right) evoked and cardiorespiratory reflex activation: function was used to compute average activity counts/min peripheral chemoreflex (NaCN, 0.03% i.a.; baroreflex, by increas- for each mare in each treatment interval. A bout analysis func- ing pump flow; diving response, 10∞C saline applied to snout. tion was used to quantify the ultradian structure of activity data Severaldifferentclassesofneuronewereidentifiableonthebasis identifying distinct bouts of high activity. Initial examination of their intrinsic electrophysiology, reminiscent of Edwards et of the actigraphs revealed distinct ultradian activity patterns al. (1995), and distinct patterns of spontaneous and reflex with a mean of 9 bouts/day (S.D.±3.1) One-way repeated meas- evokedactivities.ActiveneuronesshowedEPSPsand/oraction ures ANOVA was used to analyse; average counts/min, activ- potentials(AP)thatoccurredmostcommonlyinthepost-inspi- ity bouts/day, average bout length and percentage of activity ratoryphase.Intheseneuronesactivationofbradycardicreflexes counts/light phase (subjective day in DD) across the three treat- (baro-, chemo-, nasotrigeminal) increased EPSP frequency ments (Pasture, LD, DD). Results reveal significantly higher and/orAPwithatrialrhythmslowing.Spontaneous,vagus-and activity counts/min at pasture compared to LD and DD reflexly- evoked AP appeared mostly triggered by suprathresh- (p<0.001). Mares at pasture demonstrated reduced bouts/day old unitary EPSPs rather than by summation of subthreshold compared to LD and DD (p<0.001) and increased bout length EPSPs.VagusstimulationevokedEPSPsatalatencyof30-40ms. compared to DD (p<0.01). However, mares demonstrated a Inconclusion,itispossibletoobtainhighresolution,stableintra- greater percentage of activity within the light phase in DD com- cellularrecordingsfromphysiologicallyintactcardiacvagalgan- pared to pasture and LD (p<0.001). In addition, cosine analy- glion cells, which exhibit appropriate patterns of ongoing and sis (4) of the time series data identified a significant 24-h com- reflexexcitatorysynapticdrives.Futurestudieswillelucidatehow ponent of the activity rhythms with significantly increased cardiacvagaltransmissionmaybemodifiedinhealthanddisease. robustness (goodness of fit values) associated with DD (p<0.05). EDWARDS, F. R., HIRST, G. D., KLEMM, M. F. & STEELE, P. A. (1995) Dif- In summary, mares display activity patterns that are weakly cir- ferent types of ganglion cell in the cardiac plexus of guinea-pigs. J Phys- cadian and predominantly ultradian in nature. It is proposed iol, 486 ( Pt 2), 453-71. that the DD condition, investigated in horses for the first time, PATON, J. F. (1996) A working heart-brainstem preparation of the permits greater unmasking of endogenous circadian periodic- mouse. J Neurosci Methods, 65, 63-8. ities in the absence of environmental stimuli such as social cues. Elucidating the nature of activity rhythms in the horse will have RMcA holds an NHMRC Principal Research Fellowship; JFRP holds implications for future studies investigating diurnal variations a Royal Society Wolfson Research Merit Award; AEP is a Well- in performance parameters in the equine athlete. come Trust Advanced Clinical Fellow 1. van der Veen DR, Minh NL, Gos P, Arneric M, Gerkema MP, Where applicable, the authors confirm that the experiments Schibler U. Impact of behavior on central and peripheral circadian clocks described here conform with The Physiological Society ethical in the common vole Microtus arvalis, a mammal with ultradian rhythms. requirements. Proc Natl Acad Sci U S A 2006; 103: 3393-3398. 2. Piccione G, Costa, A., Gianetto, C., Caola, G. Daily rhythms of activity in horses housed in different stabling conditions. Biological C7 Rhythm Research 2007; 39: 79-84. 3. Berger AS, KM; Eichhorn, K; Scheibe, A; Streich, J. Diurnal and Equine activity rhythms exhibit circadian and ultradian ultradian rhythms of behaviour in a mare group of Przewalski horse characteristics under different environmental conditions (Equus ferus przewalskii), measured through one year under semi- reserve conditions. Applied Animal Behaviour Science 1999; 64: 1-17. B.A. Murphy1, A. Martin1 and J.A. Elliott2 1University College Dublin, Belfield, Ireland and 2University Of 4. Nelson W, Tong, Y.L., Lee, J.K., Halberg, F. Methods for cosi- nor-rhythmometry. Chronobiologia 1979; 6: 305-323. California, San Diego, CA, USA Activity rhythms are regulated by the hypothalamic circadian Where applicable, the authors confirm that the experiments pacemaker in response to daily changes in photoperiod. In some described here conform with The Physiological Society ethical mammalian species, both circadian (~24 h) and ultradian (< 24 requirements.

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Hoydal MA, Wisloff U, Kemi OJ, and Ellingsen O. Running speed and C8 maximal oxygen uptake in rats and mice: practical implications for exercise training. Eur J Cardiovasc Prev Rehabil 14: 753-760, 2007. The maximal rate of lipids oxidation is strain dependant and corrrelated with performance in running mice Jeukendrup AE, and Wallis GA. Measurement of substrate oxidation during exercise by means of gas exchange measurements. Int J Sports L. Mille-Hamard, E. Henry and V.L. Billat Med 26 Suppl 1: S28-37, 2005. Universite Evry-Val d’Essonne, UBIAE INSERM U902, Evry, France Lightfoot JT, Turner MJ, Debate KA, and Kleeberger SR. Interstrain variation in murine aerobic capacity. Med Sci Sports Exerc 33: 2053- In mice model, the most currently exercise performance crite- 2057, 2001. ria used in running is the maximal running speed (Vpeak) and is considered as an aerobic index (Hoydal et al, 2007), Fur- Pederson BA, Cope CR, Schroeder JM, Smith MW, Irimia JM, Thurberg BL, DePaoli-Roach AA, and Roach PJ. Exercise capacity of mice geneti- thermore links between performance and the lipid metabolism cally lacking muscle glycogen synthase: in mice, muscle glycogen is not was not explored in mice, despite the fact that the maximal rate essential for exercise. J Biol Chem 280: 17260-17265, 2005. of lipid oxidation (Lipox max) has been reported to be a major performance factor and to be training sensitive in man (Brooks Where applicable, the authors confirm that the experiments and Mercier, 1994, Jeukendrup and Wallis, 2005). Therefore, described here conform with The Physiological Society ethical the purpose of this study is to test the hypothesis that the lipids requirements. metabolism and also the anaerobic metabolism, between vVO2max and Vpeak are, as VO2max, consistent performance factors and could be highly heritable, explaining the high performance difference according the mice strains (Lightfoot et C9 al., 2001). Seven FVB (high performer strain) and 7 C57BL6 (low per- Endocannabinoids prevent lysosomal membrane former) ran an incremental exhaustive run on treadmill in a destabilisation evoked by treatment with b-amyloid in metabolic chamber until maximal speed (Vpeak) which was cultured rat cortical neurones taken as the performance criteria. The respiratory gaz exchange J.J. Noonan, R. Tanveer, S. Cunningham and V.A. Campbell (RER) allowed determination of Lipox max and the associated speed (Vlipox) Department of Physiology, Trinity College Institute of Neuroscience, We found that VO2 increased rapidly and significantly at the Dublin, Ireland beginning of the exercise with incresing speed, but VO2 plateaued early until the end of the exercise (51.7 ± 3.4 vs 48.1 Alzheimer’s disease (AD) is a neurodegenerative disease char- β β ± 3.2 ml.kg-0.75.min-1, in FVB and C57 p=0.07). Vpeak was acterised by deposition of -amyloid (A ) and loss of neurones significantly higher than vVO2max in FVB vs. C57 (167±25 vs. in the cerebral cortex and hippocampus (1). Lysosomal dys- 135±44% of vVO2max, p = 0.011). The Vpeak (rI = 0.72, g2 = function and destabilisation have been implicated in a variety 0.56), Lipox max (rI = 0.53, g2 = 0.36) and the cross over speeds of neurodegenerative events, including the apoptotic pathway β (rI = 0.83, g2 = 0.71) were highly heritable in constrast with evoked by A (2). The endocannabinoid system is emerging as VO2max ( rI = 0.30 g2 = 0.18) and the Accumula ted a promising neuroprotective target (3), thus the aim of this Oxygen Deficit (AOD) (rI=0.11 g2 = 0.05). However, the anaer- research was to explore the ability of the endocannabinoid, obic metabolism, estimated with the AOD, was a consistent anandamide (AEA), to maintain lysosomal membrane integrity factor of performance. Furthermore Lipox max (r = 0.80, p = and confer neuroprotection. 0.0006) was highly related with the performance (vPeak) while Cultured cortical neurones were prepared from one day old VO2max (r = 0.57, p = 0.04) and AOD (r = 0.56, p = 0.04) were Wistar rats. To monitor lysosomal stability, cultured cortical μ moderately. neurones were incubated with acridine orange (AO; 5 g/ml) β μ ± Thus the use of Vpeak for aerobic capacity assessment can not for 10 minutes followed by treatment with A 1-40 (2 M) be performed independently of the strain and the anaerobic anandamide (AEA; 10nM) for 6 hours. Lysosomal destabilisa- capacity is then a factor of performance eventhought AOD was tion was measured by loss of AO fluorescence intensity at not heritable. Indeed, at end exercise, RER was higher for the 633nm. Release of the lysosomal cathepsin enzymes was meas- FVB strain, indicating a greater part of glucose oxidation, and ured using a commercially available kit. Expression of the lyso- a relative higher metabolism sollicitation for this strain. In con- somal associated membrane proteins, LAMP1/2, was assessed strasts with human model, in both strain, the mice had a high by western immunoblotting and apoptosis was measured using Vlipox set between vVO2max and Vpeak. This means that the the TUNEL procedure. mice do not rely to the aerobic glycogen metabolism but rather AO fluorescence intensity at 633nm, reflective of intact lyso- ± on the lipids and the anaerobic metabolism (glycolysis and somes, was significantly reduced from 130 18 (mean fluores- ± ± phosphagene) (Craig et al., 1995, Pederson et al., 2005). In con- cence units SEM) in control cells to 52 10 in cells treated with β clusion, this study demonstrated, for the first time, that the A 1-40 (p<0.01, ANOVA, n=5) and this was prevented in cells β lipid metabolism was not only a consistent performance factor exposed to A 1-40 in the presence of AEA. Cathepsin-L activ- ± but were also highly heritable, and must also be determined ity was significantly increased from 3.7 0.5 (relative fluores- ± ± β when evaluating aerobic performance in mice. cent units, mean SEM) to 7.6 1.2 in cells treated with A 1-40 (p<0.05, ANOVA, n=5) and this was significantly reduced to Craig NP, Norton KI, Bourdon PC, Woolford SM, Stanef T, Squires B, Olds ± β TS, Conyers RA, and Walsh CB. Aerobic and anaerobic indices con- 3.5 0.9 in cells exposed to A in the presence of AEA (p<0.05 tributing to track endurance cycling performance. Eur J Appl Physiol compared to Aβ treatment, ANOVA, n=5). LAMP1 expression Occup Physiol 67: 150-158, 1993. was significantly decreased from 2.70±0.29 (arbitrary units,

9P Oral Communications mean±SEM) to 1.74±0.08 in cells treated with Aβ1-40 com- young and aged rats (p<0.05, ANOVA, n=6). An age-related pared with control (p<0.05, ANOVA, n=5) and this was pre- increase in the expression of MHC II was observed and analysis vented by co-treatment with AEA. LAMP2 expression was signi- of staining intensity in cortex identified a significant increase ficantly increased from 3.7±0.15 (arbitrary units, mean±SEM) in sections prepared from middle aged and aged rats, com- to 5.9±0.23 by Aβ1-40 (p<0.001, ANOVA, n=5) and this was pared with young rats (p<0.05, ANOVA, n=4). A significant reduced to 3.9±0.058 in cells treated with Aβ in the presence increase in CD68 immunoreactivity was observed in sections of AEA. The percentage of TUNEL positive cells was reduced prepared from aged, compared with young, animals (p<0.05, from 24.54±0.96% (mean±SEM) in cells treated with Aβ1-40 to ANOVA, n=4). These data reveal a positive correlation between 7.80±0.75% in control cells (p<0.001, ANOVA, n=5). T2 relaxation time and microglial activation in the cortex of We demonstrate that the endocannabinoid, AEA, prevents the aged rats and identify that some changes in cell surface mark- Aβ1-40 –induced lysosomal membrane destabilisation, and ers of microglial activation are evident in middle age. the subsequent release of the lysosomal enzyme, cathepsin-L. This work was supported by the Health Research Board Ireland. Similarly, AEA prevents the Aβ1-40-evoked changes in LAMP1 and LAMP2 expression. Modification of the lysosomal branch Where applicable, the authors confirm that the experiments of the apoptotic cascade by endocannabinoids could be a ther- described here conform with The Physiological Society ethical apeutic strategy of relevance for AD. requirements. 1. Terry RD et al., (1991) Physical basis of cognitive alterations in Alzheimer’s disease: synaptic loss is the major correlate of cognitive impairment. Ann Neurol 30: 572-580 2. Fogarty MP et al., (2008) A role for p53 in the beta-amy- C11 loid-mediated regulation of the lysosomal system. Neurobiol Aging Relationship between membrane potential and excitability, 3. Campbell VA and Gowran A (2007) Alzheimer’s disease; tak- and the effects of repetitive stimulation on action currents ing the edge off with cannabinoids? Br J Pharmacol 152(5): 655-662 in rat small diameter sensory neurones in vitro Where applicable, the authors confirm that the experiments J.F. Pittaway and M.D. Baker described here conform with The Physiological Society ethical requirements. Neuroscience, Queen Mary University of London, London, UK The relationship between membrane potential and excitability for C-fibres has not been established, and repetitive activation of C-fibres gives rise to a slowing of impulse conduction C10 velocity where the likely mechanism, previously ascribed to the electrogenic activity of the Na-pump, has been questioned Evidence of an inverse correlation between T2 relaxation recently by De Col et al. (2008). In order to shed light on these time and microglial activation in the cortex of aged rats issues, we have explored the effects of changing membrane R.E. Gonzalez-Reyes, K. Murphy, R. Bechara, C. Blau, C. Kerskens potential and also repetitive activation on the excitability and and M. Lynch action currents recorded in rat primary cultured dorsal root ganglion (DRG) neurones (≤ 25μm in apparent diameter), in Trinity College Dublin, Dublin, Ireland vitro. DRG neurones were maintained in culture for 24-48 hours Cellular senescence is associated with changes in the function and live stained with fluorescein conjugated IB4-lectin, allow- and structure of neurons and glia and one consistent finding is ing identification of IB4+ve and –ve populations. an increase in the activation state of microglia identified by Current-clamp experiments were performed using whole-cell increased expression of cell surface markers and increased pro- patch-clamp with quasiphysiological solutions and at 20-22∞C. duction of proinflammatory cytokines. In this study, we set out Neuronal membrane potential was initially held at -60 mV, incre- to investigate age-related changes in the expression of two cell mentally hyperpolarized to near -100 mV, and then depolar- surface markers of microglial activity in the cortex of young, ized through -60 mV until the membrane ceased to be excitable. middle-aged and aged male Wistar rats and to assess whether At all membrane potentials, threshold was measured in these changes were associated with any change in T2 relaxation response to 10 ms current steps, applied at ≥ 2 second inter- time as assessed by magnetic resonance imaging (MRI). vals. Over the potential range -100 to -20 mV, IB4 +ve neurones Young, middle-aged and aged animals were anaesthetized (iso- became more excitable with increasing depolarization until fluorane (4% induction, 1.5-2% maintenance) in 100% O2), posi- excitability was lost abruptly and completely positive to -20 tioned in a custom-built cradle and placed in 7 Tesla BioSpec mV. In contrast, IB4-ve neurones were most excitable near -55 animal scanner (Bruker BioSpin, Ettlingen, Germany). T2 relax- mV (lowest threshold at: -26.3 ± 2.3 mV and -52.9 ± 6.0 mV, ation times were obtained from high-resolution scans and at means ± S.E.M., IB4 +ve (n = 8) and IB4 –ve (n = 5), respectively; the end of the period of acquisition, animals were transcardially P< 0.01 Student’s t-test). We also report that repetitive, perfused and brain tissue was taken for preparation of cryostat suprathreshold activation affected the amplitude of action cur- sections and immunohistochemical analysis. Sections were rents. When holding the membrane potential of IB4 +ve neu- stained for the expression of MHC II (Major Histocompatibility rones at -60 mV and stimulating at 2 Hz, inward transmem- Complex Class II) and CD68 which are two markers of microglial brane current was gradually suppressed during the train, and activation. subsequently recovered at 0.5 Hz. Steady depolarization of the An age-related decrease in T2 relaxation time in the cortex was membrane potential to -50 mV caused a proportionally greater observed with a significant difference between the values in suppression of the inward current during a train, paralleling the

10P Oral Communications effect of ouabain on C-fibre conduction latencies (De Col et al. identified cortical and striatal cells which we measure, with 2008). IB4-ve neurones also showed suppression of inward experiments where the currents are generated by direct elec- transmembrane current, although the kinetics at -60 mV were trical stimulation, or by the uncaging of glutamate, close to a more variable than for IB4 +ve. Our findings show the excitabil- single spine in slices of the corticostriatal system from mice. ity of IB4 +ve neurones increases with membrane depolariza- The use of cortical and striatal cells in culture is providing us tion over a very wide membrane potential range, consistent with an excellent tool to further understand the electrophysi- with little adaptation, and also support the proposition that ology of this important synaptic input. the entry of Na+ channels into inactivated states is a major fac- Kincaid AE, Zheng T, Wilson CJ (1998) Connectivity and convergence tor in C-fibre conduction slowing during repetitive firing. of single corticostriatal axons. Journal of Neuroscience 18:4722-4731. De Col, R, Messlinger, K and Carr, RW (2008) J Physiol 586, 1089-1103. Where applicable, the authors confirm that the experiments J.F.P. is an intercalated BSc Neuroscience student described here conform with The Physiological Society ethical requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C13

C12 Delayed rectifier currents in freshly dispersed rabbit urethral myocytes are encoded by KV2.1 The corticostriatal system in dissociated cell culture B. Kyle1, E.P. Bradley1, K.D. Thornbury1, N.G. McHale1, F. Randall1, M. Garcia-Munoz1, W. Staines2 and G. Arbuthnott1 G.P. Sergeant1, S. Ohya2 and M.A. Hollywood1 1Brain Mechanisms for Behaviour Unit, Okinawa Institute of Science 1Smooth Muscle Research Centre, Dundalk Institute of Technology, and Technology, Uruma City, Okinawa, Japan and 2Faculty of Dundalk, Ireland and 2Pharmacology, University of Nagoya, Medicine, University of Ottawa, Ottawa, ON, Canada Nagoya, Japan The cortex provides a massive input to striatal neurons but some Rabbit urethral smooth muscle cells (SMC) express a delayed of its properties make it extremely difficult to study single rectifer K+ current, but its molecular identity is unknown. The synapses between cortical fibres and striatal cells. For instance, purpose of the present study was to identify which Kv channel each striatal cell may receive in the order of 5,000 cortical subtype encodes this channel in these cells. inputs, from an almost identical number of cortical neurons. Rabbits were humanely killed with pentobarbitone (I.V.) and However, the neighbouring striatal cells are extremely unlikely the urethra was removed. For electrophysiology experiments, to respond to the same set of neurons since the likelihood of SMC were isolated as previously described (Sergeant et al. sharing more than about 1% of the inputs is vanishingly small 2000), perfused with Hanks’ solution at 37 ∞C and studied (Kincaid et al., 1998). We therefore have developed methods under voltage clamp using the perforated patch configuration to culture cortical cells from green fluorescent protein express- with K+-rich pipette solutions. Following successful perfora- ing mouse embryos together with striatal neurons from wild tion of the membrane, cells were bathed in Mg-substituted type mice of the same developmental stage ( E14.5 from QBM Ca2+ free Hanks solution with 5 mM EGTA containing 100 nM Cell Science, Ottawa, Canada). It is then possible to record from penitrem A (to block Ca2+ and Ca2+ activated currents). pairs of neurons in culture and to be sure that one is cortical Freshly dispersed urethral SMC were held at –80 mV and depo- and the other striatal. We have used conventional whole cell larized to +40 mV for 500 ms to evoke the Kv currents. These patch clamp recording methods in cultures (10-28 days in vitro) were inhibited by TEA in a concentration dependent manner recorded in artificial media of composition (NaCl 136; KCl 5; (IC50 1.8 ± 0.7 mM, n=8), but were unaffected by either mar- MgCl 1; CaCl 2.5; Hepes buffer 10; Glucose 10, all mM) at 36 gatoxin or α-dendrotoxin (100 nM, n=3), suggesting that they oC. The glass micropipettes had a resistance of 6-12MΩ inter- were not encoded by Kv1.1, Kv1.2, Kv1.3 or Kv1.6 channels. nal solution (NaCl 8; K Gluconate 132; MgATP 2; NaGTP 0.4; KCl However, in 7 experiments application of the Kv2 selective 6; Hepes buffer 10, all mM). There are some striato-cortical con- blocker, stromatoxin inhibited the currents in a concentration nections (5/18 paired recordings), all are inhibitory in nature dependent manner (IC50=201 ± 39 nM) consistent with the (reversing at approximately -40mV). The currents are idea that they were encoded by Kv2 channels. 129.8±41pA at -80mV and have a latency of onset of We next cloned Kv2.1 and Kv2.2, channels from the rabbit ure- 1.9±0.23ms (mean± S.D. n=5). thra and stably expressed them in human embryonic kidney The cortico-striatal connections have been characterized by (HEK) cells. These cells were studied using the whole cell con- not being reversed near the chloride equilibrium potential but figuration of the patch clamp technique with the same proto- reversing much closer to 0mV. Thus we are able to distinguish col as above. Kv2.1 and Kv2.2 currents were blocked in a con- these excitatory synaptic currents and estimate that they are centration dependent manner by TEA with IC50s of 3.9 ± 0.75 79.3±20.7pA at -80mV with a latency of 2.4±0.5 ms to the onset mM and 10 ± 3.5 mM (n=6). Stromatoxin also inhibited these of the response (n=6). The range is large in these first few pairs, currents with IC50 of 66 ± 18 nM (Kv2.1, n=5) and 49 ± 19.5 nM but we have not yet been able to reconstruct the anatomical (Kv2.2, n=6). A two-pulse protocol was used to assess the volt- details of the connections, nor to estimate the quantal char- age dependent inactivation in cells expressing either Kv2.1 or acteristics of the connections we have seen. We will compare Kv2.2 channels. The protocol consisted of holding the cells at the currents generated by individual synaptic contacts between -60 mV and stepping to conditioning potentials ranging from

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-100 to +40 mV for 10 s before stepping to a +40 mV test pulse 3 Alvarez JL, Salinas-Stefanon E, Orta G, Ferrer T, Talavera K, Galán L, for 500ms. In 13 cells transfected with Kv2.1 only, the mean Vassort G. Cardiovasc Res. 2004;63:653-61. V1/2 of inactivation was –55 ± 3 mV compared to –30 ± 3 mV 4 Piacentino V 3rd, Gaughan JP, Houser SR. Circ Res. 2002;90:435-42. in the cells transfected with Kv2.2 (n=11). When native SMC 5 Saini HK, Tripathi ON, Zhang S, Elimban V, Dhalla NS. Am J Physiol were studied with the same protocol, the V1/2 of inactivation Heart Circ Physiol. 2006;290:H373-80. of the Kv current was –56 ± 3 mV (n=9). These electrophysiological and pharmacological data suggest I am thankful to Prof N.S. Dhalla (Winnipeg, Canada)and Prof. that Kv2.1 channels underlie the delayed rectifier Kv current W. Schreibmayer (Graz, Austria) for providing me facilties for in rabbit urethral SMC. experiments on rats and Xenopus oocytes respectively Sergeant, G.P., McCloskey, K.D., Hollywood, M.A., Thornbury, K.D. & Where applicable, the authors confirm that the experiments McHale, N.G. (2000). Journal of Physiology.Vol. 526.2, pp359-366. described here conform with The Physiological Society ethical The authors acknowledge grant support from the NIH (NIDDK requirements. Grant No: R01 DK68565). Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical C15 requirements. Electrophysiology of Rabbit Cultured Synoviocytes R.A. Large1, M.A. Hollywood1, G.P.Sergeant1, K.D. Thornbury1, C14 J.R. Levick2 and N. McHale1 ICa(TTX) is present in rat left ventricular myocytes but not 1Smooth Muscle Research Centre, Dundalk Institute of Technology, in the Xenopus oocytes expressing hNaV1.5 Dundalk, Ireland and 2Basic Medical Sciences, St George’s Medical School, London, UK O.N. Tripathi The secretion of hyaluronan into the synovial cavity is crucial Life Sciences Academy, Lucknow, Uttar Pradesh, India for normal joint function, both for tissue lubrication and syn- A slow inward current is often reported in the range of -40 mV ovial fluid conservation, but the underlying cellular mechanisms in ventricular myocytes in the presence of low (or no) extra- are poorly understood. Essentially nothing is known about syn- cellular Na+ and normal extracellular Ca2+ and is variably attrib- oviocyte electrophysiology and the purpose of this study was uted to ‘slip-mode conductance (SMC)’ of the phosphorylated to use synoviocytes cultured from the rabbit knee joint to inves- Na+ channels and to a subtype of TTX-sensitive Ca2+ channel tigate the passive electrical properties of these cells and to [INa(null) or ICa(TTX)] (1,2). It is also reported that ICa(TTX) is establish which ionic currents are expressed. The synovium was expressed specifically in the ventricular mycoytes away from microdissected from healthy New Zealand white rabbits imme- the region of chronic infract and not in those from normal heart diately after they had been killed by lethal injection of pento- (3) or that it may be due to L-type Ca2+ current overlapping barbitone. The synovial lining from lateral and medial sides of Na+ current (4). The present study was carried out on isolated the suprapatellar zone was microdissected from the underly- rat left ventricular myocytes (rLVM) obtained from the ing areolar subsynovium and chopped into 0.5-1mm3 pieces myocardium away from the infarct and on Xenopus oocytes in order to commence a primary explant culture. This was main- expressing hNaV1.5 to assess, (i) the status of ICa(TTX) in rLVM, tained until confluency was reached, whereupon tissue frag- and (ii) whether SMC contributes to ICa(TTX). The ventricular ments were removed. The synovial lining cells were cultured myocytes were isolated from the normal (n=10), sham (n=14), and reseeded and cells were used for experimental purposes and infarcted (n=19) rat heart by collagenase dispersion tech- upon reaching passage six. nique (5). Whole-cell patch-clamp experiments on rLVM from Resting membrane potential, measured in zero current clamp all the three groups showed an inward current at depolarizing mode using K+-filled electrodes immediately after establish- steps of –40 mV from h.p. of -80 mV or –130 mV in the pres- ing a gigaohm seal, ranged from –30mV to –66mV with a mean ence of 0[Na]o and 1 mM [Ca]o, or 5 mM [Na]o and 1 mM [Ca]o. of –45 ± 8.6mV (SD, n = 40). Input resistance was measured in TTX (≥10 μM) added to the bathing solution blocked this inward 33 cells in voltage clamp mode by measuring the passive cur- current. Two electrode voltage clamp experiments on Xeno- rent responses to a series of hyperpolarizing and depolarizing pus oocytes expressing hNaV1.5 (n=9), with or without beta1 voltage steps from a holding potential of -60mV. This ranged subunit, did not show any inward current in the range of –60 from 0.54 to 2.6 GΩ with a mean of 1.28 ± 0.57 (SD). Cell capac- mV to +40 mV when bathed with 0[Na]o and 0.1-65 mM [Ca]o itance averaged 97.97 ± 5.93pF (SD, n = 30) as calculated by containing solution. Injection of Sp-cAMP, an activator of PKA, integrating the capacitative current evoked by small hyperpo- into the oocytes expressing hNaV1.5 also did not facilitate Ca2+ larizing and depolarizing steps and dividing by the amplitude inward current through these channels. These observations of the voltage change. When cells were voltage clamped at –60 indicate that ICa(TTX) is present in rLVM from both the normal mV and stepped from –80 to +50mV in 10mV steps, a family heart and the heart with chronic infarction but is not observ- of outward currents was evoked which showed clear outward able in Xenopus oocytes expressing hNaV1.5. rectification. These currents were reduced from a peak of 1625 1 Aggarwal R, Shorofsky SR, Goldman L, Balke CW. J Physiol (Lond) ± 238pA at +50mV to 776 ± 127pA (SEM n= 9) in the presence 1997; 505: 353-69. of 1mM TEA. The more selective Kv1 blocker margatoxin 2 Santana LF, Gómez AM, Lederer WJ. Science. 1998;279:1027-33. (100nM) reduced the peak outward current from 1482 ± 329

12P Oral Communications to 355 ± 93pA, n=8). Alpha-dendrotoxin (100nM) reduced the by 37%. Blocking of HCN4 gave a relatively modest prolonga- peak outward current from 1189 ± 182pA to 312 ± 25pA (n=5) tion of CL by 2.5%. while 50nM kappa-dendrotoxin reduced peak outward current Conclusions: If regulates murine SAN pacemaking during the from 2450 ± 623pA to 162 ± 27pA (n=4). In conclusion we have slow diastolic depolarisation. Altough the slowly activating demonstrated that isolated cultured synoviocytes from the rab- HCN4 is the most extensively expressed, HCN2 is the isoform bit express a current that has all the properties of a delayed rec- majorly contributing to murine pacemaking electrical activity. tifier potassium current and the pharmacology indicates that On the other hand, the contribution of HCN1 is small due to this is of the Kv1.1 subtype. This current undoubtedly has a role its small conductance. The HCN isoforms have different func- in regulating cell membrane potential and its modulation is tional impacts on murine pacemaking. likely to control calcium influx and thus hyaluronan secretion Summary of model responses upon blocking of If and its individual HCN (Ingram et al, 2008). isoforms. Ingram KR, Wann AK, Angel CK, Coleman PJ, Levick JR. (2008) Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints. J Physiol. 586(6), 1715-29.

Supported by the Wellcome Trust (076752/B/05/Z) Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. Shi et al. (1999). Circ Res 85, E1–6. Moosmang et al. (2001). J Biochem 268(6), 646-1652. Ishii et al. (1999). J Biol Chem 274, 12835–12839. C16 Kaupp and Seifert (2001). Annu Rev Physiol 63, 235–257. Wilders et al. (1991). Biophys J 60 1202–1216. Contribution of Kinetically Distinct HCN Isoforms to Murine Pacemaking: A Computational Study This work was supported by Welcome Trust (UK) project grant (WT/081809/Z06/Z). S. Kharche1, J. Yu1, M. Lei2 and H. Zhang1 Where applicable, the authors confirm that the experiments 1School of Physics and Astronomy, University of Manchester, described here conform with The Physiological Society ethical Manchester, UK and 2School of Medicine, University of Manchester, requirements. Manchester, UK Aim: Pacemaking in the sino-atrial node (SAN) is modulated by hyperpolarization-activated cyclic nucleotide (HCN) gated channel, If. Several isoforms of the HCN family have been iden- C17 tified in murine SAN which show vast differences in time kinetics. This computer modelling study evaluates the functional Role of calcium-dependent and -independent reactive role of individual HCN isoforms on murine pacemaker activity. oxygen species generation in pancreatic acinar cell death Methods: Murine SAN If consists of HCN1, HCN2 and HCN4 [1]. All isoforms have similar steady state activtion [2] with a half- D. Booth1, O.H. Petersen1, A.V. Tepikin1, R. Sutton2 and activation of approximately -63.7 mV. The molecular expres- D.N. Criddle1 sion of HCN1, HCN2 and HCN4 in SAN cells has been shown to 1Department of Physiology, University of Liverpool, Liverpool, UK be 5:25:70 respectively [3]. The primary biophysical differences and 2Liverpool NIHR Biomedical Research Unit, University of in the individual isoform function have been identified to be a Liverpool, Liverpool, UK quantitative difference in time kinetics and cAMP sensitivities [4]. Based on these biophysical properties, Hodgkin-Huxley The relationship between cytosolic calcium [Ca2+]c, reactive models for each isoformal component of If were developed and oxygen species (ROS) and mitochondrial function plays an incorporated into our recently developed murine SAN mathe- important role in cellular physiology and pathology, yet is cur- matical cell model. The contributions of individual isoforms to rently poorly defined. We have previously shown that oxidant model action potential (AP) were evaluated by blocking the iso- stress promotes apoptotic pancreatic acinar cell death (Crid- formal channels individually. Model responses were defined dle et al., 2006) and have now investigated the influence of as AP features including minimum diastolic potential (MDP), [Ca2+]c on ROS generation, mitochondrial function and cell over shoot potential (OS), AP duration at 50% (APD50) and at fate in isolated murine pancreatic acinar cells. 90% repolarisation (APD90), cycle length (CL) and diastolic depo- Isolated murine pancreatic acinar cells were exposed to tau- μ larisation rate (DDR) [5]. Blocking of total If and Control cases rolithocholic acid sulphate (TLC-S; 500 M) or the quinone oxi- were also simulated. dant menadione (MEN; 30μM) and examined by confocal Results: Blocking of If or its isoforms had marked effects on CL microscopy to detect ROS (CM-H2DCFDA), [Ca2+]c (Fluo-4), and DDR with other AP features largely unaffected. The results NAD(P)H (autofluorescence, a measure of mitochondrial are summarised in the table. Blocking of total If increased the metabolism), mitochondrial Ca2+ ([Ca2+]mt; Rhod-2), and CL by 26% and the DDR reduced by 5%. Selectively blocking apoptosis (caspase activaton: R110-aspartic acid amide). The HCN1 isoform gave a small prolongation of CL by 2%. Blocking antioxidant N-acetyl-L-cysteine (NAC; 10mM) was applied as a of HCN2 however, prolonged CL by 28% and DDR was reduced ROS scavenger and BAPTA-AM (25μM) as a Ca2+ chelator.

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TLC-S caused a large, sustained rise of [Ca2+]c above basal lev- TMRE fluorescence specifically in regions (as small as 5 μm diam- els and a NAC-sensitive generation of ROS (n=16). Mitochon- eter) exposed to UV laser light for periods <1s. The regions of drial function was inhibited by application of TLC-S; a large, sus- decreased TMRE fluorescence only spread slightly over the tained increase of [Ca2+]mt was observed with a concomitant course of an experiment (~20 min), i.e. the uncoupling decrease of NAD(P)H( n=18). Both the sustained rises of [Ca2+]c remained localised to the site of photolysis. No changes in TMRE and ROS were abolished by BAPTA-AM pre-treatment, indicat- fluorescence were observed to UV light in the absence of the ing the Ca2+-dependency of TLC-S-induced ROS generation caged uncoupler . These results indicate that, in freshly isolated (n=14). In contrast, MEN-induced elevation of ROS was unaf- colonic smooth muscle cells, mitochondria are individual, elec- fected by BAPTA pre-treatment, but blocked by NAC (n=16), trically-independent units that do not move throughout the in accord with a Ca2+-independent redox cycle mechanism. cell. The caged dinitrophenol has the benefits of being mem- When cells were treated with TLC-S or MEN for 30 minutes, brane permeable (hence alleviating the requirement for micro- apoptotic cell death was increased 3.2-fold and 5.5 fold from injection or patch-clamping) and targeted to mitochondria, ΔΨ control levels (n=1132, 632), respectively, in a manner that was thus refining the capability to rapidly depolarise m in very abolished by treatment with NAC, indicating a ROS-depend- small sub-cellular regions and determine the role of those mito- ency of the actions of both compounds. chondria in cellular signalling processes. Reactive oxygen species, generated by distinct Ca2+-depend- Where applicable, the authors confirm that the experiments ent and independent mechanisms, are important mediators of described here conform with The Physiological Society ethical apoptosis in the pancreatic acinar cell. requirements. Criddle DN et al.(2006) J. Biol. Chem. 281, 52. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical C20 requirements. Calcium elevation in mitochondria is the main Ca2+ requirement for mPTP opening C19 O. Gerasimenko1, H.K. Baumgartner1,2, J.V. Gerasimenko1, C. Thorne1, T. Pozzan4, A.V. Tepikin1, O.H. Petersen1, R. Sutton3 Localised mitochondrial depolarisation evoked by sub- and A.J. Watson2 cellular release of a membrane-permeant, chemically-caged uncoupler in freshly isolated smooth muscle cells 1Physiology, Liverpool University, Liverpool, UK, 2Division of Gastroenterology, Liverpool University, Liverpool, UK, 3Division of S. Chalmers1, C. Quin2, R.C. Hartley2 and J.G. McCarron1 Surgery and Oncology, Liverpool University, Liverpool, UK and 1 2 SIPBS, University of Strathclyde, Glasgow, UK and Department 4Biomedical Sciences and CNR Institute of Neurosciences, University of Chemistry, University of Glasgow, Glasgow, UK of Padua, Padua, Italy Numerous cellular functions are influenced by mitochondrial We have investigated in detail the role of intra-organelle Ca2+ activity, including the organelle’s ability to produce ATP, accu- content during induction of apoptosis by the oxidant mena- 2+ mulate Ca and produce reactive oxygen species. Mitochon- dione while changing and monitoring Ca2+ load of ER, mito- dria are distributed throughout the cytosol and, as such, these chondria and acidic organelles. Menadione causes production μ small organelles (~1-5 m) may display particular influence of reactive oxygen species, induction of oxidative stress and over processes in their immediate neighbourhood, e.g. on subsequently apoptosis. In both pancreatic acinar and pan- 2+ nearby Ca -sensitive channels. Indeed, mitochondrial activity creatic tumour AR42J cells, menadione was found to induce within sub-cellular microdomains has been implicated in the repetitive cytosolic Ca2+ responses due to release of Ca2+ from regulation of channels both on the plasma membrane and the both ER and acidic stores. Ca2+ responses to menadione were internal calcium stores, the endoplasmic- or sarcoplasmic-retic- accompanied by elevation of Ca2+ in mitochondria, mitochon- ulum (ER or SR). We have shown previously that mitochondr- drial depolarisation and mPTP opening. Emptying of both the 2+ ial uptake of Ca promotes the activity of inositol-1,4,5- ER and acidic Ca2+ stores did not necessarily prevent mena- 2+ trisphosphate-sensitive Ca release channels (IP3R) on the SR dione-induced apoptosis. High mitochondrial Ca2+ at the time in smooth muscle cells. This suggests a localised regulation of of menadione application was the major factor determining IP3R by mitochondria located close to the channel, yet it has cell fate. However, if mitochondria were prevented from load- been difficult to study how mitochondria, acting in restricted ing with Ca2+, then apoptosis did not occur irrespectively of regions, control cell-wide activity. other Ca2+ stores’ content. These results were confirmed by We have developed a mitochondrially targeted UV-activated ratiometric measurements of intra-mitochondrial Ca2+ with caged dinitrophenol. UV irradiation in vitro caused it to undergo pericam. We conclude that elevated Ca2+ in mitochondria is the photolysis, a product of which displayed the absorbance prop- crucial factor in determining whether cells undergo oxidative erties of free dinitrophenol (DNP, an uncoupler of the mito- stress-induced apoptosis. ΔΨ chondrial membrane potential, m, from ATP synthesis). Baumgartner HK, Gerasimenko JV, Thorne C, Ashurst LH, Barrow SL, Smooth muscle cells freshly isolated from guinea-pig colon Chvanov MA, Gillies SG, Criddle DN, Tepikin AV, Petersen OH, Sutton were loaded with mitochondrially targeted UV-activated caged R, Watson AJ and Gerasimenko OV. (2007) Am J Physiol Gastrointest ΔΨ Liver Physiol 293(1), G296-307 dinitrophenol (200 nM) plus the m-sensitive dye tetram- ethylrhodamine ethyl ester (TMRE, 10 nM) for 30 min. High- Filippin L, Abad MC, Gastaldello S, Magalhaes PJ, Sandona D and Poz- speed epifluorescent imaging detected localised decreases in zan T. (2005) Cell Calcium 37(2), 129-136

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Gerasimenko JV, Gerasimenko OV, Palejwala A, Tepikin AV, Petersen OH Conclusion: According to our results, we found that the expres- and Watson AJM. (2002) Journal of Cell Science 115, 485-497 sion of p53, at least at the mRNA level, decreased at 19 dg allowing the placenta to increase in weight while at 21 dg the p53 This work was supported by an MRC cooperative grant expression increased suppressing placental growth. This was (G0300076) and MRC Programme Grant (G8801575). reflected in the weights that we obtained. Where applicable, the authors confirm that the experiments Feldman D (1997) Endocrinology 138:1777-1779 described here conform with The Physiological Society ethical Al-Bader M (2006) Reprod Biol Endocrinol. Mar 28;4:13. requirements. Hsu HH, Cheng SF, Wu CC, Chu CH, Weng YJ, Lin CS, Lee SD, Wu HC, Huang CY, Kuo WW (2006) Chin J Physiol. 49(2):110-6.

College of Graduate Studies and Graduate Research Grant C21 #YM019/07 Possible Link Between Estrogen Levels, Estrogen Receptors Where applicable, the authors confirm that the experiments and the Tumor Suppressor Gene p53 During Gestation in Rat described here conform with The Physiological Society ethical Placenta requirements. A. Elfarra, M. Al-Bader, S. Mohan and L. Jacob physiology, faculty of medicine, kuwait university, Kuwait, Kuwait C22 Introduction: Estrogen is essential for initiation and maintenance of pregnancy in the rat. However, high levels of estro- The role of plasma-mediated vascular dysfunction and PARP gens during pregnancy may have a specific growth-retarding activation in pre-eclampsia effect on the placenta (1). Consequently, there has to be a con- F. English1, S.K. Walsh1, E.J. Johns2 and L.C. Kenny1 trol mechanism that enables the placenta to proliferate regard- 1Anu Research Centre, Department of Obstetrics and Gynaecology, less of the otherwise high inhibitory levels of circulating estra- 2 diol. This may partially be mediated through a decrease in University College Cork, Cork, Ireland and Department of estrogen receptor (ER), which has been reported before (2), Physiology, University College Cork, Cork, Ireland and a parallel decrease in p53 expression as a link between the Pre-eclampsia (PE) is associated with widespread maternal vas- expression of these two genes has been reported (3). There- cular dysfunction which is thought to be mediated by one or fore, we hypothesize that a decrease in placental ER protein more circulating factors. Under normal physiological condi- expression correlates with a decrease in placental p53 expres- tions, poly(ADP-ribose) polymerase (PARP) is cytoprotective. sion during pregnancy. This study was designed to investigate However under conditions of increased oxidative stress over- whether the placental expression of p53 changes during preg- stimulation of this enzyme leads to vascular dysfunction. Inhi- nancy in rat. bition of PARP has been demonstrated to reverse the vascular Methodology: Pregnant Sprague-Dawley dams were stunned dysfunction associated with diabetes in vivo [2]. Thus the aims and killed by cervical dislocation at 16, 19 and 21 days gesta- of the present study were to 1) investigate whether circulat- tion (dg). Placental tissues from each litter were collected (four ing factors induce vascular dysfunction in the reduced uterine pregnancies at each gestational age [n = 4] and three to four perfusion pressure (RUPP) at model of PE, and 2) to examine placental tissues per pregnancy were pooled). Gene and pro- the role of PARP in any observed changes in vascular reactivity tein expression of p53 were studied using real-time PCR (ReT- in both rat and human vessels. PCR) and Western blotting and immunodetection method- On day 14 of pregnancy, animals destined for the RUPP exper- ologies. Taqman probes specific for p53 and for two imental group were anaesthetised with isoflurane (2-5% inhala- housekeeping genes, 18S and beta-glucuronidase (BGLUC) tion) and a silver clip (0.203mm ID) was placed around the aorta were used. For p53 protein expression, tissues were homoge- (above the iliac bifurcation) to reduce uterine perfusion pres- nized to obtain nuclear and cytosolic fractions (verification of sure by ~40% [3]. Arterial blood was collected from both RUPP nuclear and cytoplasmic fractions was done using Histone H4 and normal pregnant (NP) rats and plasma aliquots stored at - antibody and glucose-6-phosphate dehydrogenase (G6PD) 80oC. Resistance vessels from both virgin and NP rats were incu- assay, respectively. P53 and actin were analyzed in these frac- bated overnight in either 3% RUPP or NP plasma with or with- tions in addition to the total homogenate fraction. out the PARP inhibitor, PJ34 (3μM). Human omental arteries Results: Placentae weight increased significantly between 16 were incubated overnight with 3% NP or PET plasma, with or dg and 19 dg (p=0.001), and 16 dg and 21 dg (p=0.01), while without the PARP inhibitors, PJ34 and DR2313 (3μM). Vascu- there was no significant increase between 19 and 21 dg. Both lar function was assessed in response to the vasoconstrictor, 18S and BGLU were found to be suitable housekeeping genes U46619 and vasodilators, bradykinin and acetylcholine. as their expression was not changed with gestation. The expres- Treatment of resistance vessels with RUPP plasma resulted in sion of p53 decreased significantly by 19 dg (p<0.05) and significantly impaired vasorelaxation (to both ACh (68 ± 6% increased by 21 dg (p<0.05). As for protein expression, p53 vs. 87 ± 3%; p<0.05) and BK(36 ± 5% vs. 51 ± 3%; p<0.05)) in was detected in both homogenate and nuclear fractions with vessels from pregnant but not virgin rats. Furthermore, veryfaintbandsinthecytosolicfractionat16dgonly.There overnight incubation in RUPP plasma was necessary to induce was a trend for p53 protein to decrease by 19 dg in both this vascular dysfunction. Concomitant treatment with the homogenate and nuclear fractions, however, this was not sta- PARP inhibitor, PJ34, abrogated the RUPP plasma-induced vas- tistically significant. cular dysfunction (ACh 81 ± 3% vs. 68 ± 7%; p<0.05) (BK 53 ±

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± 3% vs. 36 7%; p<0.05). Similarly, incubation of omental ves- our previous observations that KV channels contribute to main- sels from normal pregnant women in plasma from women with tenance of chorionic plate arterial tone [2, 3]. Future studies PE resulted in impaired relaxation to BK (45 ± 7% vs. 86 ± 3%; with more specific blockers could address the contribution of p<0.01). This impaired relaxation was reversed by co-incuba- 4-AP-sensitive KV channel subtypes to regulating fetoplacen- tion of the PET plasma with PJ34 (85 ± 2% vs. 45 ± 7%; p<0.05). tal vascular resistance and blood flow in normal and compro- In contrast, treatment with DR2313 did not significantly affect mised pregnancies. the impaired vasorelaxation induced following overnight incubation with PET plasma. The present study demonstrates that plasma-derived factors appear to mediate the vascular dysfunction documented in both the RUPP rat model of PE and the clinical condition. Fur- thermore, this work demonstrates a role for the overactivity of PARP in mediating this vascular dysfunction. Sankaralingam S. et al. (2006) Acta Obstet. Gynecol. 81-86P. Soriano FG. et al. (2001) J. Mol. Med. 437-442P. Crews JK et al. (2000) Hypertension 367-372P. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Figure 1: Scatter plot represents % change in baseline diameter of chorionic requirements. plate arteries in response to cumulative concentrations of 4-AP (N=8; line at median). Hampl V et al. (2002). Am J Physiol Heart Circ Physiol, 283, H2440-9 C23 Wareing M et al. (2006). Am J Physiol Regul Integr Comp Physiol, 291, R437-46 4-Aminopyridine sensitivity in human placental chorionic Wareing M et al. (2006). Biol Reprod, 75, 518-23 plate arteries This work is supported by Action Medical Research and Tommy’s T.A. Mills, E.J. Cowley, S.L. Greenwood, C.P. Sibley and M. Wareing Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Maternal and Fetal Health Research Group, The University of requirements. Manchester, Manchester, UK

Voltagegatedpotassiumchannels(KV)altervasculartonebyeffect- 2+ ing changes in membrane potential and regulating Ca influx. C24 SeveralKVchannelsubtypesareexpressedinthefetoplacentalvas- culature,butthefunctionallyimportantKVchannelshavenotbeen Prenatalvs.postnatalinfluencesonfatandleanmassinsheep identified [1, 2]. K channel subtypes have different sensitivities V R. Coleman and D. Gardner toblockby4-aminopyridine(4-AP)andweshowedpreviouslythat thebasaltoneofchorionicplatearterieswassignificantlyincreased School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK by 1mM 4-AP [2]. Here, we test the hypothesis that different KV channel subtypes contribute to regulation of fetoplacental vas- It has been suggested that poor prenatal growth may predis- cular tone by determining the sensitivity of chorionic plate artery pose to increased adiposity later in life. In this retrospective (CPA) constriction to 4-AP,using pressure myography. analysis, a large cohort of sheep was utilised to interrogate Term placentas (N=8) were obtained post-delivery (vaginal or the a priori hypothesis that intrauterine growth restriction leads Caesarean section) from uncomplicated pregnancies. Biopsies to fatter adolescent offspring and this effect is modified by their - were placed into ice-cold HCO3 -buffered physiologic salt solu- subsequent postnatal growth rate. tion (PSS). CPAs were mounted on a pressure myograph, equil- Data for five flocks of pedigree Suffolk sheep (n=13,630) were ibrated for 30 minutes at an intraluminal pressure of 20mmHg available in which multiple variables were recorded as standard (to reproduce pressure in vivo) with intraluminal flow (20μl/min; (forinclusionintheSignetpedigreedatabase,MLC)includingbirth o 37 C, 5%O2/5%CO2). Contraction was assessed with 120mM weight, weaning weight at 8 weeks of age and weight and body potassium solution (KPSS) and U46619 (10-10-2x10-6M) added compositionat20weeksofage.Bodycompositionwasassessed to the bath. Post wash, a concentration response curve to 4- in two ways; 1) by ultrasound at the 11th rib by a skilled operator AP (1-5000μM) was performed. U46619 contraction (10-10- (n=7,671) and 2) by computed tomography (CT, n=220). Post- 2x10-6M) was then assessed in the continued presence of natalgrowthwasassessedinabsolute(g.day-1)orrelative(g.day- 5000μM4-AP. 1.kg-1birthweight)terms.Dataarepresentedasestimatedmar- Baseline arterial diameters were 272±24μM. KPSS reduced ginal means ± S.E.M. and were analysed by General Linear Mixed baseline CPA diameters by 39±8%. 4-AP reduced baseline diam- Models using Genstat v11. P<0.05 was accepted as indicating a eter of CPAs at concentrations above 5μM (Figure 1; *P<0.05 statistically significant effect. Birth weight or postnatal growth Wilcoxon Signed Rank Test). 4-AP did not affect maximum weremodelledascontinuousorcategoricalvariables;categories U46619 constriction at 10-6M (data not shown). conformingto1kgincrementsinbirthweightfrom<3kgto>8kg. In conclusion, 4-AP induced constriction of chorionic plate arter- In this cohort of sheep there was a 5-fold natural variation in ies at concentrations above 5μM, confirming and extending birth weight (Mean 5.13, range 2-10). Lambs born relatively

16P Oral Communications small (IUGR, -2S.D. or <3kgs) tended to exhibit postnatal growth Male Wistar rats were injected with 60 mg/kg of MCT or an acceleration during the first 8 weeks of life only. Ultrasound equivalent volume of saline (CON). Telemetry was used to meas- determined fat mass correlated well with CT determined fat ure the electrocardiogram (ECG) in vivo, in conscious, unre- mass (r=0.72, P<0.0001). There was a strong positive relation- strained animals on a weekly basis until day 21 then daily. Sur- ship between birth weight and/or relative postnatal growth gical implantation of telemetry devices was performed using and fat mass at 20 weeks of age (Figure 1). This effect was appar- isoflurane anaesthesia by inhalation (up to 5%) and maintained ent and similar in both sexes. Adjustment for random effects at 1-3%, using aseptic technique. Heart failure develops within in the model such as flock and shared genes from either parent 4 weeks following injection. On the presentation of clinical (ewe and ram) did not materially affect the conclusions. symptoms of heart failure, rats were killed by schedule 1 pro- In a large cohort of sheep, natural variations in birth weight as cedures, hearts removed and dissected into right ventricular a result of unknown environmental aetiologies had a pro- (RV) and left ventricular (LV) portions and free and polymerised nounced effect on body composition in the young, adolescent fractions of β-tubulin assessed using Western blot analysis. Tis- offspring. Being born of relatively high birth weight is strongly sue samples from these regions were analysed using real-time linked to increased fat mass later in life. Similarly, a tendency reverse transcription polymerase chain reaction to measure the to exhibit relative postnatal growth acceleration during early mRNA expression ratio of α-tubulin. All procedures accorded life is also, independently, associated with increased fat mass with current UK legislation. later in life. The data recapitulate observations in human epi- MCTtreatedratshadincreasedheartweight:bodyweight(CON demiological studies suggesting that the early developmental 4.1±0.2vs.MCT5.5±0.2mg/g(mean±SEM))andRVweight:LV environment is key to determining later body composition; weight (CON 0.38 ± 0.03 vs. MCT 0.68 ± 0.06 g/g), consistent observing similar effects in species with widely different meta- with the development of right ventricular hypertrophy/failure bolic function and life history gives biological plausibility to (n=6, CON and MCT t-test P=<0.001). Measurement of ECG programming of later body composition by early environment. parameters using radiotelemetry indicated modification of T- parametersinMCTtreatedanimalse.g.aprolongedQTinterval (CON49.7±2.0vs.MCT76.2±2.5ms,P<0.001,t-test)andtime from the peak to the end of the T-wave (Tpe, CON 25 ± 1.8 vs. MCT33.1±1.7ms,P=0.007,t-test)(CONn=6,MCTn=7).InMCT treated rats there was an increase in the polymerised fraction of β-tubulinintheRVcomparedtocontrolrats(CON0.55±0.02 vs.MCT0.6±0.01a.u,P=0.0032-WAYANOVA,n=6eachgroup, 3 replicates). There was a concomitant increase the expression of mRNA encoding α-tubulin in the RV of MCT rats (CON 0.6 ± 0.1 n=7 vs. MCT 17.3 ± 4.2 a.u n=9, P<0.001, 2-WAY ANOVA). We conclude that MCT treatment results in; ECG changes consistent with prolongation (QT) and increased global dispersion (Tpe) of the action potential; increased microtubule prolifera- The authors wish to acknowledge the Meat and Livestock Com- tion as evidenced by the increase in α-and β- tubulin. These mission and the School of Veterinary Medicine and Science, changes may be relevant to alterations seen in the mechanical University of Nottingham. and electrical activity of right heart failure. Cooper G (2006). Am J Physiol Heart Circ Physiol 291, H1003-H1014 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Supported by the British Heart Foundation. D.B. is an Emma requirements. and Leslie Reid Ph.D. student. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C25

ECG alterations and microtubule proliferation following monocrotaline induced right ventricular heart failure in rats C26 R. Stones1,3, M. Drinkhill2,3, D. Benoist1,3 and E. White1,3 Remodelling of the extracellular matrix in the rat sinoatrial node in congestive heart failure 1Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK, 2Cardiovascular and Neuronal Remodelling, University J.F. Yanni1, T.T. Yamanushi2, H. Dobrzynski1 and M.R. Boyett1 3 of Leeds, Leeds, UK and Multidisciplinary Cardiovascular Research 1Cardiovascular and Endocrine Science, Manchester University, Centre, University of Leeds, Leeds, UK Manchester, UK and 2Kagawa Prefectural College of Health Microtubules are load bearing and load-modulated compo- Sciences, Takamatsu, Japan nents of the cardiac cytoskeleton whose proliferation may The extracellular matrix (ECM) includes structural proteins (e.g. impede contractile function in animal and human right heart collagen types 1 and 3 and elastin), adhesive proteins (e.g. fibro- failure (Cooper, 2006). We wished to test whether there was a nectin), and matrix metalloproteinases (MMPs; responsible for proliferation of microtubules in a non-invasive model of pul- collagen degradation). MMP activity is regulated by endoge- monary hypertension induced by monocrotaline (MCT). nous inhibitors (TIMPs). Four TIMP isoforms (TIMP1-4) are

17P Oral Communications known to be present in the heart. In the heart, the ECM is resolution. The resulting 3-D image volumes were approxi- responsible for connecting myocytes, aligning contractile ele- mately 4 mm × 1 mm × 0.3 mm with isometric voxels of size (1 ments, transmitting force and preventing myocardial rupture. μm)3. A higher resolution image volume (560 μm × 560 μm × Although it is well known that there is a remodelling of the ECM 330 μm, (0.4 μm)3 voxel size) was also acquired from the mid- in the working myocardium in heart failure (Graham et al., wall of one heart of each strain at 12 months of age. 2008), it is not known whether there is a similar remodelling Collagen volume fractions were computed on each block using in the sinoatrial node (SAN; pacemaker of the heart). The aim a modified Top-hat filter that identifies regions that are bright of the present study was to investigate the effect of heart fail- relative to the local background. Specific myocardial structural ure on the ECM and the factors that control the ECM (e.g. trans- components (myocytes, blood vessels, interlaminar spaces and forming growth factor β1, TGFβ1, and tumour necrosis factor different collagen structures (endomysial, perimysial, perivas- α, TNFα). Sprague–Dawley rats, aged five weeks, received a cular, scar)) were segmented in the midwall of each image vol- single subcutaneous injection of monocrotaline (60 mg/kg), ume by digitally tracing structural boundaries. Laminar width which induced inflammation of the pulmonary artery and pul- was measured along lines perpendicular to the myolaminae in monary arterial hypertension. After three weeks, the experi- the midwall region at 200 μm intervals. To identify laminar ments were ended after the animals developed congestive remodellingindependentofchangesincellsize,thelaminarwidth heart failure as confirmed by echocardiography. All experiments wasnormalisedbytheaveragemyocytediameterineachblock. were conducted in accordance with the Japanese Act on Wel- Results are presented in Figure 1, where the collagen fractions fare and Management of Animals (Act No. 105 of October 1, (A) and layer widths (B) are expressed as group mean ± SEM. 1973). Quantitative PCR was used to measure the expression The myocyte areas (C) are expressed as box plots (first, median of transcripts in the SAN (and also the right atrium, RA, and and third quartiles, with whiskers at 10% and 90%) to avoid mak- right ventricle, RV). ing assumptions about the underlying statistical distribution. In the heart failure rats (as compared to control rats), mRNAs Subsequent analysis was performed using a 2-factor ANOVA for collagen type 1 (constitutes 80% of total collagen) and elastin with statistical significance based on a value of P ≤ 0.05. Colla- were significantly increased in the SAN, RA and RV; collagen gen fraction increases with age from a similar baseline in both type 3α mRNA was significant increased, but only in the RA and strains, but is more pronounced in the SHR. Myocyte cross-sec- RV. Fibronectin mRNA was significantly increased in all three tional area increases with age in the SHR, but not in the WKY. tissues. MMP2 mRNA was significantly increased in the RA only. Both of these measures are significant for strain and strain-age TIMP-1 was significantly increased in all three tissues; TIMP2 interaction. The layer width in terms of myocyte diameters did not show any changes; TIMP4 was significantly decreased shows a significant increase at 18 and 24 months in the SHR in the SAN and RV. Fibroblasts synthesise collagen, and vimentin (significant for strain, age, and strain-age interaction). mRNA (a marker for fibroblasts) was significantly increased in It is clear that significant morphologic changes occur in the tis- all three tissues in the heart failure rats. TGFβ1 mRNA was signi- sue structure of the SHR around 12 months with increasing col- ficantly increased in all three tissues, although TNFα mRNA was lagen and remodelling of the laminar organisation. High-reso- significantly increased in the RA only. We conclude that, in this lution analysis of the change in collagen at this time-point model of congestive heart failure, there is a remodelling of the indicates that the increase is due to both scarring and increas- ECM (fibrosis) in the SAN as well as the RA and RV. The remod- ing endomysial collagen. While some of this change occurs nat- elling may contribute to the SAN dysfunction that is known to urally with age, it is significantly accelerated in the SHR. occur in heart failure. Graham HK, Horn M, Trafford AW (2008). Acta Physiol 194:3-21. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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Age-Related Changes in Left-Ventricular Structure in the Spontaneously Hypertensive Rat G.B. Sands1, A.J. Pope2, B.H. Smaill1,2 and I.J. LeGrice1,2 1Bioengineering Institute, University of Auckland, Auckland, New Zealand and 2Department of Physiology, University of Auckland, Auckland, New Zealand High-resolution three-dimensional images of myocytes and collagen have been acquired across the left ventricular wall in ten normal Wistar-Kyoto (WKY) and thirteen spontaneously hypertensive (SHR) rat hearts using extended-volume confo- 1 cal microscopy, with at least 2 hearts of each strain imaged at Figure 1. Comparison of left-ventricular tissue structure in SHR (dark, filled) each age endpoint of 3, 12, 18 and 24 months. Tissue blocks and WKY (white, open) hearts. (A) Collagen fraction. (B) Layer width, meas- were prepared as described previously2 and imaged at 1 μm ured as number of myocyte diameters. (C) Myocyte cross-sectional area.

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Sands GB et al. (2005). Microsc Res Techniq 67, 227-239. Chase, A., et al., Coronary artery disease progression is associated with Pope AJ et al. (2008). Am J Physiol Heart Circ Physiol 295, H1243-1252. increased resistance of hearts and myocytes to cardiac insults. Crit Care Med, 2007. 35(10): p. 2344-51 Where applicable, the authors confirm that the experiments This work was supported by the NIHR and the British Heart described here conform with The Physiological Society ethical Foundation. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C28

Ischaemic Preconditioning In Hearts From Apolipoprotein E C29 Knockout Mice Fed High Fat Diet S.L. Passey1, L. Hua1, A.P. Halestrap2, G. Angelini1 and Chronic ENaC Blockade Rescues Na+ Induced High Blood M. Suleiman1 Pressure in 11b-Hydroxysteroid Dehydrogenase Type 2 Heterozygote Mice 1Clinical Science at South Bristol, Bristol Heart Institute, University of Bristol, Bristol, UK and 2Department of Biochemistry, Bristol E. Craigie, M.A. Bailey and J.J. Mullins Heart Institute, University of Bristol, Bristol, UK Molecular Physiology, University of Edinburgh, Edinburgh, UK Exposing the heart to brief periods of ischaemia followed by 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) con- reperfusion (ischaemic preconditioning) is a powerful tool for fers mineralocorticoid specificity to the mineralocorticoid protecting the heart from further ischaemic insults. Under- receptor (MR) by inactivating glucocorticoids. Mutations in standing the mechanisms involved in this phenomenon is vital HSD11B2 cause the rare hypertensive disorder of Apparent Min- for the translation of preconditioning techniques into clinical eralocorticoid Excess (AME) and polymorphisms in the general applications. Many preconditioning studies utilize healthy heart population have been linked to Na+ sensitive hypertension (1). models to evaluate the effectiveness and mechanisms of pre- We have previously shown that a high Na+ diet increases blood conditioning, however it is essential to assess the effects of pre- pressure (BP) in hsd11b2 heterozygote (hsd11b2+/-) mice (2). conditioning protocols on the compromised or diseased heart Here we have investigated the role of the epithelial sodium in order to fully appreciate the potential of preconditioning to channel (ENaC) in this response. improve outcome of patients with coronary disease. When fed Male hsd11b2+/- (n=6) & wild-type (hsd11b2+/+; n=6) mice a high fat diet for 24 weeks, male apolipoprotein E knockout were housed in metabolic cages and fed control diet (0.25% mice (apoE-/-) develop atherosclerotic lesions in the coronary Na+) for 3 days before mini-pumps delivering the potent ENaC arteries and show coronary artery disease similar to that seen inhibitor benzamil (0.7μg/day/g) were implanted under isoflu- in humans. Previous work [1] has shown that the hearts from rane anaesthesia (2% with O2 by inhalation). After recovery, these mice are resistant to ischaemic insult compared to healthy measurements were made for 2 days before high Na+ diet (2.5% apoE -/- hearts from mice fed a standard chow diet. The aim of Na+) was given: Na+ excretion was measured for a further 7 this study is to evaluate the ability of the diseased apoE -/- heart days, after which mice were moved to normal cages for a fur- to be protected by ischaemic preconditioning techniques. ther 2 weeks. Mice were then anaesthetized (Inactin, 100mg/kg Hearts from 8 month old male apoE -/- mice fed a high fat diet IP) for measurement of arterial BP. Daily sodium excretion was for 24 weeks were excised and perfused in Langendorff mode averaged over the baseline and cumulative sodium balance cal- with Krebs-Henseleit buffer. Following a 20 minute stabiliza- culated for the first 7 days on 2.5% Na diet. Data are mean ± tion period, hearts were either subjected to a preconditioning SE; statistical comparisons were made using either t-test or protocol of 2 cycles of 5 min ischaemia and 5 min reperfusion ANOVA, as appropriate. (ischaemic preconditioned, IP), or were continually perfused Baseline daily Na+ excretion was similar in hsd11b2+/- and for 20 min (control). Hearts were subjected to 60 minutes global hsd11b2+/+ mice on 0.25% Na diet (11.3±1.7 vs 12.6±2 ischaemia followed by 60 minutes reperfusion. Functional μmol/24h/g). Benzamil administration caused a significant recovery (left ventricular developed pressure, LVDP) was natriuresis in both groups (hsd11b2+/- = 16±0.7 and assessed and creatine kinase release upon reperfusion was hsd11b2+/+ = 14.3±1.3 μmol/24h/g; P<0.01 vs baseline), con- measured as an indicator of myocardial injury. There was no firming ENaC blockade. New analysis of our previous data shows significant difference in functional recovery between control the hsd11b2+/- mice in positive cumulative Na+ balance dur- and IP hearts after 60 minutes reperfusion (% LVDP 58.9 ± 8.6 ing the first week on a 2.5% Na+ diet (134.2±48 μmol; P<0.05); % for control vs. 64.5 ± 8.3 % for IP hearts, data are mean ± S.E. hsd11b2+/+ mice are in neutral balance. Benzamil treatment for n=6 hearts in each group, p=0.65 unpaired t-test). There caused a negative sodium balance in both hsd11b2+/- and was also no difference in myocardial injury as determined by hsd11b2+/+ mice after 7 days on 2.5% Na+ diet (-122±10 μmol creatine kinase release into the coronary effluent upon reper- vs -111±41 μmol). Benzamil also prevented the Na+-induced fusion. The results presented here indicate that apoE -/- hearts rise in BP in hsd11b2+/- mice, which now had a similar BP to with coronary artery disease are resistant to further cardio- hsd11b2+/+ animals at the end of the high sodium regimen protective effects of ischaemic preconditioning. These data (83.5±5 vs 83.7±4 mmHg). suggest that these diseased hearts may require optimal pre- In summary, dietary Na+ loading induces a BP rise in mice with conditioning protocols or that they could be chronically pre- reduced 11β-HSD2 activity due to impaired renal sodium excre- conditioned to resist ischaemic insult. tion. Chronic ENaC blockade prevents sodium accumulation in

19P Oral Communications repeated experiments. These data suggest ENaC may be up- group compared to the non-induced group (0.5±0.1 vs. regulated in hsd11b2+/- mice due to inappropriate MR activa- 0.95±0.16 μmol/min, p<0.05). A further incremental drop in tion by glucocorticoids. The resulting unregulated increase in sodium excretion was found after three days of induction Na+ reabsorption could be contributing towards the increased (0.36±0.09 vs. 0.95±0.16 μmol/min, p<0.05). Acute injection BP observed in hsd11b2+/- mice on a high Na+ diet. of HCTZ caused a significant natriuresis in all three groups of (1)MarinielloBetal.(2005).Analysisofthe11beta-hydroxysteroiddehy- rats: the net effect of thiazide on sodium excretion was greater drogenase type 2 gene (HSD11B2) in human essential hypertension. in the 1 day (10.8±1.5 μmol/min, NS) and 3 day (12.9±1.4 Am J Hypertens. 8, 1091-8. μmol/min, p<0.05) group than in the non-induced controls (7.2±0.84 μmol/min). Chronic administration of HCTZ signifi- (2) Craigie, E et al (2008). 11β-HSD2 Heterozygote Mice Display Blunted Natriuresis, Increased Blood Pressure & Impaired ENaC Regulation on cantly blunted the hypertensive response to transgene induc- a High Sodium Diet. Physiological Society main meeting abstract pre- tion (145.1±4.1 mmHg; n=6). sented as oral communication. These data suggest that thiazide-sensitive sodium transport contributes to the development of angiotensin II dependent Where applicable, the authors confirm that the experiments hypertension. described here conform with The Physiological Society ethical Kantachuvesiri S, Fleming S, Peters J, Peters B, Brooker G, Lammie AG, requirements. McGrath I, Kotelevtsev Y, Mullins JJ. (2001). J Biol Chem 276, 36727–36733. Liu X, Bellamy CO, Bailey MA, Mullins LJ, Dunbar DR, Kenyon CJ, Brooker G, Kantachuvesiri S, Maratou K, Ashek A, Clark AF, Fleming S, Mullins C30 JJ. (2009). J Biol Chem. Epub ahead of print.

Role of thiazide-sensitive sodium transporter in the I would like to thank Gillian Brooker for technical assistance. development of angiotensin II dependent hypertension Where applicable, the authors confirm that the experiments A. Ashek, J.J. Mullins and M.A. Bailey described here conform with The Physiological Society ethical requirements. Molecular Physiology,CVS, University of Edinburgh, Edinburgh, UK Inappropriate modulation of the renin angiotensin system (RAS) can lead to deranged blood pressure homeostasis. To identify C31 important mechanisms underlying the development of angiotensin II dependent hypertension, we inserted the mouse The impact of a low sodium diet on the renal functional Ren2 gene into the Fischer rat genome under the control of an responses to angiotension 1-7 (Ang1-7) in anaesthetised rats inducible Cyp1a1 promoter (1). Induction of the transgene causes an increase in blood pressure, associated with a reduc- A. Corbett and E.J. Johns tion in urinary sodium excretion (2). The aim of this investiga- Department of Physiology, University College Cork, Cork, Ireland tion was to establish the contribution of renal sodium handling to the development of hypertension in the Cyp1a1 Ren2 trans- Angiotension converting enzyme isoform 2 (ACE2) converts genic rat. angiotensin II (AngII) into Ang1-7 which has vasodilator and Expression of the Ren2 transgene was induced by daily gavage natriuretic and diuretic actions at the kidney. Burgelova et al of indole 3 carbinol (I3C) at the dose of 100mg/kg Bwt for either (2005) reported that the influence of Ang1-7 on renal haemo- 1 or 3 days (with a non-induced group as control), after which dynamic and excretory function was enhanced in rat models rats were anesthetized (Inactin, 120mg/kg, IP) and prepared of hypertension in which plasma renin activity was elevated. for renal clearance experiments as described (2). A urine col- This study examined whether activation of the endogenous lection was made to establish baseline sodium excretion, renal renin-angiotension system, by placing animals on a low sodium blood flow and glomerular filtration rate. In the same rats, diet, would enhance the haemodynamic and excretory actions hydrochlorothiaxzide (2mg/Kg Bwt/h) was administered to of Ang1-7 at the kidney. inhibit NCC and a second urine collection made. At the end of Groups (n=5 or 6) of male Wister rats (220-300g) were main- the experiment, a 1ml blood sample was taken and rats killed tained on a normal (0.3% Na+) or a low sodium (0.03% Na+) by an overdose of Inactin. In a separate cohort of rats, HCTZ diet for 2 weeks. Following anaesthesia, 1ml chlo- was chronically administered via osmotic minipump (4mg/d; ralose/urethane (16.5/250 mg/ml) i.p., cannulae were placed 50% DMSO in saline), implanted under halothane anaesthesia. in a femoral artery, to monitor mean arterial pressure (MAP) Baseline Systolic blood pressure (SBP) was obtained using tail and vein, to allow infusion of saline (0.9g/l NaCl) containing cuff before any induction of the Ren2 transgene and then SBP inulin (2g/l) at 3ml/h. The left kidney was exposed via the flank, was measured during the 3 days of induction by I3C daily gav- its ureter cannulated for urine collection, the renal artery age. Statistical comparisons were made by using one-way cleared to fitf an ultrasonic flow probe to measure renal blood ANOVA (Tukey posthoc test). flow (RBF), and a small cannula was inserted 4.0 to 4.5 mm into BP in the non-induced Ren2 transgenic rats was (125±4.8 the cortex to lie at the cortico-medullary boarder and saline mmHg; n=6). Induction of Ren2 expression for either 1 infused at 1 ml/h. Afterg a 2h stabilisation period, four 20 min (146.9±2.2 mmHg; n=9) or 3 (171.5±5 mmHg; n=6) days clearances were performed, two before and two 30 min after caused a significant increase in BP. Neither glomerular filtration starting an infusion of Ang1-7 at a concentration of either 9x10- rate nor renal blood flow was affected by transgene induction. 7 M or 3x10-6M. Data, means±S.E.M., were deemed significant Excretion of sodium was significantly lower in the 1 day induced when P<0.05 (Wilcoxon Signed Rank Test).

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Control values for MAP, glomerular filtration rate (GFR), RBF and Student t test for dependent or independent samples, as and urine flow (UV) were similar in all groups at 92±3 mmHg, appropriate. 2.50±0.15 ml/min/kg, 4.5±0.5 ml/min and 21.9±2.1 μl/min/kg From 16th day of HS diet, SBP became higher than in STD rats while fractional sodium excretion (FENa) was reduced (P<0.05) (163±3 vs. 140±13 mmHg, P<0.05). PNa did not change signi- in the low sodium diet group, 0.75±0.14 versus 1.45±0.32%. ficantly but Posm increased during high sodium intake. RBF, Infusion of Ang1-7 at 9x10-7 and 3x10-6 M increased GFR by CBF and IMBF (but not OMBF) tended to increase in HS rats, 20% and 28%, UV by 23% and 71% and FENa by 25% and 95%, which resulted in a reduction of the OMBF/IMBF ratio. In HS rats respectively (all P<0.05) in rats on a normal sodium diet. In the increase in CBF in response to Ach (10 μg/kg/h) was signi- the rats on a low sodium diet, both doses of Ang1-7 increased ficantly smaller than in STD rats (33±10 vs. 57±15 Perfusion (all P<0.05) GFR by 45% and 31%, UV by 200% and 225% and Units, P<0.05). For outer and inner medulla, the vasodilator FENa of 350 and 320%, respectively, which were larger (P<0.05) response to Ach (5 μg/kg/h) was visibly impaired compared to than the responses in the rats on a normal sodium intake. that in STD rats (15±10 vs. 40±18 PU for OMBF and 6±7 vs. Ang1-7 has vasodilator properties, as it raised GFR but this was 32±20 PU for IMBF, respectively). not related to the dose of peptide infused or the dietary sodium In HS rats the dose dependence of the response to NA was visibly status. By contrast, in rats on a normal sodium intake, the diminishedforRBFandcompletelyabolishedforCBF.Thedecrease diuretic and natriuretic responses to Ang1-7 were dose related in CBF in response to the higher dose of NA was significantly less suggesting a tubular action. Interestingly, the excretory in HS than in STD rats (6±1vs.25±3 %, respectively, P<0.001). responses to Ang1-7 were markedly enhanced in rats on the Our measurements of intrarenal regional perfusion confirm low sodium diet and appeared maximal as a dose-response rela- that changes in Na intake alter renal vascular reactivity to tionship was not evident. These findings demonstrate that vasoactive agents. On high Na diet the ability of the renal cor- manipulation of dietary sodium intake impacts on the ability tex and medulla to vasodilate was distinctly impaired. The rel- of Ang1-7 to determine sodium excretion. ative resistance to vasodilatation in the renal medulla, the Burgelova M et al. (2005). Kidney Int 67,1453-1461. region crucial for the control of arterial blood pressure, could contribute to the rise in the pressure observed in HS rats. On Where applicable, the authors confirm that the experiments the other hand, high Na intake appeared also to limit the cor- described here conform with The Physiological Society ethical tical vasoconstrictor response to NA, which could help main- requirements. tain glomerular filtration and renal excretion. Supported by N N401 225634 grant Where applicable, the authors confirm that the experiments C32 described here conform with The Physiological Society ethical requirements. High sodium diet modifies renal vascular responses in Wistar rats K. Olszynski, M. Kuczeriszka, B. Badzynska, A. Walkowska and C33 E. Kompanowska-Jezierska Contrast hydrotherapy following two modes of high- Laboratory of Renal and Body Fluid Physiology, M Mossakowski intensity cycle exercise: Blood lactate clearance and Medical Centre, Warsaw, Poland subsequent exercise performance High salt intake is a known risk factor in the development of D. Crampton1, B. Donne1, M. Egana1 and S. Warmington2 hypertension and related kidney damage. Changes in renal 1 blood vessel function and morphology may also be directly Department of Physiology, Trinity College Dublin, Dublin, Ireland 2 related to consequences of high salt intake other than hyper- and School of Exercise and Nutrition Sciences, Deakin University, tension, such as hypernatremia, altered reactivity to vasoac- Melbourne, VIC, Australia tive agents, oxidative stress or inflammatory process. The aim Contrast hydrotherapy comprising immersion in alternating of our study was to examine the impact of high salt diet on the cold and warm water baths during recovery from exercise is reactivity of intrarenal vasculature estimated on basis of postulated to facilitate lactate clearance by inducing fluctua- changes in intrarenal regional perfusion. tions in muscle blood flow1. This study compared a 30 min pas- The experiments were approved by the First Warsaw Ethical sive non-immersed recovery (PASS) with two separate cold Committee. In male rats maintained on standard (STD, 0.25% (8∞C) to warm (40∞C) water contrast ratios (CON 1:1 and CON Na w/w) or high sodium diet (HS 4% Na w/w) for 3 weeks, sys- 1:4) to examine blood lactate (BLa) clearance and subsequent tolic blood pressure (SBP, tail cuff method), plasma sodium performance following two different modes of high-intensity (PNa) and osmolality (Posm) were measured repeatedly (HS, cycle exercise. Ethical approval was granted by the Health Sci- n=15; STD, n=6). After 3 weeks rats were anaesthetized (sodium ences Research Ethics Committee, Trinity College Dublin. thiopental, 100 mg/kg, i.p.) and 10-min infusions of acetyl- Wingate test (WAnT) Protocol: Eight active, male volunteers choline (Ach, 5 and 10 μg/kg/h) and noradrenaline (NA, 10 and (25±3 yr; 82±6 kg; 180±9 cm) completed three trials separated 30 μg/kg/h) were given via renal artery, in random order (HS, by 7 days. For each trial, subjects completed three 30-s Wingate n=6; STD, n=4). Total renal blood flow (RBF, Transonic probe) tests. Post recovery, the Wingate tests (WAnT1, WAnT2, and cortical (CBF), outer- and inner medullary (OMBF, IMBF) WAnT3) were repeated. blood flows (laser-Doppler technique) were measured. The Repeated Intermittent Sprint (RIS) Protocol: Eight active, male mean values of all the parameters were compared using ANOVA volunteers (23±1 yr; 81±5 kg; 184±4 cm) completed three RIS

21P Oral Communications trials separated by 7 days. They completed an incremental test with an ACE insertion sequence, due to reduced ACE gene to establish Pmax. Intermittent 30-s workloads were calculated expression in muscle. as 40% Pmax (recovery) and 120% Pmax (sprint). Post recovery, Participants were untrained healthy medical students (n=12) RIS protocol was repeated to failure. recruited from the University of Berne. Aerobic performance Bothgroupscompletedthesamerandomisedrecoveryinterven- was measured and muscle biopsies collected, under a local tions:CON1:1(2.5mincold:2.5minwarm);CON1:4(1mincold: anaesthetic (lidocaine), from the Vastus lateralis muscle. Ultra- 4minwarm)andPASS(seated,non-immersedpassiverecovery). structure and gene expression were quantified in biopsies with Data were analysed using a two-way repeated measures ANOVA morphometry and cDNA microarrays. DNA was extracted and with Holm-Sidak post-hoc analysis (data presented as mean±SD). the ACE genotype identified post-hoc with specific primers. BLa concentration for the WAnT group was significantly lower Theparticipantsfellintotwocategories:DDandID.TheDD at 1 and 2.5 min of recovery in CON 1:1 and CON 1:4 compared genotype vs. the ID genotype demonstrated increased mRNA with PASS (9.6±2.4, 9.7±2.3, 13.1±2.3 mmol.l-1; P<0.001 and expression of ACE and Ang2 receptors, elevated mitochondrial 9.8±2.3, 11.1±3.2, 13.1±1.8 mmol.l-1; P<0.01). There were no density,capillarydensity,andreducedfibersize(seefig.1).Max- differences in BLa between interventions at any other time dur- imal oxygen uptake during bicycle exercise in the DD genotype ing recovery. Post recovery, mean power (MP) was significantly was elevated by 27% and correlated positively and negatively, higher (P<0.05) for WAnT1 in both CON 1:1 and CON 1:4 respectively, with mitochondrial volume density (r=0.65) and (669±71, 681±70W) compared with PASS (651±76W). For fibre area (r=-0.53) in the investigated knee extensor muscle. WAnT2, MP was significantly higher (P<0.05) in CON 1:4 The investigated, not-specifically trained population demon- (657±52W) compared with CON 1:1 and PASS (647±43, stratedashifttowardsamusclephenotypewithimprovedsub- 628±57W). A similar finding was observed for WAnT3. stratesupplyandaerobicmetabolism.Thissuggeststhatexpres- For the RIS group, there were no significant differences in BLa sional alterations in the local Ang2 signalling system intervene between recovery interventions, however total work performed in the manifestation of an endurance phenotype via the modu- post-recovery was significantly higher (P<0.001) for CON 1:1 lation of capillarity and aerobic capacity in locomotor muscle. and CON 1:4 compared with PASS (92719 ± 25765, 90503 ± Figure 1. Ultra-structural and mRNA differences between ACE DD v ID 25599, 74529 ± 22486kJ respectively). genotype DespitenooveralldifferenceinBLaclearance,contrasthydrother- apyhadapositiveeffectonexerciseperformanceinbothWAnT and RIS groups. Although BLa was lower during the initial phase of contrast water immersion for the WAnT group, this was not sustainedoverthedurationoftherecoveryperiodandnoeffect on BLa was evident during contrast hydrotherapy following RIS. 1. Fiscus KA, Kaminski TW & Powers ME. (2005). Changes in Lower-Leg Blood Flow During Warm-, Cold-, and Contrast-Water Therapy. Arch aPer fiber volume,bmm/mm3 of fiber volume, cμm2, dnormalised to total Phys Med Rehabil 86, 1404-1410. (x10-4) mRNA per array, eangiotensin receptor-1, fangiotensin receptor-2, ghonest significant difference for unequal number, hstatistical analysis of Where applicable, the authors confirm that the experiments microarrays (SAM), zonly 4 samples analysed described here conform with The Physiological Society ethical Amir O et al. (2007). Experimental Physiology 92, 881-886. requirements. Montgomery HE et al. (1998). Nature 2. Rankinen T et al. (2000). Journal of Applied Physiology 5. ScanaviniDetal.(2002).EuropeanJournalofHumanGenetics10,576-577. C34 Zhao, B., et al (2003). Life Sciences 73, 2625-2630.

Aerobic fitness in humans is under system control by the Where applicable, the authors confirm that the experiments angiotensin 2 pathway described here conform with The Physiological Society ethical requirements. D. Vaughan1, H. Hoppeler2 and M. Flueck1 1IRM, Manchester Metropolitan University, Manchester, UK and 2Institute of Anatomy, University of Bern, Bern, Switzerland C35

Bloodsupplyiscriticaltofuelmusclecontractionandlimitsexer- Maximal strength training of the plantar flexors: can changes cise performance. The presence of a silencer sequence in intron in the brain be detected using functional magnetic 16 (insertion, I) in the ACE (angiotensin converting enzyme) resonance imaging? gene, which is the enzyme that converts the vasoconstrictor H.S. Palmer, M.S. Fimland, G.M. Solstad, V. Moe Iversen, J. Hoff, angiotensin(Ang)intoitsactiveformAng2,hasbeenassociated J. Helgerud and A. Håberg with enhanced endurance capacity (Montgomery et al. 1998 andScanavinietal.2002).However,conflictingevidenceexists Department of circulation and medical imaging, NTNU, on the association of ACE genotypes with regard to the pres- Trondheim, Norway ence (or absence) of the I sequence and human performance The maximal strength training protocol used in the present (Rankinen et al. 2000, Amir et al. 2007 and Zhao et al. 2003). study has previously been shown to enhance muscular strength Adaptations of capillary supply lines in muscle contribute and and neural drive. Results suggested that these neuronal changes explain the altered endurance phenotype in ACE genotypes, were located at the supraspinal level. This hypothesis was tested

22P Oral Communications using blood oxygen level dependent functional MRI, fMRI, to endurance fatigue test to explore the contribution of central identify possible neuronal correlates to increased strength. In and peripheral limitations to short-term maximal exercise per- addition, cross education of strength in the untrained leg was formance. We hypothesized that pre-frontal cortex HbO2 would investigated. The experimental protocol was approved by the peak prior to termination of the maximal exercise, reflecting local ethics committee and all subjects gave written consent. “central fatigue”. Healthy physically active males (N=10; mean The training protocol consisted of 16 sessions in 4 weeks ± SD age, height, body mass =23.1±3.1 yr; 179.6±3.8 cm; whereby subjects performed 6 sets of 6 isometric plantar flex- 85.9±7.9 kg) volunteered to perform a maximal knee exten- ion repetitions, unilaterally on the dominant leg (determined sion/flexion isokinetic (180degrees/sec) muscular endurance using the Waterloo Footedness questionnaire). Young healthy trial of 50-repetitions. Pre-frontal cortex and muscle (right volunteers who had not undertaken strength training in the quadriceps femoris) HbO2 and HHb were monitored with a last 6 months were assigned to training (n=15) or control (n=11) multi-channel NIRS system, expired gases were collected using groups. Most subjects (n=12, n=9 respectively) consented to a ParvoMedics TrueMax 2400 metabolic cart, and heart rate MRI investigation before and after the training intervention as was monitored using a Polar HR monitor. Results (Mean±SD) well as tests for: neural recruitment at rest; maximum volun- showed that peak VO2, VE, and HR increased during testing to tary isometric contraction torque (MVIC) and electrically 2.16±0.24 L/min, 92.3±28.9 L/min, and 164±14 bpm, respec- evoked spinal reflexes. MRI investigation was performed using tively. Mean quadriceps peak torque and total work was a 3.0 T Siemens Trio system and included a T1-weighted 170.7Nm and 6392.6Nm, respectively. The fatigue index anatomical scan and fMRI during a series of isometric soleus (67±8%) correlated (Pearson R) with peak torque (r=0.74; contractions in response to a visual stimulus. During the task p<0.05). There was a significant increase (ANOVA) in pre-frontal the foot was held in place with an MR compatible device, to cortex HbO2 and muscle HHb during the 50-reps, with peak enable isometric contractions to be performed. Various meas- pre-frontal cortex HbO2 (19.6mM) and muscle HHb (13.7mM) ures were taken to reduce head motion including a vacuum pil- occurring at contraction number 38 and 36, respectively. There- low around the neck and shoulders and paradigm presentation after, pre-frontal cortex HbO2 gradually decreased to com- (Eprime, PST, Pittsburgh, PA) using VisualSystem goggles pletion of the test (50-reps), while muscle HHb reached a (NordicNeuroLab, Bergen, Norway). The fMRI images were plateau and remained at the same level until the end of the analysed using FSL (FMRIB, Oxford, UK). MVIC was analysed fatigue trial. Both variables correlated with torque output (r=- by two way analysis of variance with Bonferroni post hoc test- 0.61; -0.53, respectively). These results suggest that 1) NIRS ing (SigmaStat) and did not differ significantly across the groups can reliably track changes in pre-frontal cortex HbO2 and mus- prior to training. MVIC in the training group significantly cle HHb during isokinetic muscular endurance fatigue testing; increased from 131.8 ± 6.8 to 182.6 ± 7.8 Nm (p=0.00019) fol- and 2) the peak pre-frontal cortex HbO2 that occurred before lowing training, whilst the control group showed no change termination of the test, simultaneous with a plateau in muscle (pre 147.1 ± 11.5 Nm; post 145.5 ± 15.1). The training group HHb, would suggest that central factors were implicated in the also demonstrated a significant increase in MVIC in the maximal knee extension muscular exercise fatigue in this study. untrained leg (p=0.0087). The fMRI data from two control sub- We would like to thank the volunteer subjects for their partic- jects was excluded due to excessive head motion (>2 mm). Task- ipation in this study. Financial Support: NSERC (JPN); University related brain activation in motor and pre-motor regions was of Applied Science (MK-B). observed. However, when contrasted using standard thresh- olding (p<0.05, corrected for multiple comparisons) at the Where applicable, the authors confirm that the experiments whole brain level, a significant training effect was not detected. described here conform with The Physiological Society ethical Maximal isometric plantar flexion training enhances strength requirements. in the trained leg and also in the untrained leg through cross education. However, significant changes at the whole brain level in response to the training intervention were not observed. C37 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical How many myosin heads act on a single actin filament at any requirements. instant in working muscle? G.F. Elliott1 and C.R. Worthington2 1 C36 Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK and 2Physics and Biology, Carnegie-Mellon University, Pre-frontal Cortex and Muscle Oxygenation During Maximal Pittsburgh, PA, USA Isokinetic Endurance Exercise If N is the number of myosin heads acting on a single actin fil- J. Neary1, M. Olbert2, M. Kohl-Bareis2, W. Duff1, J. Lazorko1, ament in vertebrate striated muscle, working at any instant to S. Poloskei1, D. McLeod1 and D.G. Candow1 produce tension, stoichiometry gives 150 ≥ N ≥ 1; the actual value of N is clearly an important parameter. 1Kinesiology & Health Studies, University of Regina, Regina, SK, 2 Stiffness measurements have long been used to estimate N, Canada and Biomedical Optics, University of Applied Sciences, though a warning was sounded [1] after X-ray measurements Koblenz, Remagen, North Rhine-Westpahlia, Germany showed that a sizeable amount of the muscle compliance arose We measured pre-frontal cortex and muscle oxygenation from the actin and myosin filaments. The usual approach com- (HbO2) and deoxygenation (HHb) during a maximal muscular pares the instantaneous stiffness measured during quick stretch

23P Oral Communications or quick release in rigor and contraction. Immediately after this sity of resistance exercise in the post absorptive state (Kumar response, though, the time scales of the tension response are et al 2009). We have now tested the hypothesis that doubling very different: in rigor the tension persists for several tens of the volume of exercise would increase the MPS response in ms; in contraction it changes with a half time of a few ms. overnight fasted men, both young (n=5, 24±6 y, body mass This stiffness approach makes three inherent assumptions: (i) index (BMI) 23±2 kg.m-2) and elderly (n=6, 70±5 y, BMI 24±2 in rigor N = 150 (here resisting extension rather than produc- kg.m-2). The subjects performed, at a 3 months interval, first ing tension); (ii) the rigor stiffness is the same as when the inter- 3 and then 6 sets of 8 repetitions of isotonic unilateral leg exten- actions are producing tension; (iii) the filament compliance is sion at 75% of their one repetition maximum (1 RM). Muscle the same in contraction as in rigor. However Kawai and Brandt biopsies were taken from the vastus lateralis of the exercised showed that the stiffness of rigor (crayfish) single fibres could leg under local anaesthesia (1% lignocaine sc) before, imme- change by a factor of two or more depending on the approach diately after and 1, 2 and 4 h after exercise. After separation of to rigor [2]. Therefore the assumptions are probably over-opti- myofibrillar protein, incorporation of [1,2-13C]leucine was mistic, especially given the differences in ionic composition of measured (by gas chromatography-combustion-mass spec- the solutions used to induce the various states. trometry) and MPS calculated using plasma labeling of α- For all these reasons we think it wise to accept the warning of ketoisocaproate as surrogate precursor. Goldman and Huxley [1], that ‘stiffness cannot be used safely Figure 1. as a measure of cross-bridge attachment’, and we choose to In young men doubling the volume of exercise at 75% 1RM had discount numbers derived from stiffness measurements. It is no additional effect on the response of MPS in the post absorp- a pity that there is no independent experimental method to tive state; however, in older men, it resulted in a more rapid estimate N. and greater response. The results suggest that there is increased We present a model (derived from other physiological and bio- latency of response to exercise in muscle of older men. chemical data) where the average time between impulses on an actin filament is one or two ms and that gives an inherent natural explanation of AV Hill’s force-velocity equation from first principles [3]. The model gives physical meaning to Hill’s constants ‘a’ and ‘b’ and it also implies that the size of the individual contractile impulse in situ between an actin filament and one myosin head must be approximately 140 pN.ms (an impulse has the dimensions of force x time). The impulses nor- Figure 1. Time course of responses of MPS (%/h) to exercise at 75% 1RM of mally act sequentially in time along the filament [4]. In response 3 or 6 sets of leg extension exercise in post absorptive young and older to a fast perturbation, however, extra impulses may occur subjects. Mean ± SEM. * = P<0.05 vs. basal (ANOVA with Bonferroni post within the same time frame [5]. Our impulse model provides hoc adjustment). NB protein synthesis is measured over 2.5 h in the basal an alternative estimate of N; accumulating biochemical evi- pre-exercise state and then over the periods shown post-exercise. dence supports our view that N is of the order of 1 in normal Kumar V, Selby A, Rankin D, Patel R, Atherton P,Hildebrandt W, Williams J, Smith K, Seynnes O, Hiscock N and Rennie M J. 2009. Age-related dif- contraction and 8-9 in transient recovery after quick release. ferences in the dose-response relationship of muscle protein synthe- Goldman YE & Huxley AF (1994) Biophys J 67, 2131-2133 sis to resistance exercise in young and old men. J Physiol 587: 211-217. Kawai M & Brandt PW (1976) J Gen Physiol 68, 267-280. Supported by BBSRC, Unilever plc and the EXEGENESIS program Worthington CR & Elliott GF (1996) Int J biological macromolecules 18, of the EU 123-131 Elliott GF & Worthington CR (1997) Int J biological macromolecules 21, Where applicable, the authors confirm that the experiments 231-275 described here conform with The Physiological Society ethical Worthington CR & Elliott GF (2005) Int J biological macromolecules 35, requirements. 265-268 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical C39 requirements. Identification of aerobic-anaerobic transition in male rowers using surface electromyography during graded incremental exercise C38 N. Fleming1, B. Donne1 and N. Mahony2 Effect of doubling the volume of resistance exercise on 1Physiology, Trinity College Dublin, Dublin 2, Ireland and 2Anatomy, myofibrillar protein synthesis (MPS) and anabolic signaling Trinity College, Dublin, Ireland in muscle of postabsorptive young and old men It has recently been shown that non-linear changes in myo- V. Kumar1, A. Selby1, D. Rankin1, K. Smith1, P. Atherton1, electric data relative to exercise intensity can be used to iden- W. Hildebrandt1, N. Hiscock2 and M.J. Rennie1 tify the aerobic-anaerobic threshold (Condotti et al. 2008, Farina 1 Clinical Physiology, The University of Nottingham, Derby, UK and et al. 2007. However these studies restricted their analysis to 2 Unilever Discover R & D, Sharnbrook, UK cycling tasks. The aim of this study was to assess the use of sur- We have previously reported that older men show a blunted face electromyography (EMG) as a non-invasive determinant dose response relationship between increases in MPS and inten- of the metabolic response to incremental rowing exercise. The

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relationships between EMG threshold (TEMG) and more com- receptors (RyR) in the SR membrane and the dihydropyrimi- monly used variables for detection of the aerobic-anaerobic dine receptors (DHPR) in the cellular membrane. ECCE differs threshold namely; blood lactate threshold (TLac) and onset of from SOCE that it does not require emptying of intracellular blood lactate accumulation (OBLA) were assessed. Ca2+ stores but depends on depolarization of the membrane Eleven male club-level rowers (age 21±4yr, height 1.88±0.04m, (2). STIM1 haploinsufficiency has been shown to affect tetanic ± ± -1 -1 mass84 7kg,VO2max60.9 5.8mL.kg .min )performedgraded force in mice (5). Both SOCE and ECCE is blocked by SKF 96365 tests to volitional exhaustion on a Concept II ergometer. This eth- (1). The aim of this study was to investigate whether SOCE is icallyapprovedstudyinvolvedintermittentexerciseboutsatfixed important for the maintenance of contractile function in rat workloads(startload120W,duration3min,rest1min,increment skeletal muscle and to investigate whether SOCE or ECCE is 40W) during which EMG data were recorded from Rectus Femoris involved in the observed excitation-induced uptake of Ca2+. (RF),Vastus Lateralis (VL),Biceps Femoris(BF)andupperportionof Isolated EDL muscle from 4-week old Wistar rats were mounted Trapezius (UT).Therestperiodbetweenincrementsfacilitatedear- at fixed length on a muscle holder connected to a transducer. lobebloodsamplingforlactatedetermination.Rootmeansquare The muscles were incubated in buffer with 0 Ca2+ either at rest EMG (rms-EMG) for each muscle were calculated using a 50ms or stimulated at 1 Hz for 120 min (0.2 ms pulses, 24 V/cm). Fol- averaging window over 10 consecutive stroke cycles during the lowing incubation the muscles were allowed to recover for 40 final minute of each exercise increment. Individual loads at EMG min in either Ca2+-free buffer or normal Krebs Ringer (NKR) con- thresholdwereidentifiedusingtheV-slopemethodandcompared taining 1.27 mM Ca2+. Maximum tetanic force was measured againstloadatTLac andOBLAusingarepeatedmeasuresANOVA. prior to incubation, immediately after incubation and at 20 and Pearson’s correlation analysis showed strong association 40 min recovery (90 Hz, 0.5 sec). Mean values ± SD are given. between blood lactate and rms-EMG activity in RF (r=0.81), Statistical difference between groups was ascertained using a VL (r=0.63) and BF (r=0.70), and lower association in UT t-test for non-paired observations. (r=0.47). Analysis revealed no significant differences between Stimulation in Ca2+ free buffer led to a 60% loss of tetanic force. ± ± ± 2+ TLac (256 10W) and TEMG in RF (265 8W) and VL (267 7W), If muscles were allowed to recover in Ca free buffer no force however TEMG occurred at significantly higher workloads recovery was observed, however if muscles recovered in NKR (P<0.01) for BF (273±9W) and UT (275±11W). No significant a complete force recovery was observed. The amount of Ca2+ 45 differences were observed comparing TEMG and OBLA taken up during recovery was measured using Ca and corre- (276±12W) in any of the muscles investigated. sponded to 132±5 nmol/Ca2+ g wet wt. (n=3). Addition of the 2+ Our results suggest that TEMG is strongly associated to both cation channel blocker SKF 96365 reduced the uptake of Ca ± 2+ OBLA and TLac (r=0.75 to 0.94) and that there are differing by 60% (to 48 36 nmol/Ca g wet wt., n=3, P<0.05) and pre- recruitment strategies relative to increasing exercise intensity vented force recovery. Stimulation at 1 Hz for 120 min led to a between the muscles analysed in the current study. Our data marked uptake of 45Ca in accordance with previous studies (3). showed comparible levels of correlation with previously pub- SKF reduced this stimulation induced uptake. lished literature (Farina et al. 2007), however further exami- From this study it is concluded that SOCE can be observed in nation of TEMG in other muscles and in other dynamic tasks is whole isolated muscles and that this uptake is important for necessary in order to better assess the potential use of EMG as the maintenance of contractile function. The effect of SKF a non-invasive determinant of the aerobic anaerobic threshold. 96365 on the stimulation induced uptake of Ca2+ suggest Farina D et al. (2007) J Electromyog Kinesiol 17 393-400 involvement of SOCE or ECCE. Condotti et al. (2008) Can J Physiol Pharmacol. 86 272-278 Cherednichenko G et al.(2004). PNAS 101: 15793-15798. Gach MP et al.(2008). Biophys J 94: 3023-3034. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Gissel H and Clausen T.(1999). Am J Physiol 276: R331-R339. requirements. Lyfenko AD and Dirksen RT.(2008). J Physiol 586: 4815-4824. Stiber J et al.(2008). Nat Cell Biol 10: 688-697. This work was supported by a grant from the Lundbeck C40 Foundation. Uptake of Ca2+ is necessary for maintenance of contractile Where applicable, the authors confirm that the experiments function and is partly inhibited by the cation channel blocker described here conform with The Physiological Society ethical SKF 96365 in rat skeletal muscle requirements. H. Gissel and A. Fredsted Institute of Physiology and Biophysics, Aarhus University, Örhus, C41 Denmark It has previously been shown that excitation is associated with Are the projections from the periaqueductal grey to pontine an uptake of Ca2+ in rat skeletal muscle (3). The mechanism noradrenergic neurones excitatory or inhibitory? 2+ behind this uptake has remained elusive. Store-operated Ca L. Hickey1, E. Sher2, B. Lumb1 and A.E. Pickering1 entry (SOCE) or excitation coupled Ca2+ entry (ECCE) may serve 1 as possible entry mechanisms. The molecular basis for SOCE Department of Physiology & Pharmacology, University of Bristol, 2 has now been elucidated and involves the Ca2+ sensing protein Bristol,UKand LillyResearchCentre,EliLilly,Windlesham,Surrey,UK STIM1 in the SR membrane and the pore forming Orai in the The periaqueductal grey (PAG) is a midbrain site involved in the cellular membrane (4). ECCE seems to involve the ryanodine modulation of nociception and is believed to control nocicep-

25P Oral Communications tion, in part, by activating pontospinal noradrenergic (NA) neurones. Connections between the PAG and noradrenergic cell C42 groups have previously been demonstrated (Bajic et al., 2001), however, the neurotransmitter phenotype is as yet unknown. Acute and chronic effects of GDNF and BDNF on thermoTRPs To address this issue we have injected viral vectors to the ven- from rat dorsal root ganglion (DRG) neurons trolateral PAG (VL-PAG) to anterogradely label projections to C. Ciobanu1, A. Babes1 and G. Reid2 pontine noradrenergic (NA) neurones in combination with immunocytochemistry for glutamate (vGlut1&2) and GABA 1Department of Animal Physiology and Biophysics, University of (vGat) transporters. Bucharest, Bucharest, Romania and 2Department of Physiology, Male Wistar rats (n=3) received injections of AAV-CMV-EGFP University College Cork, Cork, Ireland (250nl) under anaesthesia (ketamine 60mg.kg-1/medetomidine 25μg.kg-1 i.p) into the VL column of the PAG. After 8 days TRPA1, TRPV1 and TRPM8 are cation channels expressed in all animals were terminally anaesthetised with sodium pento- sensory neurons involved in thermal perception and pain barbital (70mg.kg-1 i.p.) and perfusion fixed with 4% formalin, sensing. Neurotrophins (NTFs) Brain Derived Neurotrophic brains were removed and 40μm sections were cut. Sections Factor (BDNF) and Glial cell line-Derived Neurotrophic Factor were processed immunocytochemically to identify dopamine (GDNF) are essential for neuronal development and survival β-hydroxylase and visualise terminals containing EGFP and but they are also involved in pain signaling and neuronal mod- VGlut1&2 or VGat. Injection sites and terminal labelling were ulation. The aim of this study was to assess effects of chronic determined using confocal imaging and 3D reconstruction soft- and acute treatment with BDNF and GDNF on functional ware (Volocity 4, Improvision). expression and activation of TRPM8, TRPA1 and TRPV1 in rat AAV-CMV-EGFP produced strong terminal labelling in the DRG neurons. pons with a predominantly ipsilateral distribution. Terminals Calcium Green-1 imaging was used to monitor responses of showing co-localisation with Vglut or Vgat were identified neurons stimulated with specific activators for TRP channels. in A5, A6 and A7 cell groups indicating excitatory or Primary cultures of adult Wistar rat DRG were split in two; half inhibitory amino acid (EAA or IAA) projections from the VL- of the neurons were incubated with 100ng/ml NTF for 12-24h, PAG. Quantification of terminals closely apposing NA cell bod- and the other half used as control. For acute effects, neurons ies in these regions showed that the LC received the dens- were treated for 7 minutes with 100ng/ml BDNF or GDNF est innervation of EAA and IAA profiles. However after between two consecutive stimuli (MO or heat). For all experi- analysis of the density of innervation per NA neuron, the A7 ments, serum free medium (Bottenstein et al., 1980) was used. cellgroupreceivedthelargestnumberofEAAandIAApro- Chronic treatment with NTFs changed the pattern of functional files per soma (3.5±1.8 and 3.8±1.5 profiles/soma respec- expression of TRP channels. Thus, GDNF increased the popu- tively). By contrast the A6 cell group received moderate EAA lation of MO-sensitive neurons from 28% to 37% (141/513 to and IAA innervation (1.3±0.4 and 1.1±0.3 profiles/soma). 117/313, χ2 test, p < 0.01) and BDNF increased the fraction of The A5 territory received more IAA (3.5±2.2 profiles/soma) CAP-sensitive neurons from 61% to 76% (312/513 to 208/274, than EAA innervation from the VL PAG (1.5±1.0 pro- χ2 test, p < 0.001). BDNF also increased the amplitude of the files/soma). responses to Me (from ΔF/F0 = 0.20 ± 0.02, mean ± S.E.M., n = In conclusion, we show that VL PAG projections to A5, A6 and 69 up to 0.26 ± 0.02, n = 53, p<0.05, Student t test), MO (from A7 have terminals closely apposed to NA neurones. These ter- 0.46 ± 0.01, n = 141, to 0.54 ± 0.02, n = 78, p<0.01, Student t minals express vesicular transporters for EAA and IAA sug- test) and CAP (from 0.62 ± 0.01, n = 312, up to 0.68 ± 0.02, n gesting that there are both excitatory and inhibitory phe- = 208, p<0.01, Student t test). The fraction of neurons co- notypes. The A7 region is densely innervated by expressing TRPV1 and TRPA1 (neurons activated by both MO glutamatergic and GABAergic profiles, which may identify and CAP) was increased by chronic GDNF from 24% to 34% (χ2 the neurotransmitter phenotype of previously documented test, p < 0.001). Acute treatment with both GDNF and BDNF symmetrical and asymmetrical synapses made by PAG pro- significantly reduced the magnitude of TRPV1 and TRPA1 jections to A7 (Bajic et al 2001). We have also shown that the desensitization induced by repeated applications of heat and A5 region receives a strong inhibitory input from the PAG, MO. The ratio between the amplitudes of the second and the which may be responsible for the depressor response that first response to heat (used as a measure of desensitization) occurs on activation of VL PAG. These data provide evidence was 0.68 ± 0.05, n = 28 for control compared to 1.10 ± 0.13, n for a specific and differential control of pontine NA neurones = 33 for BDNF treated neurons (Student t test, p < 0.01). GDNF by the VL PAG. had the same effect (ratio control 0.74 ± 0.1, n = 15 vs. 1.20 ± 0.11, n = 36, Student’s unpaired t test, p < 0.05). GDNF also Bajic D, Van Bockstaele EJ & Proudfit HK. (2001). Ultrastructural analysis of ventrolateral periaqueductal gray projections to the A7 cate- reduced the magnitude of AITC-induced TRPA1 desensitization ± ± cholamine cell group. Neuroscience 104, 181-197. (ratio 0.81 0.07, for control vs. 1.00 0.05, p < 0.05). Our results suggest that NTFs regulate the expression and activ- BBSRC and Eli Lilly. ity of the transducer channels TRPA1 and TRPV1, leading to enhanced neuronal sensitivity to noxious stimuli. Acute treat- Where applicable, the authors confirm that the experiments ment with these neurotrophins abolishes desensitization of described here conform with The Physiological Society ethical TRPA1 and TRPV1 which are both involved in pain sensing. Thus, requirements. BDNF and GDNF may be involved in induction and maintenance of chronic pain in the peripheral nervous system. Bottenstein JE et al. (1980). Exp. Cell Res. 125, 183-190.

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical C44 requirements. P2Y activation affects the excitability of murine epidermal primary afferents through modulation of the Kv7-mediated M-current

C43 G. Bruce, R. Barrett-Jolley and R. Morris

Activation of the capsaicin receptor TRPV1 desensitises the VeterinaryPreclinicalSciences,UniversityofLiverpool,Liverpool,UK nociceptive ion channel TRPA1 in rat dorsal root ganglion Koizumi et al., (2004) demonstrated the importance of ATP and neurones P2Y2 receptors in signalling between keratinocytes and sen- C. Doran and G. Reid sory neurones. A population of neurones innervating the epi- dermis are selectively sensitive to ATP in mice (Dussor et al., Physiology, University College Cork, Cork, Ireland 2008). They also express mas-related G protein-coupled receptor D (MrgprD), activation of which inhibits the M-current in Transient Receptor Potential A1 (TRPA1) is an ion-channel recep- IB -labelled rat dorsal root ganglion (DRG) neurones (Dussor tor implicated in a number of nociceptive pathways including 4 et al., 2008; Crozier et al., 2007). The P2Y agonist, uridine cold-induced pain and the detection of noxious stimuli such 2 triphosphate (UTP), has long been known to inhibit the M-cur- as isothiocyanates. It is specifically activated by cinnamalde- rent in bullfrog sympathetic neurones (Adams et al., 1982). Pre- hyde and mustard oil. TRPA1 is often co-expressed with the viously we reported that “non-peptidergic” DRG neurones from heat/capsaicin receptor TRPV1 in primary sensory neurones. a mouse that expressed green fluorescent protein (GFP) in epi- To clarify how TRPV1 activation affects the activity of TRPA1, dermal primary afferents fired in a predominantly transient we have examined the effects of capsaicin on the cinnamalde- manner (Bruce et al., 2008). We investigated whether this fir- hyde sensitivity of dorsal root ganglion (DRG) neurones. ing pattern was due to the presence of an M-current and, if so, Adult Sprague-Dawley rats were killed by 100 % CO2 inhala- how it was modulated by endogenous P2Y agonists. tion followed by decapitation, and dissociated DRG neurones Transgenic mice carrying the thy1.2-egfp gene on a C57/Bl6 were cultured with nerve growth factor for 12-24 hours. Cap- template (SA36 strain) were deeply anaesthetised with saicin (1 μM) and cinnamaldehyde (200 μM) responsiveness of halothane and killed by decapitation. Dissociated DRG neu- individual neurones was evaluated using Calcium Green-1 rones were short-term cultured (1-3) days and used for whole- microfluorimetry (ΔF/F0). TRPA1 activation in DRG neurones cell voltage or current clamp recording. was significantly reduced as evidenced by a diminished sensi- The M-current was isolated in voltage-clamped DRG neurones tivity to cinnamaldehyde (ΔF/F0: 0.03 ± 0.003, mean ± SEM, (25-31 μm diameter) using an inactivation protocol and 1mM n=299, vs. control 0.3 ± 0.02, n=367; P<0.0001, Student’s CsCl. XE-991 (10μM) and UTP (10μM) significantly reduced the unpaired t test). conductance through K 7 channels at -30 to -50 mV and -30 to Next, we determined whether the observed capsaicin-medi- v -40 mV respectively (P<0.05, Dunnetts T). ADP (10μM) had no ated inhibition was dependent upon TRPV1 activation. DRG significant effect on M-current conductance. Under current neurones were pre-incubated with 10mM capsazepine, a TRPV1 clamp conditions, DRG neurones produced predominately tran- antagonist, before evaluating capsaicin-induced inhibition of sient responses to depolarising current injections (16/20 cells). TRPA1. Capsazepine reduced the response of TRPV1 to cap- UTP (10μM) increased afterdepolarisation amplitude and sec- saicin (ΔF/F0 decreased from 0.45 ± 0.03, n=299, to 0.14 ± 0.02, ondary spike frequency (3/5 cells). ADP (10μM) reduced or n=156; P<0.001), and reduced the capsaicin-induced inhibi- blocked the primary spike in some transiently firing neurones tion of TRPA1 (response to cinnamaldehyde increased from (2/5 cells). Bath applied ATP (10μM) elicited spontaneous activ- ΔF/F0 = 0.03 ± 0.003, n=299, to 0.16 ± 0.03, n=156; P<0.0001). ity (4/6 cells) and tonic firing in response to current injection In order to determine whether the capsaicin-induced desensi- (2/6 cells). tisation of TRPA1 occurs via a calcium-dependent mechanism, This data demonstrates that the excitability of neurones we depolarised DRG neurones using 50 mM KCl. This concen- anatomically positioned to receive input from keratinocytes tration elicits an increase in [Ca2+]i similar to that induced by can be modulated by nucleotides through P2Y/K 7 signalling. 1 μM capsaicin in TRPV1-expressing neurones, and desensi- v tised TRPA1 to a similar degree: the response to cinnamalde- Adams PR et al. (1982). J Physiol 332, 223-262. Δ ± hyde ( F/F0 = 0.02 0.002, n=35) was comparable to that fol- Bruce G et al. (2008). Proc Physiol Soc 11, C73 lowing capsaicin-mediated inhibition. We conclude that the desensitising effect of capsaicin on TRPA1 Crozier RA et al. (2007). J Neurosci 27(16), 4492-4496 is dependent on a TRPV1-mediated increase in [Ca2+]i. This Dussor G et al. (2008). J Neurophysiol 99(4), 1581-1589 mechanism could be a potential pharmacological target for modulating TRPA1-mediated nociceptive pathways. Koizumi S et al. (2004). Biochem J 380, 329-338

Financial support was from the Health Research Board, Ireland. Supported by the Pain Relief Foundation.

Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

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Supported by the Medical Research Council. C45 Where applicable, the authors confirm that the experiments KCa channels regulate stretch-evoked afferent firing from described here conform with The Physiological Society ethical muscle spindles requirements. A. Simon1, R.W. Banks2 and G.S. Bewick1 1School of Medical Sciences, University of Aberdeen, Aberdeen, UK and 2School of Biological and Biomedical Sciences, University of Durham, Durham, UK C46 Muscle spindles constantly report skeletal muscle length and movement. Mechanosensory transduction occurs at annu- Motor unit rotation in a variety of human muscles lospiral terminals around intrafusal muscle fibres. We reported P. Bawa and C. Murnaghan that Ca2+-dependent glutamate release from synaptic-like vesicles in these terminals modulates spindle firing (Bewick et al., Department of Biomedical Physiology and Kinesiology, Simon Fraser 2005). To test if voltage-gated Ca2+ channels (VGCCs) or Ca2+- University, Burnaby, BC, Canada + dependent K channels (KCa) are also functionally important, we tested whether channel-selective neurotoxins affected spin- To study the phenomena of substitution and rotation among dle afferent discharge. motor units of a muscle, motoneurone pools of seven differ- Adult Sprague-Dawley rats (male, 300-370 g) were killed ent muscles were investigated. Intramuscular motor unit activ- (Schedule 1, Animal (Scientific Procedures) Act, 1986) and 4th ity and surface EMG (electromyogram) were recorded from lumbrical nerve-muscle preparations excised and placed in one of the following muscles: abductor digiti minimi, first dorsal interosseous, extensor digitorum communis, flexor and gassed (95%O2-5%CO2) Liley`s saline. Spindle afferent dis- charges were recorded en passant with Ag wire electrodes and extensor carpi radialis, tibialis anterior and soleus. The subject firing frequency (mean ± SE, n) determined for the first 0.5 s was asked to discharge a discernible motor unit at a comfort- of the “hold” phase of stretch-and-hold cycles (~10% muscle able constant or rhythmically modulated rate with audio and length) for ‘n’ preparations. The significance of differences visual feedback. Results are reported from a total of 42 sets between pre-drug and with-drug means was evaluated by of motor units from all seven muscles. We observed that when paired t-test, with a threshold of P < 0.05. a subject fired a motor unit for a long period, frequently an Unlike inorganic Ca2+ channel blockers Co2+ and Ni2+/Cd2+, additional motor unit started to discharge after a few min- which abolish responses (Bewick et al., 2005), P/Q type VGCC utes. When the subject was asked to keep activity down to inhibitors enhanced firing. ω-Agatoxin-IVA (200 nM, P/Q type) oneunit,veryoftenitwasUnit1thatdroppedandUnit2con- increased firing to 301% of control (92.7 ± 15.8 imp/s vs 278.6 tinued to fire. While unit 2 had fired for a few minutes, Unit 1 ± 23.9 imp/s, 6; P < 0.0001). ω-Conotoxin-MVIIC (1 μM, Q type) resumed firing without any conscious effort by the subject. If enhanced firing to 202% (98.40 ± 21.83 imp/s vs 199.60 ± 24.62 the subject was then asked to retain just one unit, it was Unit imp/s, 5; P < 0.002). Conversely, ω-conotoxin-GVIA (1 μM; N 2 that dropped. Rhythmic modulation of firing rate of a toni- type) had no significant effect (176.92 ± 14.77 imp/s vs 196.92 cally firing unit showed that while the threshold of this unit ± 10.47 imp/s, 6; P = 0.1). L-type blockers Taicatoxin (50 nM, increased, the threshold of a phasically discharging unit 4) and Nifedipine (10 μM, 5) increased afferent firing (131.88 decreased substantially, and rotation occurred. Quantifica- ± 10.05 imp/s vs 302.13 ± 25.17 imp/s and 192.00 ± 13.09 imp/s tion of firing times of lower and higher threshold units showed vs 271.70 ± 9.56 imp/s, respectively) but also caused sponta- that the total discharge time was significantly longer for lower neous muscle twitching, suggesting actions on skeletal mus- threshold than for higher threshold units. This finding indi- cle dihydropyridine receptors. Thus, only P/Q type channel cates that even when lower threshold units do rest, they blockers increased afferent discharge in the absence of other recover faster to contribute for a longer time to the total con- effects. traction. The increase in threshold of a tonically discharging unit is suggested to arise from inactivation of Na+ and Ca++ P/Q type channels often regulate KCa (BK or SK) channel open- channels and the decrease in threshold of higher threshold ing (Edgerton & Reinhart, 2003). Therefore, selective KCa channel blockers were applied. Charybdotoxin (200 nM; SK & BK), units is suggested to arise from an increase in persistent iberiotoxin (200 nM; BK selective) and apamin (200 nM; SK inwards currents that may occur during prolonged contrac- selective) all increased afferent discharge (to 195%, 4; 224%, 4 tions. Whether a unit stops or starts to fire is suggested to & 160%, 6 of control, respectively; all P < 0.02). Conversely, depend on a balance between the strength of the net excita- NS1619 (BK channel activator) inhibited firing (1 μM, 179.75 tory motor command, persistent inward currents, and inacti- ± 14.35 imp/s vs 33.25 ± 18.44 imp/s, 6). This was not due to vation of voltage gated channels. Rotations among low thresh- voltage-gated potassium channel or action potential conduc- old motoneurones would ensure sustained contractions in tion block, since NS1619 did not affect Nervus saphenus com- small as well as larger muscles. pound action potential amplitude (100 μM, 4). Supported by National Science and Engineering Research Coun- The data suggest Ca2+ entry through P/Q type channels acti- cil of Canada vates KCa channels to regulate firing frequency in spindle 1a afferents. Where applicable, the authors confirm that the experiments Bewick GS et al., (2005). J Physiol 562, 381-394. described here conform with The Physiological Society ethical Edgerton JR & Reinhart PH (2003). J Physiol 548, 53-69. requirements.

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Where applicable, the authors confirm that the experiments C47 described here conform with The Physiological Society ethical requirements. Muscle movement while maintaining posture at the human wrist J. Chew1, I. Loram2 and R. Fitzpatrick1 C48 1Prince of Wales Medical Research Institute, Sydney, NSW, Australia and 2Manchester Metropolitan University, Manchester, UK Brief sacral nerve root stimulation increases somatosensory cortical activation in the rat The ability to control the static posture of a joint depends on the elastic stiffness of the load being held (Chew et al., 2008). K.M. Griffin1, C. O’Herlihy2, R. O’Connell3 and J. Jones1 Here we investigate at the wrist the muscle and tendon con- 1Physiology, UCD School of Medicine & Medical Science, University tributions to this postural load-dependence. College Dublin, Dublin, Ireland, 2Surgery, Academic Surgical Unit, The Human Research Ethics Committee of the University of St Vincent’s University Hospital, ElmPark,, Dublin 4, Ireland and New South Wales approved the studies. Subjects (N=6) used 3Obstetrics and Gynaecology, National Maternity Hospital, Holles the wrist flexor muscles to hold as still as possible three loads Street,, Dublin 2, UK of different elastic stiffness (Chew et al., 2008). One had positive-stiffness (i.e. spring like; 0.1 Nm.deg-1) so that more force Faecal incontinence in women is associated with pudendal nerve was required as the wrist flexed, one had negative stiffness damage sustained during traumatic childbirth (Snooks et al. (inverted-pendulum like; -0.1 Nm.deg-1) and the third had zero 1984). Sacral neuromodulation therapy has been used as a stiffness (isotonic). Each load was held with the wrist in the treatment for this condition, but the exact mechanism of action same posture and at this position each load required the same remains unclear. It is possible that affected individuals develop wrist torque (0.70 Nm) for static balance. Flexor carpi radialis a sensory deprivation syndrome and that sacral neuromodu- were observed by ultrasound (Acuson 128XP, L7384 linear lation increases the sensory input to the cortex. Previous stud- array), which provided a 38 mm parasagittal view of the full ies carried out by Peirce et al. (2009) showed reduced depth of the muscle with a temporal resolution of 25 Hz. Spa- somatosensory evoked potentials (SEPs) in an animal model of tial cross correlation of image luminance was used to measure pudendal nerve compression. The aim of this experiment was longitudinal tissue velocity of the proximal and distal tendon to study evoked potential following anal canal stimulation plates. Using measured changes in wrist angle and the moment before and after acute sacral neuromodulation. arm of the wrist, changes in length of the tendon and contrac- Four female virgin Wistar rats (body mass: 200-250g) were tile portions of the muscle were determined. anaesthetised with urethane (1.5g/kg i.p.). The femoral artery For the positive- and zero-stiffness loads, the ranges of wrist and vein were cannulated and the animals spontaneously movement over 30 s were approximately one degree whereas breathed room air supplemented with humidified oxygen. for the negative-stiffness load it was about five degrees. The Blood gases and respiration rate were monitored thoughout relationship between contractile length and wrist angle at fre- the experiment. A unilateral craniotomy was performed over quencies below 2 Hz varied with load stiffness. For the positive- the right hemisphere. An extradural recording array (FlexMEA, and zero-stiffness loads, the muscle was shorter at more flexed multi-channel systems: electrode diameter 30μm, electrode wrist angles, with a higher gain for the positive-stiffness rela- spacing 300μm) was placed 2mm lateral and 2mm caudal to tionship. With the negative-stiffness load, the contractile length bregma. A cathode placed in the anal canal was used to pro- was longer at more flexed wrist positions. At ~ 2 Hz, a resonant vide triggered stimulation at 1Hz (amplitude: 10volts and pulse peak in torque and wrist movement is reflected in the tendon duration 0.1ms). A concentric needle electrode was placed in length but not in the contractile portion. These load-stiffness the left S1 sacral foramen (amplitude: 6 volts, frequency 15Hz effects are not apparent at higher frequencies (> 4 Hz) where and duration 1ms). Sixteen cortical evoked potentials were sig- the inertial properties of the load dominate and torque and nal averaged (500 sweeps) before and after applying 500sec of angle are antiphase. A 10 Hz “tremor” oscillation in the EMG, sacral neuromodulation. Animals were killed on conclusion of contractile length and tendon length was minimal or absent the experiment with an overdose of sodium pentobarbitone in wrist angle because the tendon lengthened and shortened (i.v.). antiphase with the contractile portion. The latency to cortical evoked potential was 9.4 ± 0.4 ms and We conclude that there is no simple relationship between mus- the first positive deflection lasted 131±15 ms. A significant cle length and joint angle. Rather, it is a dynamic function of increase was observed in the amplitude of SEP before and after frequency strongly dependent on the elastic stiffness of the sacral stimulation in all four animals (20.4 ± 7 to 31.8 ± 6 V load. At the point of resonance, the load and joint oscillate on respectively; P=0.048, one tailed paired Student’s t test). the end of a compliant tendon without significant transfer of This 50% increase in the amplitude of an evoked potential sup- the movement to the contractile portion, thus constituting an ports the hypothesis that even brief sacral neuromodulation effective proprioceptive and control blindspot. This muscle- can increase sensory input to the somatosensory cortex. It tendon behaviour is a major determinant of the limits of human remains to be determined if chronic sacral neuromodulation postural control. can reverse the blunted SEPs observed in our models of neu- Chew JZ, Gandevia SC & Fitzpatrick RC. (2008). Postural control at the ropathic faecal incontinence (Peirce et al. 2009). human wrist. J Physiol 586, 1265-1275. Peirce, C., Healy, C.F., O’Herlihy, C., O’Connell, P.R., Jones J.F. (2009). Reduced Somatosensory Cortical Activation in Experimental Models NH&MRC Australia supported research of Neuropathic Fecal Incontinence. Dis. Colon Rectum (in the press).

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Snooks SJ, Swash M, Setchell M, Henry MM. (1984) Injury to innerva- vascular conductance is substantially lower in the SHR than the tion of pelvic floor and sphincter musculature in childbirth. The Lancet. WKR (P<0.05). 546-50. Our preliminary findings are consistent with the hypothesis This work was supported by a grant from the Health Research that the brainstem of the SHR has a lower vascular conductance Board, Ireland. than the WKR. Our findings resonate with a study of human tissue at post-mortem in which a reduction in calibre of the rv Where applicable, the authors confirm that the experiments artery was strongly and inversely correlated with ante-mortem described here conform with The Physiological Society ethical blood pressure (Dickinson, 1960). requirements. Dickinson, C et al. (1960). Clin Sci 19: 513-38. Knapp, F et al. (1965). Life Sci 4: 251-9. Krucker, T. et al. (2006). Micro. Res. Tech. 69: 138-147. C49 Paton, J et al. (2009). Exp Physiol 94(1): 11-7. Comparison of corrosion casts of brainstem vasculature of British Heart Foundation funded research. normotensive and hypertensive rats Where applicable, the authors confirm that the experiments P.D. Langton 1, A.S. Phillips1, C.R. Trappes-Lomax1, P.Verkade2, described here conform with The Physiological Society ethical A.F. James1 and J.F.R. Paton1 requirements. 1Physiology and Pharmacology, University of Bristol, Bristol, UK and 2Biochemistry, University of Bristol, Bristol, UK In the spontaneously hypertensive rat (SHR) hypertension is C50 associated with raised sympathetic outflow. Why sympathetic activity is raised is unclear but if blood flow to the brain is Over-expressed junctional adhesion molecule-1 is associated impeded, blood pressure increases (Cushing effect) and there with neurogenic hypertension in multiple rodent models is a parallel increase in sympathetic outflow (Knapp, 1965). Our hypothesis is that the brainstem is highly sensitive to a corre- H. Xu1, E.B. Oliveira-Sales2, D. Graham3, A.F. Dominiczak3, late of perfusion such that flow reduction drives a proportion- J.F.R. Paton1 and S. Kasparov1 ate increase in sympathetic outflow and arterial pressure (Paton 1Department of Physiology and Pharmacology, Bristol Heart et al., 2009). We postulate that cerebrovascular conductance Institute,SchoolofMedicalSciences,UniversityofBristol,Bristol, is reduced in the SHR compared to the normotensive Wistar UK, 2Cardiovascular Division, Department of Physiology, Federal Kyoto rat (WKR). University of São Paulo, Santos, Brazil and 3BHF Glasgow Having measured BP by tail cuff we prepared vascular corro- CardiovascularResearchCentre,UniversityofGlasgow,Glasgow,UK sion casts of age-matched WKR and SHR rats according to the method of Krucker et al. (2006). Animals were killed according Junctional adhesion molecule-1(JAM-1, or the F11 receptor) is to the UK Animals (Scientific Procedures) Act 1986. On cessa- a transmembrane protein located at the inter-cellular junctions tion of the circulation, a cannula was placed in the left ventri- of endothelial and epithelial cells (Martìn-Padura et al. 1998). cle and vascular constriction inhibited by flushing with a zero Real-time RT-PCR revealed that JAM-1 mRNA was up-regulated calcium, high magnesium phosphate buffer containing 100 μM in the nucleus tractus solitarii (NTS) of the spontaneously hyper- hydralazine. After perfusion fixation (formaldehyde, 4%), the tensive rat (SHR) and young pre-hypertensive SHR compared vasculature was filled with a urethane-based resin (Pu4ii, with age-matched normotensive Wistar-Kyoto (WKY) rats VasQtec). Perfusion pressure during flushing, fixation and resin (Waki et al. 2007). Here, we sought to compare JAM-1 levels in infiltration was the systolic pressure previously recorded from SHR with other rat models of hypertension. each animal. Resultant casts were liberated by maceration using Male WKY, SHR, stroke-prone SHR (SP-SHR) and Wistar rats sodium hydroxide (10%) and hydrochloric acid (10 mM) at 55 made hypertensive by renovascular occlusion (Goldblatt: 2 kid- oC. Casts were freeze-dried, sputter coated with gold and exam- ney 1 clip model) (n=6 for all groups) were overdosed with pen- ined in a scanning electron microscope (SEM; FEI, Quanta 400). tobarbital and perfused with Bouin’s solution. The brains were Our preliminary results relate to n=1 for each strain. Measure- harvested and immunostained with goat anti-JAM-1 (1:200) ments of arterial diameter were made using software tools antibody overnight, followed by incubation with biotinylated intrinsic to the SEM. Mann-Whitney U tests were used to make donkey anti-goat (1:500) for 1.5h and avidin-FITC (1:500) for statistical comparisons of median values. 1h. JAM-1 immunofluorescence in the NTS was analysed using Median values for basilar and vertebral artery diameter were Image-Pro Plus 6.0 software on images obtained with a Leica smaller in the SHR compared to the WKR. Similarly, the diam- confocal microscope. Fluorescence intensity of JAM-1 in WKY eters of branches arising from these arteries tended to be was 148±23 (units/section, mean±SEM), but up regulated to smaller in the SHR compared to the WKR. It should be noted 433±72 (units/section, P<0.05) in SHR and higher in SP-SHR that in the SHR, the diameter of the right vertebral (rv) artery (720±89 units/section, P<0.01). JAM-1 was also up regulated (174 μm) was very much smaller than that of the left vertebral in pre-hypertensive 3 week old SHR (144±22 vs 360±47, (lv) artery (272 μm), whereas, in the WKR, the diameters of the units/section, P<0.05). In Goldblatt rats (3 weeks post-surgery) two arteries were similar (271 vs 291 μm). The fourth power of arterial systolic blood pressure measured with radio-teleme- the vessel radius relative to the principle feeding artery (basi- try (anaesthetic for Goldblatt clamping and transmitter implan- lar, rv or lv) was calculated as an index of conductance and com- tation was a mixture of ketamine hydrochloride (50%), medeto- parisons made within and between animals. This revealed that midine hydrochloride (30%) and 0.9% saline (20%) (0.2ml/300g,

30P Oral Communications i.p.) followed after surgery with 10% atipamezole hydrochlo- ANGII (800ng/kg/min, s.c.). 2K1C model: Rats (male, 150-180g) ride (1.5ml/kg, i.m.)) increased from 124±5 to 159±2 mmHg were anaesthetised as above and radio-transmitters installed (mean±SEM, P<0.05, ANOVA) and was associated with plus the left renal artery was partially obstructed with a silver enhanced JAM-1 immunofluorescence in NTS (381±49 vs. clip of 0.2 mm width (n=6) or sham surgery (n=6). Recordings 138±17 units/section in control, P<0.05). When arterial pres- of AP and HR were made for 6 weeks. Values are means ± S.E.M., sure reached a plateau at 6 weeks (211±2 mmHg), JAM-1 compared by ANOVA. immunofluorescence in NTS was not increased further (P<0.05). Both models exhibited similar alterations in autonomic indices We next assessed whether up regulation of JAM-1 was specific especially when the hypertension plateaued. For both the 2K1C to the brain. WKY, SHR, SP-SHR, pre-hypertensive SHR and and the ANGII group AP rose to similar levels (e.g. 185±15 Goldblatt rats (male, n=4 each group) were anaesthetised with mmHg vs. 103±10 for sham rats and 150±3 vs. 91±3 mmHg halothane (5%) and decapitated. Brainstem blood vessels were pre-infusion, p<0.05 respectively). Additionally, HR was ele- isolated by ultracentrifugation (Mrsulja et al. 1976) along with vated (ANGII: 409±10 vs. 368±5 bpm pre-infusion, p<0.05; tissues from lung, liver, kidney, spleen and heart. In all animal 2K1C: 468±11 vs. 382±11 sham treated, p<0.05), very low fre- groups, JAM-1 protein levels were higher in all organs studied quency of systolic blood pressure increased (ANGII: 6.8±0.5 vs. as determined by western blotting (P<0.05, ANOVA). 5.4±0.2 mmHg2 pre-infusion, p<0.05; 2K1C: 7.0±0.3 vs. 3.9±0.3 These data indicate that up regulation of JAM-1 is a generalised mmHg2 sham treated, p<0.05), high frequency of the pulse phenomenon associated with early and pre-hypertensive stages interval was reduced (ANGII: 10.6±1.5 vs. 15.0±1.1 ms2 pre- of both genetic and renovascular hypertension and further sup- infusion, p<0.05; 2K1C: 9.6±0.5 vs. 16.7±0.9 ms2 sham, p<0.05) ports its possible causative role in this pathological condition and sBRG was reduced (ANGII: -1.1±0.3 vs. -2.0±0.1 and, hence, as a novel prognostic indicator. bpm/mmHg pre-infusion, p<0.05; 2K1C: -0.83±0.05 vs. Martìn-Padura I et al. (1998). J Cell Biol 142, 117-127. –2.5±0.17 bpm/mmHg, sham, p<0.05). Waki H et al. (2007). Hypertension 49, 1321-1327. Thus, these established models of experimental hypertension are associated with failure of the parasympathetic component Mrsulja BB et al. (1976). Brain Res 110, 361-365. of the baroreflex and increased sympathetic vasomotor activ- British Heart Foundation funded research. ity. These data suggest that chronically increased peripheral levels of ANGII, resulting from exogenous infusion or renal Where applicable, the authors confirm that the experiments hypoperfusion, may cause hypertension via mechanisms involv- described here conform with The Physiological Society ethical ing central modulation of both cardiovascular autonomic activ- requirements. ity and baroreflex function. Waki H et al (2006). Exp Physiol 91, 201-213

C51 Research funded by the British Heart Foundation Where applicable, the authors confirm that the experiments Similar cardiovascular autonomic changes during the described here conform with The Physiological Society ethical development of renovascular and angiotensin II (ANGII) requirements. induced hypertension in rats M.A. Toward1, E.B. Oliveira-Sales1,2, R.R. Campos2, S. Kasparov1 and J.F.R. Paton1 C52 1Physiology and Pharmacology, Bristol Heart Institute, University of Bristol, Bristol, UK and 2Fisiologia Cardiovascular UNIFESP, Escola Increased brainstem vascular resistance precedes onset of Paulista de Medicina Rua Botucatu, Sao Paulo, Brazil hypertension in the spontaneously hypertensive rat Hypertension remains a serious clinical problem. Numerous M. Cates, A.P. Abdala and J.F.R. Paton rodent models of hypertension have been developed to allow Department of physiology and pharmacology, University of Bristol, studies into possible causative mechanisms. Two such models Bristol, UK are the chronic ANGII infusion and renovascular hypoperfusion (two kidney one clip; 2K1C). Although both models depend on Usingthespontaneouslyhypertensiverat(SHR)asananimalmodel activation of ANGII type 1 receptors, it is not known when and of essential hypertension we are working on a hypothesis that the if the autonomic nervous system is engaged in the develop- condition is due, in part, to increased sympathetic activity that ment and/or maintenance of the resultant hypertension. occursinresponsetobrainstemhypoperfusion.Thereisgoodevi- The development of hypertension in both models was docu- dence of increased sympathetic tone in adult humans with essen- mentedusing24hourradio-telemetryrecordingofarterialpres- tialhypertensionandintheSHRevenataprehypertensive(<5week) sure (AP) and heart rate (HR) in conscious freely moving rats. age(see1).Brainstemhypoperfusioncausesanacuteriseinblood Hey Presto software (Waki et al, 2006) was used to calculate pressure in humans and other species (1,2,3) and in humans, spontaneous baroreflex gain (sBRG) and indices of autonomic chronic hypertension correlates more closely with narrowing of function from the AP and HR variabilities by spectral analysis. the vertebral arteries than any other vessel (4). There is also evi- ANGII model: Rats (male, 250-350g, n=12) were anaesthetised denceofanaerobicmetabolismintheSHRbrainstemandincreased with a mixture of ketamine (60mg kg-1) and medetomidine vertebralandbasilararterialwallthicknessattheprehypertensive (250μg kg-1, both i.m.) and radio-transmitters installed. Con- age (5,1). However, the functional significance of the latter histo- tinuous recordings of AP and HR were made for 3 days prior logicalchangesinlargevesselstoresistanceandflowremainunclear to, and 10 days during, osmotic minipump driven infusion of and were functionally investigated in the present study.

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Using a novel in vitro perfusion preparation, vascular resistance fura-PE3. All drugs were added before H2S. IPA slightly pre- within the basilar arterial tree was measured in male 4-5 week constricted with U46619 exhibited a triphasic constriction- SHR (n=7) and Wistar (n=9) rats. Under deep halothane (5%) relaxation-constriction response to H2S, which persisted in the anaesthesia, rats were decapitated at the 1st cervical level and presence of the α-adrenoceptor blocker phentolamine (10 μM, decorticated. The brainstem with cerebellum was removed in n=4) or the purinergic receptor blocker suramin (300 μM, n=3). cold (4∞C) artificial cerebrospinal fluid (ACSF) and pinned down The initial transient constriction (phase 1) and the subsequent in a perspex chamber, ventral side up. The basilar artery was sustained constriction (phase 2) were retained in endothelium- cannulated at its caudal end with a double-lumen theta glass denuded IPA (n=4); however the intervening relaxation was pipette and tied in place with a fine silk suture. The preparation suppressed by removal of the endothelium or treatment with was both superperfused with warm (32∞C) ACSF bubbled with L-NAME (300 μM; n=4&7). Phase 1 but not phase 2 was 95% O2, 5%CO2 and perfused through one lumen of the can- decreased by thapsigargin (1 μM; n=5; P<0.05) or ryanodine nula with gas bubbled ACSF (32∞C) plus 1.5% polyethylene gly- (100 μM, n=4; P<0.01). Phase 1 was unaffected by the Ca2+ col at an initial rate of 0.22ml/min. The basilar artery was then channel antagonists nifedipine (3 μM, n=2) or diltiazem (10 μM, 2+ μ clamped at its rostral end and warmed for 10 minutes. Flow n=3). In IPA bathed in Ca -free PSS (100 M EGTA), H2S elicited was increased incrementally every 10 minutes across 6 flow a rise in [Ca2+]i that was suppressed by cyclopiazonic acid (30 rates and pressure recorded via a transducer attached to the μM), n=2). In tension experiments, the Rho kinase inhibitor Y- second, fluid-filled lumen of the theta glass cannula. Average 27632 (1 μM, n=5; P<0.05) but not the broad spectrum protein pressures were significantly higher (p= <0.05, T-test) across kinase C inhibitor Gö6983 (3 μM, n=3) inhibited phase 2, while all flow rates in the SHRs and at the highest flow rate (1.2 in permeablised arteries, H2S caused a contraction additive to ml/min) were 173 ±8 vs 140 ±6 (average ± SEM) mmHg; that induced by pCa 7.1 (100 μM NaHS, n=4; P<0.05; 300 μM p=0.002. NaHS, n=6; P<0.05). Notably, the increase in tension induced These data indicate increased vascular resistance within the by H2S in IPA under slight preconstriction was not associated brainstem of the pre-hypertensive SHR, which is consistent with with a concomitant increase in [Ca2+]i. We conclude that the the reported narrowing of the basilar artery relative to age constriction-relaxation-constriction response elicited by H2S matched normotensive rats (4). Our results support the hypoth- in rat IPA is due to 1) an initial endothelium-independent con- esis of enhanced cerebral vascular resistance predisposing to traction associated with Ca2+ release from ryanodine sensitive hypoperfusion-triggered increases in sympathetic vasomotor stores, 2) an endothelium and NO-dependent relaxation, and tone. Given the relative restrictive nature of the brainstem vas- 3) finally a Rho kinase-dependent but endothelium-independ- culature to flow in the SHR, we are currently comparing different contraction that is not associated with an increase in [Ca2+]i. ences in activation of the sympathetic nervous system follow- In view of some similarities to the response to hypoxia, we spec- ing sequential occlusion of the vertebral and carotid arteries ulate that H2S elicits its response in IPA in part via inhibition of in SHR and normotensive rats. cytochrome oxidase, this mimicking hypoxia. Paton, J et al. (2009). Exp Physiol 94(1): 11-17. Olson KR, Dombkowski RA, Russell MJ, Doellman MM, Head SK, Whit- Knapp, F et al. (1965). Life Sci 4: 251-9. field NL & Madden JA. (2006). H(2)S as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation. J. Exp. Kalmar AF, et al (2005). Br J Anaesth 94(6):791-9. Biol. 209, 4011-4023. Dickinson, C et al. (1960). Clin Sci 19: 513-38. Zhao W, Zhang J, Lu Y & Wang R. (2001). The vasorelaxant effect of Toward MA et al.(2007). FASEB J 21, A1411-b. H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J. 20, 6008-6016. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Supported by the British Heart Foundation (FS/05/117/19967) requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C53

MechanismsofH2S-inducedcontractionofratpulmonaryartery C54 M. Connolly, F. Akter, J.P. Ward and P.I. Aaronson Mechanisms underlying cyclic GMP-mediated Ca2+ AsthmaAllergyandLungBiology,King’sCollegeLondon,London,UK desensitisation in rat intrapulmonary arteries

Hydrogen sulphide (H2S) has recently joined the ranks of NO S. Drndarski, A. De Silva, G.A. Knock, P.I. Aaronson and J.P.Ward and CO as the third gasotransmitter. Although H2S dilates systemic arteries (Zhao et al., 2001), pulmonary arteries respond AsthmaAllergyandLungBiology,King’sCollegeLondon,London,UK to H2S with a complex constriction-relaxation-constriction Therapies for pulmonary hypertension include the phosphodi- (Olson et al., 2006), the mechanism of which we investigated esterase (PDE) 5 inhibitor sildenafil and prostacyclin, which raise in this study using the H2S donor sodium hydrogen sulphide cGMP and cAMP respectively. Pulmonary hypertension is associ- μ ≈ μ 2+ (NaHS: 300 M, [H2S] 100 M). Rat intrapulmonary arteries atedwithaRhokinase-mediatedincreaseinCa sensitivityin pul- (IPA) were mounted on a myograph containing physiological monary arteries, and it has been suggested that cGMP and cAMP saline solution (PSS) and gassed with air/5% CO2 (pH 7.4, 37C), may suppress this pathway. We studied this hypothesis in intra- and in some cases were permeablised with α-toxin at 25C, and pulmonaryarteries(IPA)oftherat,mountedonamyograph;exper- clamped to pCa 7.1. Intracellular calcium was measured using iments were performed on intact or α-toxin permeabilised IPA.

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In intact IPA constricted with 10 μM PGF2α, 8-Br-cGMP (cGMP effect of hypoxia on GSH and GSSG in rat small intrapulmonary ± μ analogue) elicited relaxation with an EC50 of 20 13 M and max- arteries (IPA) and aorta, using the approach of Griffith (1980), imum relaxation of 102±3% (n=6). In permeabilised arteries at and investigated the effect of agents which should cause cell pCa 6.7 the potency of 8-Br-cGMP was much greater, with an oxidation on HPV and GSH/GSSG ratio. The redox potential ± ± EC50 of 18 4nM and maximum relaxation of 64 5% (n=5). 8- (expressed in mV) of the GSH-GSSG couple was calculated using Br-cGMP (100nM) also caused a rightward shift of the Ca2+ dose- the Nernst equation. ± response curve, with an increase in EC50 from 165 5 nM to Under normoxic conditions, the GSH-GSSG redox potential was 316 ± 14 nM (n=44; p<0.05), indicating calcium desensitisa- -176±4mV (n=18) in IPA and -150mV (n=16) in aorta. After 45 tion. Complete inhibition of Rho kinase with Y-27632 (10μM) min of hypoxia imposed by gassing the solution with 0, 1 and ± ± did not prevent this shift (EC50: Y-27632: 205 26 nM, n=8; Y- 2% O2, the redox potential in IPA was decreased to -159 6mV 27632+8-bromo-cGMP: 306 ± 26 nM (n=6). This suggests that (n=14, p<0.05), -162±4mV (n=20, p<0.05) and -174±4mV cGMP does not act via Rho kinase. Interestingly, 8-Br-cGMP- (n=10, ns), respectively. Conversely, the redox potential of aorta induced relaxation of permeabilised IPA was suppressed not after 45 min of hypoxia was -142±11 mV (n=12), -155±3mV μ ± only by the cGMP antagonist Rp-8Br-cGMP (25 M) (control: 77 (n=12) and -171 4mV (n=10, p<0.01), in 0, 1 and 2% O2, respec- ± 3%; Rp-8Br-cGMP: 44 ± 3%, n=4-8, p<0.01), but also by the tively. Thus, hypoxia tended to oxidize IPA. cAMP antagonist Rp-8Br-cAMP (25μM) (43 ± 5%; n=4; p<0.01); Glutathione reductase (GR) converts GSSG to GSH, whilst oxidiz- there was no increase when these were applied together (43 ± ingNADPHtoNADP+;NADPHisreformedfromNADP+byglucose- 9%, n=3), suggesting that cGMP and cAMP may be acting 6-phosphatedehydrogenase.TheconversionofGSHbacktoGSSG sequentially. ismediatedbyglutathioneperoxidase(GPx);peroxideisreduced cGMP inhibits cAMP-selective PDE 3, and the PDE 3 inhibitor mil- towaterinacoupledreaction.Carmustine(100μM),aGRblocker, rinone proved to be a powerful vasorelaxant in intact IPA (EC50 21 completelyabolishedthesustainedphaseofHPV(n=8),andshifted ± 7 μM, maximum 100 ± 13%, n=4). To determine whether cGMP thecellredoxpotentialduringnormoxiafrom-172±3mV(n=8)to elevatesintracellular[cAMP],weusedanELISAandculturedsmooth -154±6mV (n=6). Conversely, mercaptosuccinate (MS; 100μM), muscle cells derived from rat IPA. 8-Br-GMP (100μM) increased ablockerofGPx,increasedthesustainedphaseofHPVby47±13% cAMP from 25 ± 3 pmol/mg protein to 48 ± 6 pmol/mg protein (n=8,p<0.05).NeithercarmustinenorMSaffectedtheintitialtran- (n=16, p<0.001), whereas 10μM milrinone increased it to 43 ± 6 sientphaseofHPV.Ebselen(30μM),aGPxmimic,attenuatedboth pmol/mg protein (n=10, p<0.01). As 10μM milrinone, which acts transient (44±9%, n=3) and sustained phases (39±18%, n=3) of via cAMP, causes ~50% relaxation of intact IPA, the elevation in HPV.Dehydroepiandrosterone(100μM),whichblocksglucose-6- cAMPcausedby8-bromo-cGMPisclearlysufficienttoaccountfor phosphatedehydrogenase,shiftedthenormoxiccellredoxpoten- asignificantcomponentofvasorelaxation.ThissuggeststhatcGMP- tial to -154±6mV (n=6, P<0.05 vs control) and blocked sustained mediated Ca2+ desensitisation may involve cAMP and PKA; con- HPVby43±13% (n=3). Diethylmaleate (1 mM), which depletes sistent with this, the protein kinase A inhibitor H-89 (10μM) abol- glutathione, shifted the cell redox potential to -105±6mV (n=3), ished the effects of 8-Br-cGMP on the Ca2+ dose-response curve, andblockedsustainedHPVby36±6%(n=6).Insummary,hypoxia although H-89 itself caused a significant shift in response (EC50: causedanoxidizingshiftintheredoxstateinintactIPA.Moreover, H-89: 533 ±81 nM; H-89 + 8-Br-cGMP: 550 ±57 nM (n=4). In con- several substances which reduce total glutathione levels in tissue clusion,weshowthatcGMP-inducedCa2+ desensitisationofIPAis and caused cell oxidation consistently inhibited sustained HPV. independent of Rho kinase, but that a significant component of Griffith OW (1980) Determination of glutathione and glutathione disul- relaxationmayberelatedtoacAMPandPKA-dependentpathway. fide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207-12. British Heart Foundation and Wellcome Trust Postdoctoral Grant MEC-2007-1180 and the Wellcome Trust Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

C55 C56 Relationship Between Glutathione Redox State And The VasoconstrictorResponseToHypoxiaInRatPulmonaryArteries Potential selective role for CXCR7/CXCL12 signalling in the J. Prieto-Lloret, J.P. Ward and P.I. Aaronson lung in response to hypoxic stress 1 1 2 3 4 Asthma,AllergyandLungBiology,King’sCollegeLondon,London,UK C. Costello , K. Howell , S. Doherty , F. Martin , J. Belperio , M. Keane1, S. Gaine2 and P. McLoughlin1 Hypoxia elicits constriction of pulmonary arteries (hypoxic pul- 1 monary vasoconstriction, HPV) but dilation of systemic arter- School of Medicine and Medical Science, Conway Institute, UCD, 2 ies. It is proposed that hypoxia-induced changes in the pul- Dublin, Ireland, Department of Respiratory Medicine, Mater 3 monary artery smooth muscle cell redox state and/or reactive Misericordiae University Hospital, UCD, Dublin, Ireland, School of oxygen species trigger HPV, although there is controversy as BiomolecularandBiomedicalScience,ConwayInstitute,UCD,Dublin, 4 to whether this involves a net oxidation or reduction. Glu- Irelandand DivisionofPulmonaryandCriticalCareMedicine,David tathione is the major thiol-disulfide redox buffer of the cell, so GeffenSchoolofMedicine,UCLA,LosAngeles,CA,USA measurement of GSH and GSSG levels should provide an indi- Pulmonary hypoxia is a common complication of chronic lung cation of overall cytoplasmic redox state. We measured the diseases leading to the development of pulmonary hyperten-

33P Oral Communications sion. The underlying sustained increase in vascular resistance month after AOE, there is an increase in the cellular excitabil- in hypoxia is a response unique to the lung. Thus, we hypoth- ity within the dorsal cochlear nucleus (DCN) and this is con- esized that there must be genes whose expression is altered sidered as a possible neural correlate of tinnitus. The origin of selectively in the lung in response to alveolar hypoxia. Previous this phenomenon is still unknown but it is suggested that it is experiments using a subtractive array-based strategy to com- triggered by plastic adjustments within the DCN arising at the pare gene responses of primary human microvascular endothe- early stages of deafness (Kaltenbach JA. 2007). The purpose of lial cells from the lung and systemic circulations under normoxic the present study was to determine how DCN excitability was (21% O2) and hypoxic conditions (1% O2) showed that a recently affected 3-4 days after AOE. Wistar rats (14-16 days old) were identified CXCL12 receptor, namely CXCR7, was selectively anesthetized (0.15mg/kg fentanyl; 5.1 mg/kg fluanisone; 2.5 upregulated in response to hypoxia in the lung. This finding was mg/kg midazolam I.P)and exposed to a 110 dB SPL 15-kHz noise of interest given that in many chronic lung diseases, vascular stimulus for 4 hours (AOE). In vivo auditory brainstem response loss and damage is a key step associated with disease progres- recordings (anaesthesia as above) and in vitro whole-cell cur- sion and CXCL12 is a potent secreted pro-angiogenic rent-clamp recordings from brainstem slices containing the chemokine. DCN were made 3-4 days post AOE. Auditory brainstem To investigate this finding further, adult male C57Bl6 mice were response recordings showed that the hearing thresholds were housed in normoxic (inspired oxygen 21%; n=8) or hypoxic condi- significantly elevated (between 20-30 dB SPL) for frequencies tions (inspired oxygen 10% for 48hrs; n=8). For TaqMan studies, above 15 kHz. Control fusiform cells (the main output of the mice were killed by cervical dislocation and organs snap-frozen in DCN) fired with a regular firing pattern as assessed by the coef- liquid nitrogen. For immunohistochemical studies, mice were ficient of variation of the interspike interval distribution of 0.19 housed as above (n=10 per group), euthanised (sodium pento- ± 0.11 (n=5). Three to four days after AOE, 30% of fusiform cells barbitone,60mg/kgi.p.)andtheheartandlungsremoveden-bloc. exhibited bursting irregular discharge patterns (coefficient of Lungs were fully inflated via the trachea with 4% paraformalde- variation of the interspike interval distribution of 1.8 ± 0.6, n= hyde overnight and then embedded in paraffin. CXCL12 levels in 5; unpaired T test p<0.05). Control granule cells (interneurones plasma from patients with chronic hypertensive lung disease or projecting onto fusiform cells) fired with a high gain (slope of normal controls were determined by ELISA and immunohisto- 2.5 ± 0.4 Hz/pA, n=10) that was decreased 3-4 days after AOE chemistrywascarriedoutonexplantedhumanhypertensivelungs. (to 1.4 ± 0.2 Hz/pA, n=9; unpaired T test p<0.05). This was In vitro experiments examined the ability of CXCL12 to induce accompanied by a decrease of their membrane resistance (from wound healing in human microvascular endothelial cells. 1.9 ± 0.3 GΩ, n=10 to 1.1 ± 0.2 GΩ, n=10; unpaired T test p<0.05 TaqMan analysis of a panel of murine tissues showed a >2-fold ) and more hyperpolarized resting potentials (from -43 ± 4 mV, increase in CXCR7 mRNA only in lung tissue. A second CXCL12 n=9 to -57 ± 4 mV, n=10; unpaired T test p< 0.05 ). Data receptor, CXCR4, was down-regulated in the lung in the same obtained in fusiform cells and in granule cells recorded in con- animalmodel.ImmunohistochemistrydemonstratedthatCXCR7 trol condition and after AOE were fitted with a leaky integrate- protein was significantly increased and CXCR4 was significantly and-fire model. In conclusion we suggest that the DCN excitabil- decreasedinthehypoxiclunginvivo.Invitroexperimentsshowed ity changes occurring 3-4 days post AOE trigger the increased that CXCL12 induced wound healing in microvascular endothe- DCN excitability observed at a later stage and this represents lial cells. Finally, CXCL12 was significantly elevated in the plasma the initial stages of tinnitus. of patients with chronic hypertensive lung disease and CXCR7 The dorsal cochlear nucleus as a contributor to tinnitus: mechanisms wasalsohighlyexpressedinexplantedhumanhypertensivelungs. underlying the induction of hyperactivity. Kaltenbach JA. (2007). Prog These novel results suggest that signalling via CXCR7/CXCL12 Brain Res. 166: 89-106. Review. pathway is selectively up-regulated in the lung in response to hypoxia and may play a role in pulmonary vascular disease. This work was supported by GlaxoSmithKline, Medisearch and Future studies will now decipher the exact role of CXCR4 and the Wellcome Trust. CXCR7 receptors in mediating angiogenic responses. Where applicable, the authors confirm that the experiments Funding: HRB, HEA, SFI, NIH HL080206. described here conform with The Physiological Society ethical requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C58

C57 Synaptic release evoked by depolarization in mouse cochlear inner hair cells imaged in situ in the isolated temporal bone Acoustic over-exposure changes the firing pattern of dorsal cochlear nucleus neurons J. Ashmore1,2, J. Boutet de Monvel2, C. Petit2 and S. Safieddine2 N. Pilati1, M. Mulheran2, R. Naud3 and M. Hamann1 1NPP,UCL,London,UKand 2Neuroscience,InstitutPasteur,Paris,France 1 CellPhysiologyandPharmacology,UniversityofLeicester,Leicester, The ribbon synapse of cochlear inner hair cells (IHCs) is the first 2 UK, Medical&SocialCareEducation,UniversityofLeicester,Leicester, relay point of the auditory system. Each contact made by a 3 UK and Ecole Polytechnique Fédérale, Lausanne, Switzerland single IHC onto the afferent nerve can operate with a high Acoustic over-exposure (AOE) triggers deafness in animals and degree of temporal precision. To understand the underlying in humans and provokes auditory nerve degeneration. One mechanisms, we have developed techniques to image vesicle

34P Oral Communications release at the IHC ribbon using the styryl dye FM1-43 in a man- of this work was to characterise the cholinergic effects on mor- ner similar to that reported previously (Griesinger et al., 2005), phologically identified giant cells of the CN, using electro- but now adapted to the mouse in order to permit access to a physiology, Q-PCR and immunohistochemistry. In vitro exper- wide range of transgenic models with altered synaptic func- iments were conducted on thin slice preparations from the tion. By voltage-clamping the IHC we show that it is also pos- brainstem of 10-14-day-old Wistar rats. The protocol was sible to control the calcium currents triggering vesicle release. authorised by the Committee of Animal Research of the Uni- Mouse cochleas were imaged in situ in the isolated temporal versity of Debrecen, and it was in accordance with the appro- bone after their removal in accordance with current national priate international and Hungarian laws. Application of the guidelines. The bone was attached to the base of a chamber cholinergic agonist carbachol increased the spontaneous activ- containing artificial perilymph (in mM): NaCl,140; KCl,4; ity of the giant cells (from 2.4 ± 0.5 Hz to 7.3 ± 1.3 Hz; avg ± CaCl2,2; MgCl2,1.5; Hepes, 10 to pH 7.3). A small opening made SEM; n = 36). The increased activity was partly the consequence in the apical turn exposed the 10-20kHz region of the organ of of the reduction of a K+ conductance, and this effect was medi- Corti. All ages of preparation can be used. FM1-43 (2 μM) was ated via M4 and M3 receptors. Cholinergic modulation affected added to the bath to be taken up rapidly by IHCs from their the synaptic transmission targeting the giant cells, too. Exci- intact apical surface and trafficked to the cell base at an effec- tatory synaptic currents evoked by the stimulation of the super- tive rate of 0.08 μm-s-1. After 300 s regions corresponding to ficial and deep regions of the CN were sensitive to cholinergic ribbon sites were identified using a 2 photon (2P, 840nm) laser modulation: the amplitude of the first postsynaptic current was scanning microscope. Images of FM1-43 ‘hotspots’ were reduced, and the otherwise present short-term depression was acquired as a series of frames (61 ms/frame) or as lines scans also affected (from 45 ± 10 pA to 32 ± 12 pA; the paired pulse (1.8 ms/line). ratio (PPR) changed from 0.72 ± 0.09 to 1.02 ± 0.05; n = 8). On stimulation by transepithelial extracellular current designed These changes were mediated via M3 receptors alone and via to depolarise the basolateral terminal, hotspots destained by the combination of M4, M2, and M3 receptors, when the super- up to 15% during 200ms 160 μA pulses. This result is consistent ficial and deep layers were activated, respectively. Inhibitory with vesicle exocytosis. IHCs were also stimulated during con- synaptic currents evoked from the superficial layer showed ventional whole-cell tight-seal recording from the basolateral prominent short-term depression (PPR = 0.5 ± 0.1; n = 8), but surface. The pipette contained 140 mM Cs+ to reduce large out- they were unaffected by carbachol. In contrast, inhibitory cur- ward K+ currents. Under such conditions inward calcium currents triggered by the activation of the deep parts exhibited no rents of up to 80 pA, with peak currents at -15mV, were significant short-term depression (PPR = 0.93 ± 0.1; n = 7), but recorded. Simultaneous 2P imaging showed FM1-43 localised they were highly sensitive to cholinergic activation, which was at the presumed ribbon sites. Cell depolarization by 60mV (from mediated via M3 receptors (the amplitude of the first peak ± ± Vh = – 70 mV) decreased the fluorescence by 1-2%. The fluo- decreased from 119 34 pA to 33 13 pA; n = 7). Our results rescence intensity recovered with a time constant of 220 ms. indicate that pre- and postsynaptically expressed muscarinic In a few cases there was also a small fluorescence rebound, sug- receptors have important roles in mediating the effects of gesting redistribution of dye. The imaged destaining rate is cholinergic modulation of the CN, and they strongly influence consistent with the inference (Goutman & Glowatzki, 2007) the excitability and firing characteristics of the giant cells. that only a small number of vesicles (10-20) may be released at This work was supported by grants from the Hungarian Scien- each site on short 200 ms depolarizing commands. tific Research Fund (OTKA K-72812, NK-61412). Griesinger CB, Richards CD & Ashmore JF (2005) Nature 435, 212-215. Where applicable, the authors confirm that the experiments Goutman, JD & Glowatzki,E (2007) Proc natl Acad Sci USA 104, 16341-16346. described here conform with The Physiological Society ethical requirements. Supported Eurohear LSHG-CT-20054-512063, Chaires Blaise Pascal (Île de France) and Collège de France (JBM). Where applicable, the authors confirm that the experiments C60 described here conform with The Physiological Society ethical requirements. Fast NMDAR-mediated EPSCs in the auditory brainstem of mousecontributetonitricoxidesignallinginmaturedsynapses J.R. Steinert, M. Postlethwaite, M. Jordan, T. Chernova, C59 S. Robinson and I.D. Forsythe MRC Toxicology Unit, University of Leicester, Leicester, UK Targets, receptors and significance of muscarinic neuromodulation on giant neurones of the rat dorsal Synaptic NMDARs provide voltage-dependent depolarization cochlear nucleus and a Ca2+ influx signal allowing downstream activation of Ca2+- B. Pal, A. Koszeghy, P. Pap, G. Bakondi, K. Pocsai, G. Szucs and dependent processes. In tandem with AMPARs which mediate Z. Rusznak most fast glutamatergic EPSCs, NMDARs mediate a slow EPSC in this dual component glutamatergic synapse. Synaptic Department of Physiology, University of Debrecen, MHSC, Debrecen, NMDAR currents are large during early development but decline Hungary in magnitude as synaptic pathways mature. This has lead to the Although the cholinergic modulation of the cochlear nucleus suggestion that NMDAR have no role at mature auditory brain- (CN) is important, neither its cellular consequences, nor the stem synapses. We have shown that NMDAR-mediated nitric types of receptors conveying it are precisely known. The aim oxide production occurs at the mature (P35) calyx of Held

35P Oral Communications synapse (1). Therefore, the present study characterises the than ~7. We recently identified H532 as the sensor coupling changing properties of NMDAR from P9 to P35 and shows that extracellular acidification to complete closure of the channel they do contribute to synaptic signalling at the mature synapse. (Niemeyer et al., 2009). This extracellularly-facing histidine is CBA/CaJ mice (P9–P35) were killed by decapitation in accor- located at the N-terminus of transmembrane helix Q that, dance with the UK Animals (Scientific Procedures) Act 1986. according to a homology molecular model of the channel, con- Brainstem slices containing the superior olivary complex were nects via a short loop with Y555, a conserved residue that has prepared as previously described (2) and patch clamp experi- been suggested to take part in the gating of certain ClC pro- ments were performed at 36∞C on principal neurons of the teins at an intracellular gate located at the cytoplasmic end of medialnucleusofthetrapezoidbody(MNTB).Synapticactivity the pore (Bell et al., 2006; Jayaram et al., 2008). We have now was achieved by midline stimulation using a bipolar electrode. investigated whether amino acids in the environment of H532 We confirm the decline in NMDAR-mediated EPSC magnitude are important in the acidification-induced channel closure and by P18, and show that this maturation process can be divided whether the H532 sensing function depends upon inner pore into two phases. First, there is an early decline in NMDAR cur- residue Y555. The homology model for ClC-2 suggests that rent amplitude (P9: 3.7±0.8nA to P35: 0.3±0.1nA*, n=4-5) and extracellular loop I-J is in the vicinity of H532. Several charged acceleration in kinetics, so that NMDAR-mediated EPSCs decay residues in I-J were neutralised without any effect upon the inhi- with a dominant fast time-constant (P9: 36.3±3.3ms to P35: bition of ClC-2 by acidification (mutants examined E302V, 16.7±4.1ms*, n=4-5) at mature synapses. Quantitative PCR E301Q, R311Q and D315N). Three aromatic residues of loop I- showed increased NR2A mRNA expression after P10, consis- J, however, had a marked effect upon acidification-induced inhi- tent with faster kinetics and a declining contribution from NR2B bition of ClC-2. These were F308, F312 and F316. Double subunits, since EPSCs from P18/P21 showed no block by the mutants F308A-F312A and F308A-F316A were completely NR2B-prefering antagonist ifenprodil (10μM, Ctrl: 0.48±0.06nA indifferent to acidification. As shown in Figure 1, when Y555 vs ifen: 0.44±0.1nA, n=4). Second, the NMDAR-mediated EPSC was mutated to F there was a complete disappearance of ClC- 2+ exhibits decreased voltage-dependent block by [Mg ]o, con- 2 inhibition by acidification. sistent with incorporation of NR2C subunits into synaptic chan- Our present data highlight the importance of extracellular nels. In addition, sensitivity to extracellular Zn2+ was not residues of the I-J loop F308, F312 y F316. Given the closeness detected, and the Zn2+ chelating agent, TPEN had no effect. of H532 to these residues and their chemical nature, it is tempt- We conclude that NMDARs are not eliminated from the calyx ing to speculate that some aromatic-aromatic, perhaps of the of Held synapse, but that channel expression evolves from an π-π type, interaction occurs between H532 and the mentioned immature state where large conductance channels with slow F residues. Protonation of H532 might lead to the disruption of kinetics are replaced by small conductance channels with fast this putative network of interactions and to channel closure. kinetics and reduced Mg2+ block. These results confirm the rel- Finally our result showing that ClC-2-Y555F fails to be blocked evance of NMDAR channels throughout development and sug- by extracellular acidification suggests that the effect of H532 gest that the declining NMDAR-EPSC is a shift from ‘gross’ elec- charge change might be exerted through a long-range action trical signalling to a Ca2+-dependent intracellular signalling (e.g. on a putative intracellular gating mechanism located near the activation of tightly coupled neuronal nitric oxide synthase). intracellular pore entrance. Results are reported as mean±SEM. Significance was tested using two-tailed Student’s t-test. Differences were considered statistically significant (*) at p<0.05. (1) Steinert JR, Kopp-Scheinpflug C, Baker C, Challiss RA, Mistry R, Haustein MD, Griffin SJ, Tong H, Graham BP, & Forsythe ID (2008). Nitric oxide is a volume transmitter regulating postsynaptic excitability at a glutamatergic synapse. Neuron 60, 642-656. (2) Wong AY, Graham BP, Billups B, & Forsythe ID (2003). Distinguish- ing between presynaptic and postsynaptic mechanisms of short-term depression during action potential trains. J Neurosci 23, 4868-4877. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

C61

An extracellular-facing sensing histidine closes the ClC-2 Fig. 1. Effect of extracellular pH on Y555F mutant of ClC-2.The data (triangles) areareaverageswithSEMs,n=3.Theintra-andextracellularsolutionscontained chloride channel through a long-range effect on the pore 35and140mMchloriderespectively.Thepotentialusedwas-130mV.Datafor Y.R. Yusef1,R.Briones1,L.Cid1,M.I.Niemeyer1andF.V.Sepúlveda1,2 ClC-2-H532F are from Niemeyer et al. (2009) and are shown for comparison. 1CentrodeEstudiosCientíficos(CECS),Valdivia,Chileand 2CIN,Centro Bell SP et al. (2006) Biochemistry 45, 6773-6782 de Ingeniería de la Innovación asociado al CECS, Valdivia, Chile Jayaram H et al. (2008) Proc Natl Acad Sci USA 105, 11194-11199 The activity of the ClC-2 chloride channel has a biphasic Niemeyer MI et al. (2009) J Physiol 587, 1387-1400 response to extracellular pH, with activation by moderate acidification followed by abrupt channel closure at pH values lower Supported by Fondecyt 1070722

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Where applicable, the authors confirm that the experiments O’Kelly, I. et al., (2002) Cell 111 577-599. described here conform with The Physiological Society ethical requirements. This work was supported by the BBSRC. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C62

Acid-sensitive K2P channel heterodimerisation E. Lowry1, N. Bulleid1 and I. O’Kelly2 C63 1 2 University of Manchester, Manchester, UK and University of The Cl- channel bestrophin-1 can also mediate cation Southampton, Southampton, UK transport Acid-sensitive background two-pore potassium channels (K2P) I.D. Millar1, A.E. Davidson2 and P.D. Brown1 are leak channels that are open at all membrane potentials and 1 are important contributors to the control of cellular excitabil- Faculty of Life Sciences, University of Manchester, Manchester, UK and 2Faculty of Medical and Human Sciences, University of ity. The members of acid-sensitive K2P channels; K2P3.1 and Manchester, Manchester, UK K2P9.1 share 54% homology with each other, and 51% homology with K2P15.1 (Kim et al., 2000). K2P3.1, K2P9.1 and K2P15.1 Several visual degenerative diseases (Best disease, autosomal are widely expressed, and demonstrate co-expression in an dominant vitreoretinochoroidopathy, and autosomal recessive array of tissues including the pancreas, placenta, kidney and bestrophinopathy) result in macular visual loss in adulthood lung. K2P3.1 and K2P9.1 show functional expression in het- and are associated with mutations in the VMD2 gene. The pro- erologous expression systems, they are potassium selective tein product of the VMD2 gene, bestrophin-1, is expressed in and are strongly inhibited by acidic pH. A single nucleotide poly- the basolateral membrane of the retinal pigment epithelium morphism was found at position 95 of the selectivity filter of and has been shown to form functional Cl- channels when K2P15.1, when cloned in 2001 (GYG to EYG), and to date both expressed heterologously (Hartzell et al, 2008). Here, we isoforms have been shown to be non-functional when describe experiments that examined the interactions of expressed in COS-7 cells (Kim et al., 2001). bestrophin-1 with cations. Here we investigated the interaction between both K2P15.1 HEK293 cells were transfected with cDNA for wild-type human - EYG and K2P15.1 GYG and other acid-sensitive K2P channels. We VMD2. Cl channel activity was measured using whole cell patch used two-electrode voltage-clamp to measure the functional clamp and a bath solution containing a high NaCl or NMDG-Cl - activity of the acid-sensitive K2P heterodimers. Xenopus laevis concentration. The pipette contained 38.4 mM Cl and the prin- + + oocytes expressing K2P3.1 or K2P9.1 exhibited a pH-dependent ciple intracellular cation was either Cs or NMDG . Intracellu- μ ± μ 2+ current, typical of acid-sensitive K2P channels (4.6 A( 2.0 A, lar Ca was buffered to 500nM. μ ± μ n=25) and 5.2 A( 3.8 A, n=27) respectively at a membrane Cells transfected with WT bestrophin-1 exhibited large out- μ ± voltage potential of 60mV at pH 7.6). A small current (1.2 A wardly rectifying Cl- currents in response to step potentials (Vm 1.1μA,n=9)wasseenforK 15.1 EYG when expressed in 2P = -120 to +80 mV) from a holding Vm of -50 mV. Gout was μ ± μ + oocytes, but K2P15.1 GYG (0.45 A 0.28 A, n=13) was no dif- 20.7±4.5 nS and V of -19.0±2.0 in Na bath solution. How- μ rev ferent from water injected controls at 60mV in pH 7.6 (0.35 A, ever, when NMDG+ was used to replace all Na+ and Cs+ in the ± μ 0.24 A, n=20). Co-expression of K2P15.1 EYG or K2P15.1 GYG bath and pipette solutions respectively, the outward conduc- alongside K2P3.1 or K2P9.1 did not alter the current recorded. tance was reduced to 9.5±2.7nS (n=13 and 15 respectively, Δ AK2P3.1 V411 mutant channel tagged with green fluorescent P<0.05). Untransfected cells showed no significant Cl- current. protein (eGFP) was used as a tool to look at the heterodimeri- These data suggest that the Cl- conductance of bestrophin may sation using immunofluorescence microscopy. The be modulated by the cationic environment. Δ + K2P3.1 V411 mutant is unable to bind cytosolic protein 14-3- In a further series of experiments, performed with the Na bath 3, and this loss of interaction has been shown to prevent sur- solution, we observed an increase in inward current when step face expression of the channel (O’Kelly et al., 2002). Here the potentials were evoked from a holding Vm of +80mV rather Δ mutant K2P3.1 V411 channel was expressed in COS-7 cells, and than -50mV. Thus, the inward conductance was 11.6±3.1nS was able to overcome intracellular retention by co-expressing from -50mV and 26.9±9.6nS from +80mV (n=10). The magni- with wild type K2P3.1, K2P9.1 or K2P15.1. COS-7 cells transfected tude of this inward current developed over time so that the Δ with the eGFP-tagged K2P3.1 V411 mutant alongside wild type maximum current was observed 78±9s after changing holding channels were analysed using flow cytometry to quantify the Vm. The increased inward current was also associated with a level of surface expression. In addition the interaction between ± positive shift in Vrev (+19.1 4.2mV). One explanation for this + the three acid-sensitive K2P channels was characterised at a bio- observation is that Na may be permeating the bestrophin chan- chemical level using co-immunoprecipitation. Together this nel. This conclusion is further supported by the finding that data provides the first evidence that K2P15.1 EYG is functional, there was no significant increase in inward current when record- + and that K2P15.1 interacts with K2P3.1 and K2P9.1. Given that ing from cells bathed in NMDG -containing solutions. these channels are co-expressed in a number of tissues the phys- These data suggest that cations influence the Cl- conductance iological significance of these results is far reaching. of bestrophin-1 and may, under certain conditions, permeate Kim, D et al.,(2001). Biochem Biophys Res Commun 284 923-930. this channel. Kim, Y, et al., (2000) J Biol Chemistry 275 9340-9347. Hartzell, H.C. et al. (2008) Physiol Rev 88: 639-672.

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This work was supported by grants from The Wellcome Trust Cao L, Young MT, Broomhead HE, Fountain SJ, North RA. (2007) J Neu- and BBSRC rosci 27, 12916-12923.

C64 C65 P2X receptor operates as a symmetrical trimer 2 Lysophosphatidic acid promotes keratinocyte migration L. Cao and R. North through Orai1- and lipid raft-mediated calcium mobilization Faculty of Medical and Human Sciences, University of Manchester, and NFAT activation Manchester, UK R. Jans, A.M. Brown, K. Ross and N.J. Reynolds

P2X receptors are ligand-gated ion channels gated by extra- Dermatological Sciences, Newcastle University, Newcastle upon cellular ATP. Several approaches indicate that P2X receptors Tyne, Tyne and Wear, UK have a trimeric structure; this is different from other ion channel families, such as glutamate and nicotinic receptors. Whole Lysophosphatidic acid (LPA) enhances cell motility in many cell cell and single channel patch clamp recordings were used to types and is thought to promote metastasis as well as wound study the functional contribution of each channel subunit in rat repair. LPA is well known to elicit mobilization of intracellular 2+ P2X2 receptors expressed in HEK293 cells. We used a previously calcium ([Ca ]i) but the physiological consequences down- described reporter mutation [T339K] in the second trans- stream of this phenomenon remain incompletely explored. membrane domain of the receptor (Cao et al., 2007). This muta- Here we tested the wound healing-related hypothesis that LPA- tion markedly reduced unitary conductance (7.3 ± 0.4 pS, n = activated calcium fluxes affect the motility of normal human 8, at -120mV), compared to wild type (49.3 ± 4 pS, n = 7, t-test, epidermal keratinocytes through the calcium-responsive NFAT p < 0.01) channels. Wild type P2X2 receptors typically show signalling pathway. Moreover, we characterized LPA-evoked strong inward rectification when activated by ATP (at sub-EC50 calcium mobilization, investigating if LPA-induced calcium sig- μ concentrations, 3 M); however, P2X2[T339K] channels exhibit nalling was modulated by the extracellular calcium concen- 2+ both inward and outward rectification in whole cell recording. tration [Ca ]o and analyzing the involvement of Orai1 chan- First, we co-expressed wildtype P2X2 and P2X2[T339K] sub- nels and lipid rafts. The effect of LPA on keratinocyte migration units in HEK293 cells, and we measured single channel con- was assessed over 14h using a 2-dimensional wounding motil- ductance and rectification. After co-expression of wild type and ity assay and a 3-dimensional chemotactic migration assay. As [T33K] subunits, we observed four levels of unitary conduc- expected, 10 μM LPA significantly promoted both 2- and 3- tance (I: 8.7 ± 0.1; II: 17.3 ± 1; III: 29.4 ± 1.2; IV: 52.8 ± 1.9 pS, dimensional cell migration. Pre-treatment for 1h with the NFAT n = 18). The appearance of new levels (II and III) intermediate pathway inhibitor cyclosporin A (CsA, 1 μM) significantly between those of wild type and [T339K] homomers suggests impaired LPA-induced migration, indicating that LPA induces the assembly of two species of heteromeric channels. Second, keratinocyte migration through the NFAT pathway. As moni- we expressed the series of P2X2 trimeric concatamers that con- tored by Fluo-4 imaging, LPA stimulation of keratinocytes in 60 μ 2+ ± 2+ tained the [T339K] substitution in one or more of the first, sec- M [Ca ]o evoked short-lived ( 3 min) transient [Ca ]i eleva- 2+ ond or third subunit of the concatamer (i.e. position 1, 2 or 3). tions due to store release, while in 1.2 mM [Ca ]o, LPA trig- 2+ Concatamers with three wild type or three [T339K] subunits gered a peak elevation of [Ca ]i followed by a plateau eleva- had unitary conductance similar to those observed with the tion extending over 10 min, indicating store release coupled to corresponding monomers (i.e. Thr in position 1, 2 and 3: 45.1 extracellular calcium influx. As expected, manganese quench- ± 2 pS (n = 9) and with Lys in position 1, 2 and 3: 7.9 ± 0.3 pS, n ing blocked calcium influx. Interestingly, calcium influx was = 7). Concatamers with one Lys-containing subunit had a uni- blocked by diethylstilbestrol (DES) but not by the SK&F96365 tary conductance similar to level III (in position 1: 26.4 ± 0.8 pS; or MRS-1845. Transient expression of dominant/negative position 2: 29 ± 1 pS; position 3: 22.9 ± 1.4 pS, n = 4 - 8, ANOVA Orai1R91W and lipid raft disruption using methyl-β-cyclodex- p > 0.05). Concatamers with two Lys-containing subunits had trin treatment also impaired LPA-evoked calcium entry. NFAT unitary conductance similar to level II (positions 1+2: 15.4 ± activity was assessed using a luciferase reporter assay and by 1.1 pS; positions 1+3: 13.2 ± 0.7 pS; positions 2+3: 13.5 ± 2.3 monitoring the nuclear translocation of GFP-tagged NFAT2. pS, n = 4 – 8, ANOVA p > 0.05). Thus, the opening levels of the Both assays revealed modest activation of NFAT by LPA-evoked concatamers were independent of the position (i.e. subunit) store release. LPA-evoked store release coupled to influx in 1.2 2+ that carried the mutation [T339K]. Measurement of the recti- mM [Ca ]o resulted in a much more robust activation of NFAT, fication shown by concatamers also indicated a progressive which was blocked by addition of DES, expression of mutant contribution by each subunit that was also independent on the Orai1R91W and lipid raft disruption. Our data thus indicate that 2+ position of mutation. In summary, these experiments show that LPA promotes keratinocyte migration by triggering [Ca ]i the rat P2X2 receptors operates as a symmetrical trimer in which influx, which activates a NFAT signalling cascade via Orai1 and each of three subunits contribute equally to the permeation lipid rafts, firstly suggesting an involvement of NFAT in epider- properties of the open channel. mal wound healing.

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Where applicable, the authors confirm that the experiments the plasma membrane that differ, not only in terms of their described here conform with The Physiological Society ethical lipid composition (raft vs. non-raft), but also by their expo- requirements. sure to distinct local Ca2+ signals, serves to optimise interplay between dynamic Ca2+ events and AC8-mediated cAMP production. 1. Willoughby D & Cooper DMF (2006). J Cell Sci 119: 828-836. C66 2. Fagan K et al (1996). J Biol Chem 271: 12438-12444. 3. Martin et al (2009). Mol Pharmacol 75: 830-842. Selectivity of adenylyl cyclase type 8 for specific modes of Ca2+ increase is attributable to its localization in a protected 4. Tallini et al (2006). Proc Natl Acad Sci USA 103: 4753-4758. microdomain Where applicable, the authors confirm that the experiments D. Willoughby, S. Wachten, N. Masada and D.M. Cooper described here conform with The Physiological Society ethical requirements. Pharmacology, University of Cambridge, Cambridge, UK

The Ca2+-stimulated adenylyl cyclase type 8 (AC8) generates dynamicpatternsofcAMPsignalinresponsetolocalCa2+ events to coordinate the actions of Ca2+ and cAMP (1). Previ- C67 ous studies have revealed that AC8 is uniquely sensitive to sub- μ 2+ 2+ MCa rises mediated by capacitative Ca entry (CCE). In Clopidogrel-induced calcium transients in isolated rat dorsal 2+ contrast, AC8 displays little sensitivity to Ca releasefromER root ganglia neurons stores and to other forms of Ca2+ entry, including ionophore- mediated entry (2). Part of the selectivity of AC8 for CCE is E. Alcin1, M. Ozcan2, S. Kutlu1, A. Ayar3 and H. Kelestimur1 thought to rely on its targeting to lipid rafts alongside CCE chan- 1 nels (3). Physiology, Firat University Faculty of Medicine, Elazig, Turkey, 2 Here, we have tethered a genetically-encoded Ca2+ sensor, Biophysics, Firat University Faculty of Medicine, Elazig, Turkey and 3 GCaMP2 (4), to the N-terminus of AC8 to directly monitor Ca2+ Physiology, Karadeniz Technical University Faculty of Medicine, changes within the immediate vicinity of the AC when exposed Trabzon, Turkey to a range of Ca2+ stimuli in HEK293 cells. Comparisons were Extracellular signalling by purine nucleotides exerts a wide made between the GCaMP2-AC8 sensor and GCaMP2-AC2 (AC2 range of biological effects including platelet aggregation, is a Ca2+-insensitive, non-raft targeted AC). All experiments exocrine and endocrine secretion and nociceptive transduc- were performed at RT using a CCD-camera imaging system. In tion. Yet, some subtypes of ionotropic purinergic receptors situ calibrations provided Kd values of 316nM for GCaMP2-AC8 have been shown to be predominantly localised in the sub- and 240nM for GCaMP2-AC2 with Hill coefficients ~3.0 (con- population of small nociceptive sensory neurons in dorsal root sistent with previous data for untagged GCaMP2 (4)). To com- ganglia (DRG). The aim of this study was to investigate the pare the response of the targeted sensors to different modes effects of clopidogrel, the potent antithrombotic agent with of Ca2+ increase the muscarinic agonist, carbachol, was used high P2Y12 receptor antagonist action on intracellular calcium to trigger ER store depletion and CCE was then evoked upon signalling ([Ca2+]i) in isolated rat sensory neurons. addition of 2mM external Ca2+. GCaMP2-AC2 detected a 7.26 DRG neurons were loaded with 5 μmol Fura-2 AM and Ca2+ ± 0.91 fold larger Ca2+ signal during IP -mediated Ca2+ release 3 responses were assessed by using the fluorescent ratiometry than during CCE (n=34 cells). In GCaMP2-AC8 expressing cells (excitation at 340 and 380 nm, and emission at 510 nm). All this ratio was decreased to 1.73 ± 0.20 (n=87; p<0.01) with data were analyzed by using an unpaired t test, with a 2-tailed ~50% of cells detecting no Ca2+ rise during IP -mediated 3 P level of <0.05 defining statistical significance. Clopidogrel release. Furthermore, the rate of signal increase during CCE was caused a dose-dependent increase in the [Ca2+]i. The mean faster for GCaMP2-AC8 (26.8 ± 2.0s to peak) than for GCaMP2- 340/380 nm ratio was 0.75±0.02 (baseline, n=6), 0.89±0.01 AC2 (77.4 ± 5.2s to peak) suggesting that AC8 resides much (10 nM clopidogrel, P<0.01, n=6); 0.85±0.03 (baseline, n=11), closer to sites of CCE. Consistent with this hypothesis pre-incu- 1.10±0.01 (100 nM clopidogrel, P<0.01, n=11); and 0.76±0.02 bation with 10μM EGTA-AM did not significantly reduce the ini- (baseline, n=32), 1.08±0.02 (1 μM clopidogrel, P<0.001, n=32), tial CCE-induced Ca2+ rise detected by GCaMP2-AC8 (n=40). In respectively. contrast, detection of CCE by GCaMP2-AC2 was attenuated by These results indicate that antagonism of the ADP receptors EGTA (n=15; p<0.01). Neither sensor detected any Ca2+ rise fol- P2Y12 by clopidogrel cause a dose dependent increase in lowing BAPTA loading (n=16). Finally, we examined whether [Ca2+]i indicating involvement of P2Y12 receptor subtype spe- GCaMP2-AC8 could detect ionophore-mediated Ca2+ entry. In cific purinergic signalling in these sensory neurons which might all HEK293 cells tested, GCaMP2-AC8 was unable to detect the have importance in nociceptive and neuropathic pain. non-specific Ca2+ entry during ionomycin treatment (n=75). Our findings suggest that AC8 resides in a discrete This study was financially supported by a grant from Firat Uni- microdomain that is exposed to rapid Ca2+ changes during versity Research Fund. CCE but is uniquely ‘shielded’ from other modes of Ca2+ rise. Differential distribution of AC8 and AC2 into sub-domains of

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Where applicable, the authors confirm that the experiments White C, Li C, Yang J, Petrenko NB, Madesh M, Thompson CB & Foskett described here conform with The Physiological Society ethical JK. (2005). Nat Cell Biol 7, 1021-1028. requirements. This work was supported by the Schweppe Foundation. Where applicable, the authors confirm that the experiments C68 described here conform with The Physiological Society ethical

2+ requirements. Modulation of InsP3 receptor-dependent Ca signalling by the antiapoptotic proteins Bcl-2 and Mcl-1 E.F. Eckenrode and C. White C69 Rosalind Franklin University of Medicine & Science, North Chicago, IL, USA Isoflavone induced activation of endothelial nitric oxide synthase is associated with cellular ROS production: is F-actin Members of the Bcl-2 protein family play a central role in the pulling the strings? regulation of apoptosis. Some of their physiological effects are mediated by modulation of the ER localized inositol trisphos- S. Chapple1, D. Rowlands1, V. Snetkov2, R. Siow1 and G. Mann1 phate receptor Ca2+ release channel (InsP R). In previous stud- 3 1Cardiovascular Division, King’s College London, London, UK and ies we determined that Bcl-XL, an antiapoptotic Bcl-2 protein, 2Asthma, Allergy & Lung Biology Division, King’s College London, bound to the InsP R to increase its sensitivity to InsP and 3 3 London, UK enhance spontaneous Ca2+ signalling (White et al., 2005; Li et al., 2007). The present study tested the hypothesis that the The risk of cardiovascular disease is increased in post- structurally related family members Bcl-2 and Mcl-1 had simi- menopausal women and growing concerns over the use of hor- 2+ mone replacement therapy (HRT) have prompted a search for lar effects on InsP3R-dependent Ca handling. Bcl-2 or Mcl-1 were stably transfected into wild type DT40 cells endogenously alternative therapy. We have shown that dietary phytoestro- gens (genistein, daidzein and its metabolite equol) modulate expressing all three InsP3R isoforms, or into DT40 cells genet- endothelium dependent relaxation in vitro and lower blood ically deficient in InsP3Rs (DT40-InsP3R-KO). The effect of Bcl- 2+ pressure in vivo (Mahn et al., 2005). More recently, we reported 2 and Mcl-1 on ER store content and InsP3R-dependent Ca release was monitored directly in permeabilized cells loaded that physiological concentrations of equol (100nM) rapidly with the low affinity indicator mag-fura-2. In the DT40-WT (2min) activate eNOS via phosphorylation of Akt and ERK1/2 2+ (Joy et al., 2006). In the present study, we have investigated background, the steady-state [Ca ]ER was significantly lower in Bcl-2 and Mcl-1 expressing cells; 30.3 ± 1 μM and 28.8 ± 1μM whether equol (100nM) induced eNOS activation is associated respectively (mean ± SEM), compared to vector only express- with (i) increased production of reactive oxygen species (ROS), ing controls 53.9 ± 3 μM (P < 0.001, ANOVA, n ≥ 36). The low- (ii) eNOS dissociation from caveolin-1 and (iii) changes in the 2+ F-actin cytoskeleton. ering of steady-state [Ca ]ER by Bcl-2 and Mcl-1 was partially μ Human umbilical vein endothelial cells (HUVEC) were incubated reversed by application of heparin (100 gml-1), an InsP3R antagonist, during store loading. In addition, the filling state in Krebs Henseleit buffer containing lucigenin (5μM), vehicle (DMSO) or equol (100nM) in the presence or absence of NADPH of the ER stores in DT40-InsP3R-KO cells expressing Bcl-2 (55.6 ± 2 μM) and Mcl-1 (52.7 ± 2 μM) was not significantly different oxidase inhibitors apocynin (10μM) and diphenylionium (1μM) ± μ ≥ or the mitochondrial complex-I inhibitor rotenone (5μM). from DT40-InsP3R-KO controls (59 2 M; n 69). These data HUVEC were also serum deprived for 4h before treatment suggest that InsP3Rs are required for Bcl-2 and Mcl-1 to regu- (2min) with vehicle (DMSO) or equol (100nM) and confocal late store content. The effect of Bcl-2 and Mcl-1 on InsP3-medi- 2+ 2+ immunofluorescent staining for eNOS (475 515 ). Mito- ated Ca release was determined by recording [Ca ]ER in ex em μ chondrial superoxide production was detected in serum response to 0.1 or 10 M InsP3 and examining the first order 2+ deprived HUVEC loaded with Mitosox dye for 30min. Cells were derivative of the release phase (d[Ca ]ER/dt) as a function of 2+ 2+ then treated for 20min with vehicle (DMSO), equol (100nM) or [Ca ]ER. Cells expressing Bcl-2 and Mcl-1 displayed faster Ca μ μ the mitochondrial complex-III inhibitor antimycin-A (100ng ml- release in response to 0.1 M InsP3 but not 10 M. Thus, Bcl-2 1). Similarly, Phalloidin was used to visualize F-actin by confo- and Mcl-1 increase the apparent sensitivity of InsP3R-depend- 2+ cal fluorescence microscopy using wavelengths (nm) 560 ent Ca release to low levels of InsP3. Fura-2 imaging experi- ex ments were carried out to assess the effect of Bcl-2 and Mcl-1 625em for Mitosox or Phalloidin, and 375ex 450em for nuclear expression on whole cell Ca2+ signalling. In the absence of stim- Hoechst staining. Inhibition of NADPH oxidase and mitochondrial complex I abro- ulation, intact DT40-WT cells display spontaneous InsP3R- dependent Ca2+ oscillations. In cells expressing empty vector gated equol stimulated ERK1/2, Akt and eNOS phosphoryla- the mean oscillation frequency was 3.45 ± 0.1 mHz; however, tion (n=4 cultures, p<0.05 Student’s t-test). Equol increased oscillation frequency was significantly higher in both Bcl-2 (6.18 mitochondrial superoxide production, with basal fluorescence ± 0.2 mHz) and Mcl-1 (8.19 ± 0.3) mHz expressing cells (P < (96 ± 5 arbitrary units, mean ± SEM, n=4 cultures) elevated by 0.001, Mann-Whitney test, n ≥ 313). Taken together, these data ~50% (141 ± 7, n=4 cultures, p<0.01 Student’s t-test) and stim- 2+ ulated ROS production was attenuated by inhibitors of NADPH demonstrate that Bcl-2 and Mcl-1 regulate [Ca ]ER handling 2+ oxidase and rotenone (n=4 cultures, p<0.05 Student’s t-test). and InsP3R-dependent Ca signalling in ways that are qualitatively similar to Bcl-XL. Immunostaining for F-actin and eNOS revealed alterations in Li C, Wang X, Vais H, Thompson CB, Foskett JK & White C.(2007). Proc their intracellular distribution. eNOS was found to translocate Natl Acad Sci USA 104, 12565-12570. from the membrane to the cytosol and was accompanied by

40P Oral Communications the appearance of F-actin stress fibers, highlighting that equol increased in groups treated with DWW and DRW for 12 weeks may alter intracellular eNOS trafficking. In summary, equol stim- (83 ± 10, 122 ± 10 and 125 ± 18 light units/mg protein/min for ulated mitochondrial ROS production may be important for control, DWW- and DRW-treated mice, respectively, n=8, S.E.). activation of eNOS, with equol induced alterations in the F-actin The presence of nitrotyrosine compounds in aortic tissue was cytoskeleton modulating mitochondrial ROS production (Felty decreased in mice treated with DRW for 20 weeks (76%, n=6). et al., 2005) and subsequent eNOS activation. HO-1 expression was increased in HUVEC incubated with 1% Felty et al. (2005). Biochemistry 44,6900-6909. and 3% DRW for 8 h (197 and 684% for 1% and 3% DRW, respec- Joy et al. (2006). J. Biol. Chem. 281,27335-45. tively, n=3). The present findings suggest that physiological levels of endogenous NO and O2.- production may be involved Mahn et al. (2005). FASEB J. 19,1755-1757. in the protection afforded by wine polyphenols against the pro- Supported by British Heart Foundation, Wellcome Trust and EU gression of atherosclerosis via their ability to upregulate expres- COST ACTION B35 sion of the antioxidant enzyme HO-1. Stocker R & O’Halloran RA (2004). Am J Clin Nutr 79,123-130. Where applicable, the authors confirm that the experiments Halliwell B. (1989). Br J Exp Pathol 70,737-757. described here conform with The Physiological Society ethical requirements. Supported by EU COST ACTION B35, Heart Research UK. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical C70 requirements.

Involvement of nitric oxide, superoxide anion and heme oxygenase-1 in cytoprotective actions of white and red wine C71 polyphenols in atherosclerosis N. Martinez1, B. Bonacasa2, R.C. Siow2, G.E. Mann2 and Effects of cigarette smoke extracts on endothelial migration M.T. Mitjavila1 are independent of oxidative stress 1Fisiologia, Universitat de Barcelona, Barcelona, Spain and D. Acheampong, E. Bishop and I.M. Fearon 2Cardiovascular Division, School of Medicine, King’s College London, Group R&D, British American Tobacco, Southampton, UK London, UK Endothelial damage is an early step in atherosclerotic lesion for- Previous evidence suggests that the content of polyphenols in mation, which is enhanced by pro-atherogenic inhibition of alcoholic beverages are responsible for their beneficial effects endothelialrepair.Previousinvitrostudieshavesuggestedarole in cardiovascular diseases such as atherosclerosis (1) characr- forincreasedproductionofoxygenfreeradicalsinendothelial terised by increased oxidative stress (2). The aim of the pres- damage repair. Since cigarette smoke extracts induce oxidative ent study was to examine the effects of wine polyphenols on stress,wehaveexaminedthepotentialroleofadditionalsmoke- the production reactive oxygen radicals and the expression of induced oxidative stress in mediating inhibition of endothelial antioxidant enzymes in vascular tissue in atherosclerosis. repairinamigration(scratchwound)assay.Methods.Confluent ApoE-deficient mice, that spontaneously develop atheroscle- monolayers of human umbilical vein endothelial cells (HUVEC) rosis were fed a control diet or diets containing dealcoholized were scratched with a pipette tip, creating a ~800μm wound. white (DWW) or red wine (DRW) (25 ml wine/ kg body MigrationofHUVECacrossthewoundwasassessedover20hours weight/day) for 12 and 20 weeks. Other groups of mice received by image capture and computer analysis of wound width using L-NAME (50 mg/kg body weight/day) in their diet for 20 weeks. theIncuCyteplatform(EssenInstruments).Cigarettesmoketotal At the end of the treatment mice were anesthetized with 150 particulate matter (TPM) from University of Kentucky 3R4F ref- mg/kg ketamin: 10 mg/kg xilacin and thoracic aorta was erence cigarettes was trapped on a Cambridge filter pad, eluted opened lenghtwise to atherome plaque quantification. Expres- in DMSO, and added to cells immediately after wounding. Free sion of endothelial (eNOS) and inducible (iNOS) nitric oxide syn- radical production was examined by loading cells with the non- thases and p22phox (subunit of the NADPH oxidase) were then fluorescent indicator dyes 2’,7’ dichlorofluorescein (DCFH) or probed in the aortic root sections by immunofluorescence. Gen- dihydroethidine (DHE) 4 hours after wounding and measuring eration of superoxide anion (O2.-) in thoracic aorta rings was free radical generated fluorescent dye products by fluorescence determined by lucigenin chemiluminiscence and nitrotyrosine microscopy. In experiments with ascorbic acid, cells were incu- compounds by immunofluorescence in aortic root. Further- bated with the antioxidant both for 5 hours prior to and during more, human umbilical vein endothelial cells (HUVEC) were exposure to TPM. Results. Contrary to previous reports, fluo- incubated with 1% and 3% DWW or DRW for 4, 8 and 12 h, and rescence emitted from cells loaded with DCFH or DHE was not expression of the antioxidant enzymes heme oxygenase-1 (HO- greateratthewoundedgewhencomparedtocellslocatedaway 1) and NAD(P)H quinone oxidoreductase 1 (NQO1) determined from the wound. (Table 1). Incubation of cells with the antioxi- by immunoblotting. dant L-ascorbic acid alone (200μM) had no effect on endothelial A decreased formation of lesions was detected after 20 weeks migration in the endothelial wound repair assay and was also dietary treatment with wine polyphenols (23 and 62%, n=8 without effect on the ability of TPM to inhibit endothelial repair for DWW and DRW, respectively), yet this was prevented in in this assay (Table 2). Conclusion. Ourdatasuggestthatoxida- mice treated with L-NAME. Although expression of eNOS, iNOS tive stress is unimportant in endothelial wound repair in vitro, and p22phox were not changed, the production of O2.- was

41P Oral Communications and may argue against a role for oxidative stress in the inhibition the body of the pericyte. Ureteric microvascular pericytes were ofendothelialwoundrepaircausedbycigarettesmokeextracts. totally resistant to caffeine (10mM) and PhE (10-100μM) but Table 1. Comparison of fluorescence at and away from the wound edge in cells loaded with DCFH and DHE. , P>0.05 vs wound edge. All data are readily responded to ET-1 (10nM). Precapillary pericytes 2+ means±s.e.m. n=24 in all cases. responded with a single spike-like Ca transient resistant to ryanodine and removal of [Ca]o but which was abolished by SERCa pump inhibitor cyclopiazonic acid (20μM) or inhibitor of μ 2+ IP3R 2-APB (50 M). The Ca transient induced by ET-1 consisted of an initial fast component followed by a slowly decay- ing sustained component producing local constriction of the Table 2. Effects of TPM on endothelial wound recovery. All data are microvessel which lasted for 2-3 minutes. Transmitted light means±s.e.m. , P>0.05 vs control. *, P>0.05 vs TPM alone. imaging revealed that contraction of precapillary pericytes produced complete closure of the microvessel blocking the flow of red blood cells. The data obtained suggest that precapillary pericytes are likely to act as local sphincters controlling capillary blood flow by responding to local factors. This work was supported by the British Heart Foundation. Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

C73 C72 Mechanisms underlying cell toxicity of IMD-0354, an 2- and 3-dimentional imaging of ureteric precapillary inhibitor of IkB kinase, in isolated rat ventricular myocytes pericytes: morphology, Ca2+ signalling and function A. Neep, M. Hardy, K. White, L. Hull and S. Harrison L. Borisova1, D. Eisner2, S. Wray1 and T. Burdyga1 Multidisciplinary Cardiovascular Research Centre, Institute of 1The Physiological Laboratory, University of Liverpool, Liverpool, Membrane and Systems Biology, University of Leeds, Leeds, UK UK and 2Unit of Cardiac Physiology, University of Manchester, Tumour necrosis factor alpha (TNF-α) and interleukin 1β (IL- Manchester, UK 1β), have been implicated in Ca2+ dysregulation and depressed It is well known that the control of local blood flow in capillar- contractility in ventricular tissue (Duncan et al., 2007). These ies is controlled by the “precapillary sphincters” (PS) although cytokines stimulate several signalling pathways, one of which κ κ their nature and the mechanism controlling their function results in activation of I B kinases, phosphorylation of I B and κ κ remain controversial. Precapillary pericytes are good candi- subsequent translocation of nuclear factor- B (NF- B) to the κ dates for regulating blood flow and perform sphincter func- nucleus to modulate gene transcription. Inhibition of NF- B tion. However, little is known about the mechanisms control- translocation with IMD-0354 (IC50 ~250 nM) during ling Ca2+ signalling and their contractile activity in situ. We used ischaemia/reperfusion injury reduces infarct size and improves confocal imaging of in situ rat ureteric microvessels (17 ves- functional recovery (Onai et al., 2004). Our aim was to investi- κ sels from 15 rats) loaded with the Ca2+-sensitive indicator Fluo- gate the role of NF- B signalling in altered Ca2+ regulation α β 4 in order to investigate morphology, Ca2+ signalling and induced by TNF- and IL-1 in ventricular myocytes. mechanical activity of precapillary pericytes. The effects of cen- Rats (~250g) were sacrificed humanely and ventricular tral (Phenylephrine, PhE) and local (endothelin-1, ET-1) factors myocytes prepared using a standard collagenase/protease dis- as well as caffeine on Ca2+ signalling and contraction of pre- persion technique. Cells were loaded with fura-2 AM, super- ∞ capillary pericytes and their effect on the diameter of the vas- fused with Tyrode solution (30 C) and stimulated electrically ± cular wall of the precapillary branches of the ureteric microves- (1 Hz). Data are mean SEM and statistical comparisons made sels (i.d.<10μm) have been investigated. with paired or unpaired t-tests as appropriate or Friedman The ureteric microvascular tree consists of 3 generations of Repeated Measures ANOVA on ranks. microvessels. The last (third) generation of the microvessel has In contrast to Onai et al. (2004), our initial experiments showed μ a monolayer of smooth muscle coat in its proximal part fol- IMD-0354 to be toxic; exposure to 0.1 M IMD-0354 led to an ± lowed by a coat of precapillary pericytes running circumferen- initial increase in contractility (by 60 13%, n=11; P<0.001) fol- tially around the endothelium in its distal part. Third order lowed by failure of both contractions and Ca2+ transients (Fig. branches give off side branches of microvessels which have only 1A) before the cell entered rigor, reminiscent of complete meta- a coat of precapillary pericytes (pericytic microvessels). Pre- bolic inhibition (Lancaster & Harrison, 1998). Time from appli- ± ± ± capillary pericytes formed an asymmetrical coat with a thick cation to rigor was dose-dependent; 335 28, 417 93, 499 32, ± μ body located on one side of the vessel giving 2-3 fingers like 751 78 s at 1.0, 0.3, 0.1 and 0.03 M IMD-0354, respectively. processes which tightly wrapped around the endothelium. Each The initial increase in contractility was associated with an precapillary pericyte occupied a length of 10.10±0.48 μm of increase in myofilament sensitivity (P<0.05, n=11) assessed the vessel (n=15). Live staining of nuclei with propidium iodide from plots of cell length vs fura-2 fluorescence (Spurgeon et revealed that pericytic nuclei were curved and were located in al., 1992). During the phase of contractile failure myofilament

42P Oral Communications sensitivity was significantly decreased (P<0.05, n=11), time to predominantly, but not exclusively, across the t-tubule mem- peak contraction accelerated (P=0.002), time to half relaxation brane (1). However the location of the sarcolemmal Ca2+ ATPase reduced (P=0.029) whereas time to half decay of the Ca2+ tran- is unknown. We have therefore investigated the distribution of sient was increased (P=0.005, Fig. 1B), consistent with an intra- Ca2+ efflux via the Ca2+ ATPase between the t-tubule and sur- cellular acidosis. face membranes. DMEM and Tyrode supplemented with foetal calf serum (FCS) Ventricularmyocyteswereisolatedfromtheheartsofmale protected cells from IMD-0354 toxicity (contractility was sta- Wistar rats, and detubulated as described previously (2). Intra- ble after >38 min exposure to 0.1 μM IMD-0354 with FCS pres- cellular Ca2+ was recorded using fluo-3 in conjunction with con- ent). Time to the development of a rigor contracture was not focal microscopy during electrical stimulation at 0.5 Hz. Fol- delayed significantly by cyclosporin A (P=0.214). lowing a train of stimuli, 20 mM caffeine was used to release These data illustrate that IMD-0354 is toxic at concentrations Ca2+ from the sarcoplasmic reticulum (SR); this was repeated 10-fold below the IC50 for IκB kinase inhibition unless super- in the presence of 10 mM NiCl, to inhibit NCX, or following 8- fusion solutions contained FCS. This may suggest that IMD- 10 minutes incubation with 20 μM carboxyeosin, to inhibit the 0354 toxicity does not result from blockade of NF-κB activity. sarcolemmal Ca2+ ATPase, in control and detubulated myocytes. The proportion of Ca2+ removedfromthecytoplasmbydiffer- ent pathways was calculated from the rate constants of decline of the electrically stimulated Ca2+ transient, and those obtained in the presence of caffeine, as described previously (3). The rate constant of decline of the systolic Ca2+ transient was 2.985±0.254s-1 in control myocytes, and 2.8±0.218s-1 in detubulated myocytes (n=22/26; p=0.5810; unpaired t-test). The rate of decline of the caffeine-induced Ca2+ transient was slower: 0.520±0.115s-1 in control myocytes and 0.188±0.031s- 1 in detubulated myocytes (n=9/11; p=0.0109); this was decreased further by NiCl, from 0.520±0.115 to 0.179±0.022s- 1 in control myocytes (n=9/12; p=0.0057), and from 0.188±0.031 to 0.083±0.012s-1 in detubulated myocytes (n=11/12; p=0.0212). The rate of decline of the caffeine- induced Ca2+ transient was also slowed by carboxyeosin in control myocytes, from 0.520±0.115 to 0.294±0.047s-1 (n=9/9; p=0.0484), but not in detubulated myocytes (0.188±0.031 vs. 0.203±0.36s-1; n=11/7; ns). In control myocytes, therefore, the SR appears to be responsible for ~83% of Ca2+ removal, NCX for ~11%, and the sarcolemmal Ca2+ ATPase for ~7%, while in detubulated myocytes, the SR is responsible for ~93%, NCX for ~4% and the Ca2+ ATPase Figure 1. A, cell length and fura-2 fluorescence ratio (Fr) during exposure to for 0%. Thus Ca2+ efflux via the sarcolemmal Ca2+ ATPase 0.1 μM IMD-0354. B, fast time base traces from points a, b and c in panel A. appears to occur only across the t-tubule membrane, so that Grey lines are control traces from a for comparison. the slower Ca2+ extrusion in detubulated cells is likely to be due Duncan DJ et al. (2007). Br J Pharmacol 150, 720-726. to partial loss of NCX and complete loss of Ca2+ ATPase. Lancaster MK and Harrison SM (1998). Expt Physiol 83, 349-360 Despa et al (2003) Biosphys J 85, 3388-3396. Onai Y et al. (2004). Cardiovasc Res 63, 51-59 Brette et al (2002) Am J Physiol Heart Circ Physiol 283, H1720-H1728. Spurgeon HA et al. (1992). J Physiol 447, 83-102 Negretti et al (1993) Cardiovascular Research 27, 1826-1830.

Supported by the British Heart Foundation. This work was supported by the British Heart Foundation. Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

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Ca2+ efflux via the sarcolemmal Ca2+ ATPase occurs only in the t-tubules of rat ventricular myocytes A. Chase and C. Orchard Physiology, University of Bristol, Bristol, UK Ca2+ influx into cardiac ventricular myocytes, via the L-type Ca2+ current, appears to occur mainly across the t-tubule membrane. Ca2+ efflux, via Na+/Ca2+ exchange (NCX), also appears to occur

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FTY720, a sphingosine 1-phosphate analogue, prevents ischemic/reperfusion-induced cardiac arrhythmias in an ex vivo rat heart model via activation of p21-activated kinase/protein kinase Akt signaling E. Eroume A Egom1, Y. Ke2, H. Musa1, T. Mohamed1, T. Wang1, E. Cartwright1, R. Solaro2 and M. Lei1 1Division of Cardiovascular and Endocrine Sciences, University of Manchester, Manchester, UK and 2Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA Recent studies demonstrated a role of Sphingosine-1-phosphate (S1P) in protection against the stress of ischemia/reperfusion injury (I/RI). In experiments reported here, we have investigated the signaling through the S1P cascade by FTY720, a sphingolipid drug candidate displaying structural similarity to S1P, underlying the S1P cardio-protective effect. In ex vivo rat heart and isolated sino-atrial node models, FTY720 significantly prevented I/RI induced arrhythmic events including premature ventricular beats, VT and sinus bradycardia as well as A-V conduction block. Real-time PCR and Western blot analysis demonstrated the expression of the S1P receptor transcript pools and corresponding proteins including S1P1, S1P2 and S1P3 in tissues dissected from sino-atrial node, atrium and ventricle. FTY 720 (25 nM) significantly blunted the depression of the levels of phospho-Pak1 and phospho-Akt with ischemia and with reperfusion. There was a significant increase in phos- Jin ZQ, Zhang J, Huang Y, Hoover HE, Vessey DA, Karliner JS. A sphingosine kinase 1 mutation sensitizes the myocardium to pho-Pak1 levels by 35%, 199%, 205% after 5, 10 and 15 mins of ischemia/reperfusion injury. Cardiovasc Res 2007;76:41-50. treatment with 25 nM FTY720 compared with control non- treated myocytes. However, there was no significant difference Karliner JS. Sphingosine kinase regulation and cardioprotection. Car- in the levels non-phospho-Pak1 expression between non- diovasc Res 2008;1093/cvr/cvn309. treated and FTY720 treated. Phospho-Akt levels were increased We thank Dr James Tellez for his support. The project was sup- by 44%, 63%, and 61% after 5, 10 and 15 min of treatment with ported by The Wellcome Trust (ML), The British Heart Founda- 25 nM FTY720 respectively. Our data provide the first evidence tion (ML, EJC) and National Institute of Health grants RO1 HL that FTY720 prevents the arrhythmias induced by I/RI, and indi- 64035 and PO1 HL 62426 (Project 1) (RJS). cate its potential significance as an important and new agent protecting against ischemia/reperfusion induced arrhythmias. Where applicable, the authors confirm that the experiments The cardio-protective effect of FTY720 is likely to involve acti- described here conform with The Physiological Society ethical vation of signalling through the Pak1 and Akt cascade. requirements.

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Diurnal variation in excitation-contraction coupling in rat ventricular myocytes: sensitivity to b-adrenergic stimulation H.E. Collins and G. Rodrigo Cardiovascular Sciences, University of Leicester, Leicester, UK Many pathological cardiovascular events show morning prevalence, possibly reflecting diurnal changes in cardiac haemodynamics, metabolism and sympathetic activity. Whilst around 10% of rat cardiac gene expression displays diurnal variations, little is known of whether this impacts on cardiac excitation- contraction (E-C) coupling. We have, therefore, set out to determine whether cardiac E-C coupling shows diurnal variation and if this influences the arrhythmia threshold of ventricular tissue to sympathetic stimulation.

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Single ventricular myocytes were isolated from hearts excised direct effects on muscle contraction. Therefore, the primary at two opposing timepoints, ZT3 and ZT15, where ZT0 refers aim of this study was to investigate the effects of clenbuterol 2+ to “lights on”. [Ca ]i was measured using Fura-2, cell short- on the contractile properties of isolated intact mouse fast and ening with a cell-edge detection system and L-type Ca2+ cur- slow twitch skeletal muscle fibres. rent density using the whole cell, patch clamp technique. The All the experiments were performed at 20 ± 0.1∞C on small 2+ data show that basal percent cell shortening and systolic [Ca ]i muscle fibre bundles isolated from either the extensor digito- was significantly higher in ZT3 than ZT15 myocytes; with a % rum longus (fast twitch muscle) or soleus (slow twitch muscle) cell shortening of 12.4±0.3 in ZT3 myocytes (n=209) vs of CD-1 mice 46 ± 1.6 (n=9; S.E.M) days old. The mice were 11.0±0.2 (n=216) in ZT15 myocytes (S.E.M; P<0.05), and a peak humanely killed and all the experiments conformed to the Ani- 2+ ± ± systolic [Ca ]i of 422 12nM (n=166) in ZT3 vs 341 9nM mals (Scientific Procedures) Act 1986. The fibre bundles were (n=176) in ZT15 myocytes (S.E.M; students t-test, P<0.01). The mounted horizontally between a force transducer and a ser- SR Ca2+ content revealed by the application of 20mM caffeine vomotor and were continuously perfused with mammalian was significantly higher in ZT3 myocytes, with a peak Ca2+ of Ringer’s solution. Twitch and tetanic contractions were then 672.8 ± 20.5nM (n=71) vs 550.9±12.9 (n=97) in ZT15 myocytes recorded in Ringer’s solution with or without any added cleneb- (S.E.M; students t-test, P<0.001). We looked at β-adrenergic uterol. In another experiment, the fibre bundles were treated stimulation with isoproterenol (ISO) to simulate sympathetic for 1hr with the standard Ringer’s solution or the standard activity. We found no significant difference in EC50 of ISO-stim- Ringer’s solution with clenbuterol. Proteins isolated from these ulation of systolic Ca2+, but a significant reduction in the steady- bundles were then immunoblotted for the levels of phospho- state response at concentrations >3nM in ZT15 myocytes, with rylated AKT, extracellular signal regulated kinases 1&2 (ERK1&2) 2+ ± a maximum systolic [Ca ]i, recorded in 100nM ISO, of 2,330 and phospholamban. 402nM (n=33) in ZT3 myocytes vs. 1,384 ± 109nM (n=36) in At all concentrations investigated (100ηM - 250μM), clen- ZT15 myocytes (S.E.M; students t-test, P<0.01). A similar diur- buterol decreased twitch and tetanic contractions in both fibre nal variation was shown in the response of the L-type Ca2+ cur- types. Used at concentrations <150μM, its effects were com- rent to ISO, with a maximal percentage increase in current den- pletely reversible. However, above this concentration they were sity of 67.0%±8.5 (n=15) in ZT3 myocytes compared to not. For example, 50μM clenbuterol reversibly decreased twitch 37.4%±5.2 (n=16) in ZT15 myocytes, (S.E.M; students t-test, tension to 72 ± 4% and tetanic tension to 23 ± 8% (n=4; S.E.M) P<0.01,) in 100nM ISO. We also looked for any diurnal variation of controls in slow twitch fibres. The corresponding values in in the arrhythmia threshold to ISO. The percentage of ZT3 the fast twitch fibres were 81± 6 and 55 ± 4% (n=5; S.E.M), myocytes that developed arrhythmias was significantly greater respectively, of controls. In addition to its effects on force, clen- than ZT15 myocytes, with 43.3±4.8% vs. 13.4±1.9% in 3nM ISO, buterol also increased the phosphorylation of ERK 1&2 and the and 61.6±4.4 vs. 35.8±2.7 in 10nM ISO (n=15-18, S.E.M; one- dephosphorylation of AKT in both fibre types. In contrast, it way ANOVA and Bonferroni post hoc test, P<0.01). increased the phosporylation of phospholamban in slow twitch Our data shows the existence of diurnal variation in E-C cou- fibres but decreased it in fast twitch fibres. From these results pling, at rest and in response to β-adrenergic stimulation. The we suggest that clenbuterol decreases force production in data also show diurnal variation in the threshold for arrhyth- mammalian fast- and slow-twitch skeletal muscles by regulat- mogenesis to sympathetic stimulation. ing the phosphorylation of phospholamban. Lynch GS & Ryall JG (2008). Role of β-adrenoceptor signalling in skele- HECollinshasaDepartmentofCardiovascularSciencesstudentship tal muscle: Implications for muscle wasting and disease. Physiol Reviews Where applicable, the authors confirm that the experiments 88, 729-767. described here conform with The Physiological Society ethical This research was funded by the University of East Anglia. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C77

The b2-adrenoceptor agonist, clenbuterol, decreases force C78 in isolated intact mouse fast and slow skeletal muscle fibres D. Treen and G. Mutungi Skeletal muscles from mice lacking Cu,Zn superoxide dismutase show accelerated loss of muscle mass and Biomedical and Clinical Research Institute, University of East Anglia, function and attenuated responses to contractile activity Norwich, UK L. Zibrik1, A. Vasilaki1, H. Van Remmen3, A.G. Richardson3, β 2 1 1 Clenbuterol is a 2-adrenoceptor agonist, structurally similar J.A. Faulkner , M.J. Jackson and A. McArdle to adrenaline, used in the treatment of asthma. Although its 1School of Clinical Sciences, The University of Liverpool, Liverpool, primary action is bronchodilation, there is growing evidence UK, 2Institute of Gerontology, University of Michigan, Michigan, that it, like other β -adrenoceptor agonists, may also have ana- 2 MI, USA and 3Health Science Centre, Department of Physiology, bolic effects when used for prolonged periods (Lynch & Ryall, University of Texas, San Antonio, TX, USA 2008). However, the mechanism(s) underlying the anabolic β effects of 2-adrenoceptor agonists is still poorly understood. Reactive oxygen species (ROS) are increased in skeletal mus- β It is also uncertain whether 2-adrenoceptor agonists have any cles by isometric contractions and muscle adapts to this by

45P Oral Communications increasing transcription of cytoprotective proteins though acti- formance and its control by diet and/or genetic background, vation of redox-sensitive transcription factors such as Nuclear we have constructed an ergometer to measure the output of Factor kB (NF-kB) and Activator protein-1 (AP-1). ROS may play the jump muscle (tergal depressor of trochanter) of the fruit a crucial role in ageing and increased mitochondrial superox- fly, Drosophila melanogaster (1). The muscle executes a twitch ide generation is implicated in this process. We previously contraction in response to one action potential in its single showed aberrant DNA binding activity of NF-kB and AP-1 in motoneuron, which can be driven by single stimuli to the muscles of old mice compared with adult both at rest and fol- descending giant fibre (2). In our ergometer, the movement lowing contractile activity. Mice lacking Cu, Zn superoxide dis- of the leg is transduced by a flexible optical fibre illuminating mutase (SOD1; Sod1-/- mice) show accelerated loss of skeletal a quadrant photodiode. muscle mass and function (1). The aim of this study was to In wildtype flies, the energy output of the jump muscle declines examine ROS in isolated skeletal muscle fibres from adult Sod1- more rapidly than the population size: energy output is reduced /- mice and the effect of a lack of SOD1 on the adaptive to 50% with flies 18 days old, when 80% of the flies are still alive. responses in muscle following contractile activity. Only by 26 days have 50% of the flies died (Fig. 1). Although all Adult C57/BL6 wild type (WT) mice and Sod1-/- mice were anaes- jump muscles contract in 20 day old flies, by 30 days, 43% of thetised (65mg/100g pentobarbitone sodium) and the hind the jump muscles fail to contract. However, other muscles limbs subjected to an isometric contraction protocol (2). Imme- driven by the giant fibre still contract, suggesting failure of diately following the contractions mice were killed according the jump motoneuron or its neuromuscular junction. to UK legislation. Single muscle fibres were isolated from the The composition of the diet affects the fly lifespan, with both flexor digitorum brevis (FDB) and intracellular superoxide activ- poor and rich foods reducing the median lifetime (3). We find ity monitored using dihydroethidium (DHE) at rest and after that enriching the food with yeast, or restricting nutrient avail- electrically stimulated contractions (0.5 sec every 5 sec at 50 ability by mixing the food with agar, both reduce the age at Hz for 15 min). Apocynin (an inhibitor of NAD(P)H oxidases) which the 50% of the energy output occurs. Already by day 15, was used to determine the potential source of ROS. Gastroc- when an average of 13% of the flies have died, the difference nemius muscles were analysed for NF-kB and AP-1 DNA bind- in jump performance is significant (ANOVA, F3,61df=11.4, P < ing activity by Electrophoresis Mobility Shift Assay and for com- 0.001). ponents of the NF-kB activation pathway by western blotting. Drosophila provide an exceptional transgenic toolbox with Data indicated aberrant DNA binding activity of AP-1 and NF- which to examine aging, including neurodegenerative dis- kB in muscles of Sod1-/- mice, similar to that seen in muscles of ease like Parkinson’s disease. A small proportion of Parkin- old WT mice (2) and identified p50 and p65 as components of son’s disease patients express mutations of the LRRK2 gene. the NF-kB complex. Phosphorylated IkBa was increased by In transgenic flies in which the kinase domain of dLRRK is inac- ~60% in muscles of Sod1-/- mice. Electrical stimulated contrac- tivated (4) the lifespan at 29 ∞C is reduced to 54% (Kaplan- tions of FDB fibres from WT mice induced a significant increase Meier test, Log Rank χ2 =98, P <0.001), and jump muscle per- in ethidium fluorescence that was abolished by apocynin treat- formance is reduced by 25% throughout the lifespan - ANOVA: ment. There were no differences in ethidium fluorescence in significant effect of age (F 3,186df = 13.4, P < 0.001) and geno- quiescent muscle fibres from 6 month Sod1-/- mice compared type (F 2,186df = 20.8, P < 0.001) but no interaction of age with with fibres from WT mice. These findings indicate that a lack of genotype (F 6,186df =1.5, P = 0.16) indicating a uniform loss of SOD1 causes attenuation of adaptive responses of skeletal mus- muscle output. cle to contractile activity analogous to that seen in muscle from We conclude that the fly, like the human (5), shows progres- aged mice, but this is not associated with increased cytosolic sive sarcopenia, with loss of contractile ability preceding loss superoxide activity measured by oxidation of DHE. of functional innervation and population mortality. However, Muller FL et al. (2006).Free Rad Biol Med 40, 1993-2004 our apparatus provides for a medium-throughput assay to Vasilaki A et al. (2006). Mech Ageing Dev 127, 830-839 record the consequences of dietary or genetic manipulation.

The authors would like to acknowledge the financial support of the US National Institute on Aging. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

C79 in vivo measurement of the decline in performance of individual muscle twitches with age A. Lopez-Rodriguez, A. Grant and C.J. Elliott Fig. 1. Decline in the percentage of flies alive (N=109) and in output of the Biology, University of York, York, UK jump muscle with age (N= 130, mean ± SE). As muscles age, their contractions become weaker. In order to 1. Harvey J et al. (2008). Invert Neurosci 8, 63 . understand the neuromuscular basis of this decline in per- 2. Allen MJ et al. (2006). Sem cell Dev Biol 17, 31 .

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way, key regulator of differentiating process, has a main role 3. Partridge L et al. (2005). Mech Ageing Dev 126, 938 . also for HMG-CoAR activation durind the early phase of L6 4. Imai Y et al. (2008). EMBO J 27, 2432 . differentiation. 5. Doherty TJ (2003). J Appl Physiol 95, 1717 . Pathologies characterized by muscle degeneration might benefit from therapeutic programmes committed to muscle func- We thank Sean Sweeney and John Sparrow for their advice and tion restoration, such as modulation and planning myoblast encouragement, Yuzuru Imai for fly stocks, and the Parkinson’s differentiation. Thus, the important role of HMG-CoAR in mus- Disease Society for support. The ergometer is now available cular differentiation providing new molecular basis for the con- commercially from Digitimer. trol of muscle development could be helpful in the design of therapeutic treatment committed to potentiation of regener- Where applicable, the authors confirm that the experiments ative ability of muscle tissue in degenerative myopathies and described here conform with The Physiological Society ethical in diseases characterised by the weakening of muscular fibres requirements. and aging-related disorders (sarcopenia). Allal C., Favre G., Couderc B., Salicio S., Sixou S., Hamilton A.D., Sebti S.M., Lajoie-Mazenc I., Pradines A. (2000) RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element tran- C80 scription. J Biol Chem 275:31001-31008. Christopher-Stine L. (2006) “Statin myopathy: an update” Curr Opin HMG-CoA reductase role in rat skeletal myoblast Rheumatol 18: 647-53. differentiation Goldstein J.L., Brown M.S. (1990) Regulation of the mevalonate path- L. Trapani, C. Martini, M. Marino and V. Pallottini way. Nature 343:425-430. Ogura T., Tanaka Y., Nakata T., Namikawa T., Kataoka H., Ohtsubo Y. University Roma Tre, Rome, Italy (2007) Simvastatin reduces insulin-like growth factor-1 signaling in differentiating C2C12 mouse myoblast cells in an HMG-CoA reductase 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMG- inhibition-independent manner J Toxicol Sci 32: 57-67. CoAR) is the key and rate limiting enzyme of cholesterol biosyn- thetic pathway (Goldstein and Brown, 1990). Besides choles- Prendergast G.C., Oliff A. (2000) Farnesyltransferase inhibitors: anti- terol, HMG-CoAR end-products such as neoplastic properties, mechanisms of action, and clinical prospects. Semin Cancer Biol 10:443-452. farnesyl-pyrophosphate (FPP) and geranyl pyrophosphate (GGPP) are essential compounds for survival, proliferation and Where applicable, the authors confirm that the experiments differentiation of cells (Ogura et al., 2007) through the acti- described here conform with The Physiological Society ethical vation of small GTPases, such as Ras and RhoA (Allal et al., 2000; requirements. Prendergast and Oliff, 2000). Although HMG-CoAR activity has already been related to the differentiation of some cellular lines there are no stud- C81 ies that analyse the role of HMG-CoAR, and the pathway it is involved with, in a fully characterized muscle differentia- The effect of IL-6 on insulin secretion, nitric oxide release, tion model. redox status and signal transduction in a clonal pancreatic Thus, the aim of this work is to evaluate such role and delineate beta cell line the pathway involved in foetal rat myoblasts (L6) induced to differentiate by insulin, a standard and feasible model of the myo- M.D. Krause1,2, P.H. Bittencourt Júnior1 and P. Newsholme2 genesis, a dynamic process characterised by the proliferation, the withdrawal from the cell cycle and the fusion of mononu- 1Department of Physiology and Department of Physical cleated undifferentiated myoblasts into myotube. The aim is Education, Federal University of Brazil, Porto Alegre, Rio Grande supported by experimental and clinical studies that demon- do Sul, Brazil and 2UCD School of Biomolecular and Biomedical strate how HMG-CoAR strong inhibiting statins, widely used Science, UCD Conway Institute, University Collegue Dublin, Dublin, in therapies against hypercholesterolemia, could cause myopa- Ireland thy characterized by weakness, pain, and elevated serum creatine phosphokinase (Christopher-Stine 2006), thus demon- IL-6 is often correlated with many disease states and conditions strating the important role of HMG-CoAR in muscular such as obesity and metabolic syndrome, all marked by abnor- physiology. mal inflammatory mediators. However, given that contracting To this end the differentiation was monitored both by bio- skeletal muscle produces and releases substantial amounts of chemical and morphological approaches; protein levels were the IL-6 then it is likely that this cytokine has positive effects on analysed through western blotting assays; cholesterol and endocrine regulation. Thus the main objective was to deter- prenylated protein cellular content have been analysed through mine the effects of IL-6 on pancreatic beta cell metabolism, TLC and radioisotopic assay respectively. insulin secretion, nitric oxide release and redox status. We used The results obtained by biochemical and morphological a clonal b-cell line (BRIN-BD11) to check these variables in the approaches demonstrate that (i) HMG-CoAR increase is cru- presence of an exercise IL-6 concentration (50pg/ml) for 24h cial for differentiation induction, (ii) p21waf, whose increase incubation. BRIN-BD11 cells were maintained in RPMI-1640 tis- is a necessary requisite for differentiation to occur, rises sue culture medium with 10% FCS, 0.1% antibiotics and downstream HMG-CoAR activation, (iii) p38/MAPK path- 11.1mmol/l D-glucose, pH 7.4. Cells were then washed with

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PBS after which they were incubated in fresh media, contain- Therefore, the present study determined the trans-cerebral ing 11.1mM D-glucose, 2mM L-glutamine, in the absence or exchange kinetics of oxidative-nitrative stress biomarkers in presence of IL-6 (50pg/ml) added. After 24h incubation, a media response to hypoxia. We hypothesised that hypoxia would aliquot was removed and used for quantization of insulin, D- increase the net cerebral output of free radicals that would inac- glucose, glutamate, urea and nitrites; while the cells were used tivate NO as indicated by a decrease in the net uptake or confor the measurement of glutathione metabolism, AMPK/AMPK- sumption of nitrite (NO2’) and increased output of 3-nitroty- P e iNOS expression. After the 24h incubation, the cells were rosine (3-NT). stimulated acutely for 40 min in the presence of 1.1mmol/l glu- Ten healthy males aged 27 (mean) ± 4 (SD) years were exam- cose followed by 20 min in the presence of 16.7mM glucose ined in normoxia and following 9h passive exposure to hypoxia and 10mM alanine, when an aliquot of the incubation medium (12.9%O2). Global cerebral blood flow (CBF) was measured was removed and analyzed for acute insulin secretion. At lest using the Kety-Schmidt technique with paired samples three different experiments were made (*P≤0.05). IL-6 incu- obtained from the radial artery and internal jugular vein. Global bations increase insulin secretion over 38% (1379±162ug/mg cerebral plasma flow (CPF) was determined as CBF x (1-haema- protein/24h against 994±151ug/mg protein/24h from the con- tocrit). The serum concentration of spin-trapped α-phenyl-tert- trol group). Moreover, IL-6 not only increase the chronic insulin butylnitrone (PBN)-adducts was assessed via X-band electron secretion but also induced changes in the basal and acute stim- paramagnetic resonance spectroscopy. Plasma NO2’ was deter- ulated levels. Basal levels of insulin secretion were increased by mined by ozone-based chemiluminescence using modified tri- almost 100% in the presence of IL-6 (4.8±2ug/mg protein/20min iodide reagent and 3-NT via ELISA. Trans-cerebral net exchange from the control group to 9.6±3.2ug/mg protein/20min with was calculated as the arterio-jugular venous concentration dif- IL-6) indicating an improvement on b-cell sensitivity. AMPK lev- ference x CPF. Data were analysed with a two-way repeated els were decreased by 75% with a concomitant increased measures ANOVA and post-hoc Bonferroni-corrected paired expression of AMPK-P by 84%. We also found a raised iNOS samples t-tests. ± ± expression together with an intensified nitric oxide production, Despite a marked reduction in PaO2 (107 6 to 46 3 mmHg, ± as measured by nitrite release (0.34 0.12umol nitrite/mg pro- P < 0.05), the cerebral metabolic rate for O2 was preserved (2.43 tein/24h from the control groups to 3.59±0.86umol nitrite/mg ± 0.54 in normoxia vs. 2.49 ± 0.34 in hypoxia, P > 0.05) due to ± protein/24h with IL-6 incubation). Both enzyme activities have an increase in global CBF [85 15 in normoxia vs. (PaCO2-cor- been suggested as mediators for the increased insulin secre- rected) 123 ± 24 mL/100g/min, P < 0.05). Hypoxia increased tion. IL-6 did not induce redox changes as measured by the glu- the net cerebral output of PBN-adducts identified as lipid- tathione metabolism. Those results indicate that IL-6 can exert derived alkoxyl radicals (-73 ± 192 vs. -360 ± 253 AU/g/min, P positive effects on the b-cell metabolism, protection and func- < 0.05). This was associated with an attenuation in the net ± ± tion. IL-6 may act as a communication factor between skeletal uptake of NO2’ (126.4 93.9 vs. 16.0 46.7 nmol/g/min, P < muscle cells and pancreatic b-cells after exercise, so elevating 0.05) and increased output of 3-NT (-3.1 ± 9.0 vs. -10.7 ± 18.2 insulin secretion to achieve optimal concentrations for glucose nmol/g/min, P < 0.05). uptake and metabolism by contracting muscle. These findings provide the first direct evidence for increased oxidative-nitrative stress in the hypoxic human brain. The Where applicable, the authors confirm that the experiments regional loss of NO ’ likely reflects the combined effects of NO described here conform with The Physiological Society ethical 2 “consumption” to support the observed increase in CBF to pre- requirements. serve cerebral O2 delivery and NO “loss” subsequent to oxidative inactivation by superoxide. Bailey DM et al. (2009). J. Physiol. 15, 73-85. C82 Where applicable, the authors confirm that the experiments Hypoxiatriggersoxidative-nitrativestressinthehumanbrain described here conform with The Physiological Society ethical requirements. D.M. Bailey1, S. Taudorf2, R.M. Berg2, C. Lundby2, J. McEneny3, I.S. Young3, K.A. Evans1, P.E. James4, J.M. McCord5, B.K. Pedersen2 and K. Moller2 1Faculty of Health, Science and Sport, University of Glamorgan, South Wales, UK, 2Department of Infectious Diseases, University of Copenhagen, Copenhagen, Denmark, 3Centre for Public Health, Queen’s University Belfast, Belfast, Ireland, 4Wales Heart Research Institute, Cardiff University, Cardiff, UK and 5Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Denver, CO, USA Oxidative stress may be responsible for the reduction in systemic nitric oxide (NO) bioavailability and impaired neurovascular reactivity recently observed during acute exposure to inspiratory hypoxia (Bailey et al., 2009). However, the brain’s contribution to these systemic changes in redox-homeostasis remains unknown.

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Effects of systemic inflammation on arterial hypoxaemia and the alveolar-arterial oxygenation gradient during acute inspiratory hypoxia in healthy humans R.M. Berg1, S. Taudorf1, D.M. Bailey2, C. Lundby3, B.K. Pedersen1 and K. Møller1,4 1Centre of Inflammation and Metabolism, Department of Infectious Diseases, University Hospital Rigshospitalet, Copenhagen, Figure1.AagradientduringinspiratoryhypoxiaandLPSinfusion(mean±SEM) 2 Denmark, Neurovascular Research Laboratory, Faculty of Health Preas HL II et al (2001). Am J Respir Crit Care Med 164, 620-626. and Science, University of Glamorgan, Mid-Glamorgan, South Wales, UK, 3Copenhagen Muscle Research Centre, University of Where applicable, the authors confirm that the experiments Copenhagen, Copenhagen, Denmark and 4Department of described here conform with The Physiological Society ethical Cardiothoracic Anesthesia and Intensive Care Unit 4131, University requirements. Hospital Rigshospitalet, Copenhagen, Denmark Systemic inflammation is associated with mild arterial hypoxaemia by compromising oxygen (O2) diffusion (Preas II et al, C84 2001) and may therefore augment the widening of the alveolar-arterial oxygen (Aa) gradient observed upon exposure to Haemostatic response to inspiratory hypoxia and physical hypoxia. In the current study, we tested the hypothesis that exercise; interpretive implications of plasma volume shifts lipopolysaccharide (LPS) infusion, a human experimental model of systemic inflammation, would further compound arterial L. Fall, M. Gelsei, A. Sinnott, K.A. Evans and D.M. Bailey hypoxaemia and increase the Aa gradient during acute expo- Neurovascular Research Laboratory, University Of Glamorgan, sure to inspiratory hypoxia in healthy humans. Pontypridd, Wales, UK Twenty four healthy male volunteers aged 25 (mean) ± 4 (SD) years old were enrolled in the study after ethical approval and During physiological stress, plasma is shifted to the extravas- informed consent. In a double-blind fashion, subjects were ran- cular space resulting in a concentration of diffusible blood con- domised to stituents. This haemoconcentration is important to most 1) hypoxia for 12 hours (12.9% O2) and a four-hour intravenous haemostasis factors because they are too large to pass through infusion of saline from 4-8 hours, n=11 endothelial pores with plasma. Research has established that 2) hypoxia for 12 hours (12.9% O2) and a four-hour intravenous blood is hyper-coagulable after exercise in normoxia, as seen infusion of LPS from 4-8 hours (total dose of 0.3 ng/kg), n =13 by a shortening of activated partial thromboplastin time (aPTT). Arterial blood samples were obtained and the Aa gradient was However results for prothrombin time (PT) & thrombin time calculated at 0, 4, 9 and 12 hours as FIO2(pAtm – pH2O) - (TT) remain equivocal. In hypoxia, studies have shown both (PaCO2/RQ) + (1-RQ / RQ) - PaO2, where FIO2 denotes the activation & suppression of coagulation. Reasons for these dif- inspired fraction of O2, pAtm is the prevailing atmospheric pres- ferences have been attributed to the use of varying protocols sure, pH2O is the partial pressure of water, PaCO2 is arterial & study populations. Both hypoxia & exercise independently PCO2, PaO2 is arterial PO2, and the respiratory quotient (RQ) contract plasma volume (PV), yet interpretive implications of is assumed to be 0.8. this remain unexplored. The purpose of the present study was Inspiratory hypoxia reduced PaO2 and oxygen saturation and to evaluate & compare the independent effects of hypoxia & induced a hyperventilatory response with decreases in PaCO2 exercise on coagulation times with & without correction of PV. (P < 0.0001 for all, ANOVA), whereas the Aa gradient increased 18 healthy males were recruited & administered 6 hours pas- (P < 0.01, ANOVA). LPS infusion induced systemic neutrocyto- sive exposure to normobaric hypoxia (Fraction of inspired oxy- sis, increased the core temperature (P < 0.01 for both, random gen [FIO2] 12%). After exposure, they performed an incre- mixed model) and potentiated the effect of inspiratory hypoxia mental cycling test to exhaustion. Blood was sampled at three on the Aa gradient (P < 0.01, random mixed model; Figure 1), time points: FIO2 21% [Normoxia rest], after 6 hours exposure but affected neither the severity of arterial hypoxaemia nor the [Hypoxia rest] & post hypoxic exercise [Hypoxia exercise]. It was hyperventilatory response (both NS, random mixed model). then tested for plasma levels of Fibrinogen (FB), PT, TT & aPTT. In conclusion, the present findings indicate that systemic inflam- Comparing all results to normoxia rest, in uncorrected data (PV- mation may augment the effects of acute inspiratory hypoxia ) there were no significant differences in any marker at hypoxia on the pulmonary circulation. rest. However, FB was significantly increased and aPTT significantly shortened at hypoxia exercise. PT and TT remained unchanged. Upon correction for PV (PV+) the changes in FB and aPTT were abrogated & there were significant elongations of PT & TT at hypoxia exercise. FB & aPTT remained unchanged. These data suggest blood coagulation is affected by PV. The question of whether or not to correct for plasma volume remains a clinically important concept that deserves consideration.

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Table 1 The time constant was significantly larger (P<0.05) in the diabetic group compared with the non-diabetic control group at 50% VT (43.9±14.6 s diabetics, 29.3±11.7 s non-diabetic controls) and 80% VT (47±7.8 s diabetics, 35.4±6.3 s non-diabetic ± controls); but not at 50% [peak VO2–VT] (47.7 10.1 s diabetics, 47.4±6.5 s heavy controls). Cardiac output responses during exercise (both at 30 and 220 s) were not different between the two groups. The results confirm that type 2 diabetes mellitus slows the dynamic response of VO2 during light and moderate exercise in females; but that this is probably not related to a slowed cardiac output. Regensteiner JG et al. (1998). J Appl Physiol 85(1) 310-7. Bauer TA et al. (2007). Diabetes Care 30(11) 2880-2885. ± Data table. Data are expressed as means standard deviations. Implications Where applicable, the authors confirm that the experiments in bold signify a significant difference (P < 0.05) compared to baseline data. described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

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C85 Analysis of Timed Up and Go (TUG) test in young and elderly humans and in elderly fallers Oxygen uptake kinetics and cardiac output during cycling C. Smith, J. Bergmann and K. Cook in females with type 2 diabetes mellitus Applied Biomedical Research, King’s College London, London, UK O. Mac Ananey1, J. Malone1, D. O’Shea2, S. Warmington3, S. Green4 and M. Egaña1 The TUG test is widely used in the clinical assessment of postural stability - with some health authorities asking for an annual 1Physiology, Trinity College Dublin, Co Dublin, Ireland, assessment for all patients over 65. The subject has to stand up 2Endocrinology, St Colmcilles Hospital Loughlinstown, Co Dublin, from a chair, walk round an object 3 metres in front of the chair Ireland, 3School of Exercise & Nutrition Sciences, Deakin University, and sit again. Clinically, the test has both high sensitivity and Melbourne, VIC, Australia and 4Physiology, University of Otago, high selectivity with TUG times of greater than 14 seconds being Dunedin, New Zealand associated with a high risk of falling for older adults living in the The dynamic response of oxygen uptake during submaximal community (Shumway-Cook et al., 2000 – but note recent dis- cycle exercise appears to be slowed in subjects with type 2 dia- sent from the Tromso study: Thrane, 2007). Our study com- betes mellitus compared to non-diabetic controls1,2. This pared 34 young subjects (mean 28 SD 10 years) 43 elderly sub- slowed VO2 response may be due to slowed responses of car- jects (72 SD 5) and 20 elderly fallers (75 SD 4, who self-reported diac output, limb blood flow and/or oxygen conductance, an average of 1.8 trips in the previous year). The TUG test was although human data are lacking. completed in a laboratory setting and was measured by CODA This human study examined the effect of type 2 diabetes mel- mpx30 (Charnwood Dynamics, UK) 3D motion analysis. The litus on cardiac output and the dynamic response of VO2 dur- test was undertaken five times by each subject with data aver- ing three intensities of cycling. aged from the last three. Nine middle-aged type 2 diabetic and nine healthy non-dia- TUG times were progressively slower in the elderly fallers (12.3s betic females were recruited for the study. Initially, the venti- SEM 1.0) compared to the elderly (9.2s SE 0.3) and the young latory threshold (VT) and peak VO2 were determined using a subjects (7.7s SEM 0.2). These changes were related mainly maximal graded cycle test. Then subjects completed a series to changes in the stride length rather than cadence. A regres- of constant-load exercise bouts (7 min long) on separate days, sion of TUG times against the ABC balance confidence scale during which the dynamic response of VO2 was assessed three showed an equal increase in TUG times with decreasing confi- times, and cardiac output once, at each of three intensities dence with both the elderly and elderly fallers but accounting (50%VT, 80%VT & 50%[peakVO2–VT]). Cardiac output was for only a minor part of variance of TUG times (R 0.27 and 0.34 recorded by a closed circuit inert gas rebreathing technique at respectively). Young subjects showed only very small variance rest and at 30th & 220th sec of the bout. Dynamic response in confidence. parameters (e.g., time constant) of the VO2 response were Regression of the sit to stand transition (S2S) at the start of the determined by fitting a monoexponential function to the VO2 TUG test showed a strong and roughly equal contribution to data. Ethical approval was granted by the Trinity College Dublin, TUG times in all groups (R 0.73 fallers, 0.55 elderly, 0.52 young), Faculty Research Ethics Committee. Results were analyzed using S2S being about 10% of the TUG time. However elderly fallers a one-way ANOVA and are shown as mean±SD. were distinguished by choosing a wider stance during standing (18.1cm SEM 1.4 fallers, 14.5cm SEM 0.5 elderly, 14.3cm

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SEM 0.8 young). It is thus clear that specific aspects of the TUG to highlight the diameter of each fibre. The areas of 450 fibres test help distinguish fallers whilst other aspects relate more to were measured form each sphincter muscle. The cross sectional age or to fear of falling. Analysis of the turn in the TUG was not area of fibres in the EAS was on average 376 ± 5 μm2 while in possible using the CODA system as individual markers are lost the urethra it was 187 ± 4 μm2 (P<0.0001). The smaller specific from vision during the turn. We have thus subsequently moved force and fibre size of the urethra coupled with its fatigue resist- to measurements based on inertial units (Bergmann et al., ance indicates that it has a higher oxidative capacity than the 2009) which allow analysis of the full range of the TUG test. anal sphincter. This was confirmed by succinate dehydrogenase Bergman J, Alexiou C & Smith C (2009) J. Physiol Abs KCL (SDH) histochemistry. In conclusion, the voluntary muscle of Shumway-Cook A, Brauer S & Woollacott M (2000) Phys Ther. 80, 896 urinary continence in the rat is composed of small, fatigue resistant muscle fibres in comparison to the more powerful but fati- Thrane G (2007) BMC Geriatr. 12,1 gable voluntary sphincter of the anal canal. GKT Charitable Foundation for financial support. This work was supported by the Health Research Board and Uni- Where applicable, the authors confirm that the experiments versity College Dublin. described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

C87 C88 Fibre size and oxidative capacity of the external anal and Restoration of bile bilirubin excretion rate after cessation of urethral sphincters obstructive cholestasis is suppressed by hyperprolactinemia M. Buffini1, C. O’Herlihy1,2, R. O’Connell1,3 and J. Jones1 in rats 1School of Medicine and Medical Science, UCD, Dublin, Ireland, N. Kushnareva1 and O.V. Smirnova2 2Obstetrics and Gynaecology, National Maternity Hospital, Holles 1Biological Department, Moscow State University, Moscow, Russia Street, Dublin, Ireland and 3Professorial Surgical Unit, St. Vincent’s and 2BiologicalDepartment,MoscowStateUniversity,Moscow,Russia University Hospital, Dublin, Ireland Background: Key liver function is maintenance of bile flow and The external urethral (EUS) and anal (EAS) sphincters are two bilirubin excretion rate. Some liver pathologies (e.c., cirrhosis) striated muscles which play a fundamental role in the mainte- are associated with alteration of bile flow and hyperpro- nance of urinary and faecal continence. Despite their common lactinemia. Female prevalence of obstructive cholestasis (OC) embryological origin and synchronised electrical activity in vivo, development was assumed is related to high prolactin (Prl) and these muscles display different contractile properties. The aim its hepatic receptors level in women. of this study was to explain the findings from in vitro functional Aims:toinvestigatePrlinfluenceonliverbilirubinexcretoryactiv- experiments by examining some of the structural characteris- ity using rat model of hyperprolactinemia combined with OC. tics of the EAS and EUS. Methods: Obstructive cholestasis was induced by common bile The fatigue indices of sphincters from eight female Wistar rats duct ligation and hyperprolactinemia by donor pituitary trans- (200-250g) were measured. The EAS and EUS were mounted plantation under kidney capsule of recipient. Surgical proce- as ring preparations in tissue baths containing Tyrode’s solu- dures were conducted under diethyl ether anesthesia. Biliru- ο tion maintained at 37 C and bubbled with 100% O2. Tubocu- bin concentration in bile, blood, and urine, bile flow and bilirubin rarine (10-4M) was added to the bath solution to ensure nerve excretion rates in early (3 h) period of decompression (EPD) independent muscle contraction. The fatigue stimulation pro- were tested. tocol consisted of 50 200ms trains at 50Hz which were 4 sec- Results: OC induced elevation of bile bilirubin concentration onds apart. Data are expressed as mean ± SEM and statisti- in females and its depletion in males and elevation of blood and cally analysed with a students t-test. urine bilirubin level in both sex. Bile flow rate was elevated in The EAS was much more susceptible to fatigue than the EUS. EPD as comparied with normal conditions with male predom- At the end of the fatigue protocol the final contraction of the inance so that bilirubin excretion rate was the same in both sex. EAS had fatigued to 41.86 ± 2.73% of the initial contractile force. OC combined with hyperprolactinemia (4 weeks) resulted in Conversely, the contractions of the EUS were relatively unaf- sharp decreasing of bile bilirubin level and appearance of “white fected by this particular stimulation protocol, the final con- bile” in females and additional decreasing in males. Bilirubin traction of the EUS was 94.93 ±3.09% of the first, p<0.001 (n=8). concentration in females was firstly elevated in blood and urine The absolute force produced by the muscle per cross sectional and then decreased in bile, in males there were no evident alter- area or the specific force was measured. At 50Hz the specific ations of blood and urine bilirubin concentration. Depending force of the EAS was twice that of the EUS, 4.7 ± 0.6 mN.mm-2 on hyperprolactinemia duration restoration of bile flow after vs. 2.4 ± 0.3 mN.mm-2, p=0.014. This suggested that the EAS bile duct decompression was firstly suppressed and then not is composed of larger muscle fibres than the EUS. In order to restored in females and essentially suppressed in males. Hyper- determine the average area of the striated muscle fibres in the prolactinemia without bile duct obstruction had no marked EAS and EUS, the sphincters from three animals were oriented influence on investigated parameters. so that the muscle fibres were cut transversely. These sections Conclusions: Hyperprolactinemia deteriorates liver functions were then incubated with antibodies against laminin (1:500) suppressing bile flow restoration after OC cessation, however

51P Oral Communications due to decreasing bilirubin entry into bile it may participate in p<0.001). The present data suggest that CCK participates in redistribution of bilirubin pool and promote kidney bilirubin the hypophagic effect during endotoxemia through CCK-1 excretion. receptor. This effect is likely to be mediated by an activation of CRF neurons in the PVN but not α-MSH in the ARC. Supported by RFBR (grant 07-04-00319-a) Technical assistance: Maria V.A. dos Santos, Marina Holanda, Where applicable, the authors confirm that the experiments Milene Mantovani Lopes e Lilian E.C.M. da Silva. described here conform with The Physiological Society ethical requirements. Financial support: FAPESP, CAPES and CNPQ. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C89

Cholecystokinin participates in the LPS-induced hypophagia through corticotrophin-releasing-factor neuron activation C90 R.C. Rorato, J. Antunes-Rodrigues and L.L. Elias Desensitization of hypophagic effects in the endotoxin Physiology, School of Medicine of Ribeirao Preto, University of Sao tolerance is associated with a decrease of STAT-3 Paulo, Ribeirao Preto, Sao Paulo, Brazil phosphorylation not mediated by changes in SOCS-3 mRNA expression in the arcuate nucleus Administration of lipopolyssacharide (LPS) decreases food intake. However, the mechanisms underling this hypophagic B.D. Borges1, R.C. Rorato1, L.E. Silva1, M. Castro2, J. Antunes- effect are not well established. LPS increases peripheral pro- Rodrigues1 and L.L. Elias1 duction of cytokines that in turn induce cholecystokinin (CCK) 1Physiology,SchoolofMedicineofRibeiraoPreto-UniversityofSao secretion, a satiety-related peptide. CCK induces an activa- Paulo, Ribeirao Preto, Sao Paulo, Brazil and 2Internal Medicine, tion of brainstem neurons that show reciprocal projections with School of Medicine of RibeiraoPreto, Ribeirao Preto, Sao Paulo, Brazil hypothalamic neurons. Corticotrophin-releasing-factor (CRF) and alpha-melanocyte stimulating hormone (α-MSH) are neu- Desensitization of hormone response and hypophagic effect ropeptides with anorexigenic properties. This study aimed to to repeated exposure to endotoxin has been described. Leptin, investigate the effects of LPS on food intake, CRF and α-MSH produced by adipose tissue, reduces food intake and boosts neuron activation in rats pretreated with devazepide (CCK-1 energy expenditure via activation of JAK/STAT signaling path- receptor antagonist). Adult male Wistar rats (220–300g) were way in hypothalamic neurons, via a phosphorylation of STAT- singly housed in metabolic cages with free access to chow 3, which in turn stimulates SOCS-3 (an intracellular molecule offered in a metal container, to verify food consumption. On that triggers a negative feedback loop) gene expression in the the day of the experiment the chow were removed and arcuate nucleus (ARC). We evaluate the effects of exogenous rats(n=7-11) were weighed and pretreated with devazepide leptin administration in rats under single or repeated exposure (1mg/Kg, ip) or vehicle (0.5/0.5/9, tween80/DMSO/saline0.9%) to LPS on food intake and p-STAT-3 expression in the ARC. Male 30 minutes before LPS (100μg/Kg, ip) or saline (0.1ml/100g Wistar rats (250-300g) received single or repeated injections bw, ip) injection. Thereafter, food was offered for 2h (18:00- of LPS (100μg/Kg ip) or saline (0.15M NaCl, 1ml/Kg), between 20:00h). Another set of rats (n=5-7) was pretreated with 0400h-0500 PM, during 6 days. Two hours after the last injec- devazepide or vehicle followed by LPS or saline injection and tion,rats received intracerebroventricular (icv) injection of lep- 4h after they were anaesthetized with tribromoethanol tin (10μg) or vehicle (saline). The anaesthetic for cannulation (1ml/100g bw, ip) and transcardially perfused with s 4% was tribromoethanol (1ml/100g bw, i.p.). Food intake was paraformaldehyde for brain collection. In rats saline injected, determined during 24h (n=8). Another set of rats, subjected to devazepide (3.85±0.9 g/100g; p<0.01) increased food intake the same protocol, were anaesthetized (as above) and tran- compared to group pretreated with vehicle (2.89±0.4 g/100g). scardially perfused with 4% formaldehyde. After 20 minutes Food intake was decreased after LPS in vehicle (1.1±0.6 g/100g; the brains were collected for p-STAT-3 immunolabeling by p<0.001) pretreated animals, which was reversed by devazepide immunohistochemistry (n=6). A third set of rats were treated (2.2±0.9; p=0.06) pretreatment. Devazepide and vehicle pre- with single or repeated LPS and two hours after the brains were treatement in saline rats resulted in similar immunoreactivity collected by decapitation for determination of SOCS-3 mRNA to Fos/α-MSH (devazepide; 0.5±0.5 and vehicle 0.7±0.9) in the expression by real time PCR (n=7). ANOVA one or two way, fol- ARC and Fos/CRF in the medial (PaMP; devazepide 4.2±2.4 and lowed by Student-Newman-Keuls, was used to analyze the data vehicle 4.3±1.1) and posterior parvocellular subdivisions of the (mean±SEM). Single LPS decreased food intake and body PVN (PaPo; devazepide 3.2±1.6 and vehicle 3.8±1.4). In the weight(p<0.05), compared to control rats. In turn, repeated group pretreated with vehicle, LPS treatment increased Fos/CRF LPS did not change the food intake and body weight.Icv leptin double labeled neurons in the PaMP (22.9±6.4; p<0.001) and reduced(p<0.05) the food intake and body weight in 6 saline PaPo (9.1±1.6; p<0.001). No change in the Fos/α-MSH double treated rats, but no changes in these parameters were observed labeled neurons in the ARC was observed after LPS stimulus in animals under single or repeated LPS. We observed an (devazepide 0.3±0.7 and vehicle 0.3±0.7). Pretreatment with enhancement(p<0.05) of p-STAT-3 expressing neurons in the devazepide decreased the LPS-induced Fos/CRF double label- ARC after a single LPS followed by vehicle treatment compared ing in the PaMP (9.5±3.6; p<0.001) and PaPo (4.8±5.5; to control (64.5 ± 19.4 vs 31.1 ± 19.8), with no changes in p-

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STAT-3 in 6 LPS group(17.8 ± 16.9). Leptin treatment increased sion in the ARC and compact zone of the dorsomedial nucleus p-STAT3 positive neurons in the ARC of 6 saline rats(62.1 ± 25 in obese high-lard fed rats was lower than in low-lard, and high- vs 5.3 ± 30.6)compared to vehicle. There was no effect of lep- PUFA fed rats. The GALP expression increased by about 50% and tin on p-STAT-3 expression in the 6 LPS group. Compared to 40% in the ARC in high-sunflower oil and high-fish oil groups, controls, there was an increase in the SOCS-3 mRNA expres- respectively vs. rats fed the corresponding low-fat diet. POMC sion in the ARC (p<0.05)after a single LPS, with no changes in expression in the ARC was increased in both high-PUFA fed rats the SOCS-3 mRNA in the repeated LPS-treated rats. In conclu- vs. that fed the corresponding low-PUFA and high-lard diets. sion, these data demonstrated a desensitization of hypophagic The POMC expression in the whole hypothalamus was effect in response to repeated exposure to endotoxin. This increased, and the pre-orexin and MCH mRNA transcript levels effect is associated with an insensitivity to leptin in activating in the LHA were decreased exclusively in high-fish oil fed rats. STAT-3, however SOCS-3 is unlikely to underlie this resistance The expression of MC4R in the PVN was lower in high-lard than to leptin signaling in the ARC in the endotoxin tolerance. in low-lard fed rats. The high-fat fed rats had a significantly increased leptin, insulin and glucose levels independently of Maria Valci dos Santos, Marina Holanda, Milene Mantovani, Fer- the kind of dietary fat. nando C. do Amaral, FAPESP, CNPq. Summarizing, high ingestion of fish oil originate PUFA favoured Where applicable, the authors confirm that the experiments anorexigenic POMC expression in the hypothalamus and down- described here conform with The Physiological Society ethical regulated orexigenic peptides in the LHA. The high-saturated requirements. feeding failed to up-regulate POMC expression and down-regulated MC4R expression in the PVN. Our study indicates that hyperphagia caused by diet high in saturated fat may impair melanocortin transmission in the hypothalamus. C91 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Diet high in saturated fat impairs the hypothalamic requirements. neuropeptide and melanocortin-4 receptor expression B. Dziedzic1, J. Szemraj2, J. Bartkowiak3 and A. Walczewska1 1Department of Cell-to-Cell Communication, Medical University of C92 Lodz, Lodz, Poland, 2Department of Molecular Neurochemistry, Medical University of Lodz, Lodz, Poland and 3Department of Effect of leptin on the LH secretion depends on estrogen Medical Biochemistry, Medical University of Lodz, Lodz, Poland modulation The hypothalamic neuropeptide neurons play the pivotal role B. Del Bianco-Borges and C.R. Franci in energy balance. The arcuate nucleus (ARC) neurons respond Physiology, Medicine School of Ribeirao Preto - University of Sao to peripheral hormones and nutrients through neuropeptide Paulo, Ribeirao Preto, SP, Brazil Y/agouti-related peptide (NPY/AgRP) and pro-opiome- lanocortin/cocaine-amphethamine regulated transcript There is an interaction between the reproductive axis and the (POMC/CART). These neurons project to the paraventricular energy homeostasis. One possible mediator of this interaction nucleus (PVN) and lateral area of the hypothalamus (LHA) where is the leptin, a hormone that acts in the control of metabolism, activate the second order of neurons producing e.g. orexins feed behavior and reproduction. Homozygous animals for the and melanin-concentrating hormone (MCH). The aim of this gene “ob” (ob/ob) do not produce leptin, and present obesity study was to investigate the effect of feeding with different fats and infertility. These effects are reversed after systemic admin- on neuropeptide expression in the ARC and LHA, and istration of leptin, but the gonadotropin-releasing hormone melanocortin recepror-4 (MC4R) in the PVN. (GnRH) neurons not express the leptin receptor (Ob-R) and a Three groups of rats were fed six weeks a low-fat diet (10% trasynaptic action of leptin on GnRH neurons is more likely. energy from fat), next three a high-fat diet (40% energy from On the other hand, the estrogen may modulate the secretion fat) prepared with the same fat; lard - source of saturated fatty of leptin by adipocytes and the expression of the Ob-R. The aim acids, sunflower oil - source of n-6 polyunsaturated fatty acids of this work was verify the effect of leptin on the luteinizing hor- (PUFA), or fish oil rich in n-3 PUFA (8 rats/group). Throughout mone (LH) secretion under estrogen modulation in female rats. the experiment body weight and food intake were monitored. Methods: Wistar female rats with regular estrous cycles were Before decapitation and brain removal rats were i.v. anaes- anaesthetized (tribromoethanol, 250mg/kg body wt, i.p.) and thetized with ketamine (20 mg/kg) plus xylazine (10 mg/kg) received a cannula in the 3V. After two regular cycles estrous and blood was drawn by heart puncture. Plasma level of glu- the rats received estrogen antagonist, tamoxifen subcutaneous cose, leptin and insulin were measured. The ARC, PVN and LHA (s.c.) (TMX, 3mg/rat), in the metestrous and diestrous. In the were punctured from frozen brain sections. NPY, galanin-like day of proestrous, these animals received rat leptin intracere- peptide (GALP), POMC, pre-orexin, MCH, MC4R expression was broventricular (i.c.v.) (0,3;1;3μg/μl) and were killed by decap- measured by RT-PCR. The whole hypothalamus was assayed for itation at 5:00 pm and the blood was collected . Results: Our NPY and POMC expression by in situ hybridization (4 results showed that the injection of 3ug of leptin in the animals rats/group). without TMX amplified the plasma LH concentration on the The lard diets fed rats gained the highest body weight and ate proestrous afternoon (F(3,82) = 2,64 p<0,05; Duncan p<0,05; the most energy vs. rats fed the PUFA diets. The NPY expres- vehicle: 28,66 +/- 7,13 ng/ml; 3μg leptin: 83,90 +/- 27,38

53P Oral Communications ng/ml). Furthermore, TMX blocked the LH secretion in the after- GKA50 addition. Glucokinase is a critical enzyme in beta cell noon of proestrous (F(1,82) = 39,28 p<0,05; Duncan p<0,05; physiology and its activity has important implications for vehicle: 28,66 +/- 7,13 ng/ml; TMX: 4,74 +/- 0,5 ng/ml) the injec- metabolism and insulin secretion. However an increase in gluco- tion of leptin did not restore the plasma LH concentration in kinase activation does not alter DNA fragmentation responses the animals treated with TMX. Conclusion: Our results indicate or insulin secretion after incubation in either glucolipotoxic or that the central action of leptin on LH secretion depends on pro-inflammatory cytokine conditions. However our results estrogen modulation. support the hypothesis that the detrimental effects of cytokines or glucolipotoxicity occurs via a post-glycolytic event. Where applicable, the authors confirm that the experiments Danial et al. Dual role of proapoptotis BAD in insulin secretion and beta described here conform with The Physiological Society ethical cell survival. Nature Medicine 14, 144-153 (2008). requirements. Park et al. Inhibitory effects of Streptozotocin, Tumor Necrosis Factor- α and Interleukin-1β on Glucokinase activity in Pancreatic Islets and Gene Expression of GLUT2 and Glucokinase. Biochemistry 362, 217- 224 (1999). C93 Kim et al. Exposure to chronic high glucose induces beta cell apoptosis through decreased interaction of glucokinase with mitochondria. Effects of a glucokinase activator on glucolipotoxic and Diabetes (2005). cytokine-induced beta cell dysfunction and cell integrity Where applicable, the authors confirm that the experiments N. Mullooly and P. Newsholme described here conform with The Physiological Society ethical University College Dublin, Dublin, Ireland requirements. A pathological feature of both type 1 and type 2 diabetes is loss of pancreatic beta cell mass through increased apoptosis. The molecular mechanisms mediating increased apoptosis are C94 unknown but new evidence links the beta cell glucose sensor “glucokinase” to beta cell survival. The proapoptotic protein b-secretase modifies insulin signalling and GLUT4 BAD is localised with glucokinase in beta cells potentially link- translocation in skeletal muscle cell lines ing glucose homeostasis with beta cell functional integrity1. Exposure of islets to proinflammatory cytokines or glucolipo- P.J. Meakin, K.A. Bannon, D.F. Bestow and M.L. Ashford toxic conditions activate beta cell apoptotic pathways with con- Biomedical Research Institute, University of Dundee, Dundee, UK commitent decreases in beta cell insulin secretory function. Glucokinase has been implicated as a mediator of beta cell dys- β-secretase (β-site APP Cleaving Enzyme 1 (BACE1)) is a key function in both cytokine and glucolipotoxic conditions2,3. player in the development and progression of Alzheimer’s dis- Molecular compounds which bind to an allosteric activation ease (AD). Sequential cleavage of amyloid precursor protein by site on glucokinase have recently been developed. GKA50 is a β- and γ-secretase produces β-amyloid peptides, the major com- potent activator of glucokinase which increases enzyme activ- ponent of plaques in AD patient brains. BACE1 has been vali- ity with enhanced insulin secretion in vitro and in vivo. Given dated as a therapeutic target for AD as BACE1 knockout (KO) the probable role of glucokinase in the regulation of beta cell mice have low β-amyloid levels. BACE1 KO mice also exhibit a survival/apoptosis we have used GKA50 to elucidate the effects lower body weight and better glucose disposal than their wild of increased glucokinase activation on glucolipotoxic and type littermates. Using C2C12 (mouse) and L6 (rat) cultured cytokine induced beta cell integrity and insulin secretion (using myoblasts we show that pharmacological inhibition of BACE1 the rat-derived beta cell line BRIN-BD11). Apoptosis was enhances insulin signalling, as measured by protein kinase B induced by culturing cells in the presence of a lethal proin- phosphorylation (p-PKB). Treatment of C2C12 cells with 5 nM flammatory cytokine cocktail (IFNγ, IL-1β, TNFα) or chronic high insulin increased p-PKB levels by 6.07 ± 1.27 fold (P < 0.001, n glucose and lipid levels (25mM glucose,100μM palmitate) for = 10), compared to un-stimulated cells. A 24-hour pre-treat- 24hr with or without GKA50. A thorough analysis of cell viabil- ment with 250 nM BACE1 inhibitor (1) enhanced insulin-stim- ity and apoptosis was performed. Cell apoptosis was examined ulated p-PKB levels, 10.27 ± 1.52 fold (P < 0.001, unpaired Stu- by DNA fragmentation, LDH release and Trypan blue staining. dent’s t-test, mean ± SEM; n = 10) compared to control cells. Cell viability was determined by WST and neutral red assays Comparison of insulin-stimulated p-PKB levels in the presence and mitochondrial membrane potential. Chronic and acute and absence of the BACE1 inhibitor showed that BACE1 inhibi- stimulated insulin secretion was measured by ELISA. Significant tion significantly enhanced insulin stimulated p-PKB levels (P < cell death and impairment of insulin secretion occurred in pro- 0.05). L6 myoblasts were more insulin sensitive, with 0.3 nM inflammatory or glucolipotoxic experimental conditions insulin increasing p-PKB levels by 3.18 ± 0.55 fold (P < 0.01, n (p<0.05). When viability was examined after exposure to either = 4) compared to control cells. L6 myoblasts pre-treated with cytokines or glucolipotoxic conditions those cells treated with BACE1 inhibitor increased p-PKB by 5.15 ± 0.79 fold in response GKA50 were protected from loss of membrane integrity as cells to insulin, (P < 0.001 versus control; n = 4) and a significant treated with GKA50 had a significantly reduced level of LDH enhancement in insulin sensitivity (P < 0.05). In cultured L6- release following incubation in glucolipotoxic conditions GLUT4myc cells, treatment with BACE1 inhibitor per se (p<0.05). However, DNA fragmentation which was enhanced increased translocation of the glucose transporter (GLUT4; 1.53 by in pro-inlammatory cytokine or glucolipotoxic conditions, ± 0.08 fold; P < 0.001, n = 5). Insulin (100 nM) increased GLUT4 or suppressed levels of insulin secretion, were not altered by translocation by 1.40 ± 0.11 fold (P < 0.01, n = 5) in untreated

54P Oral Communications cells and by 1.82 ± 0.20 fold (P < 0.05, n = 5) in cells pre-treated suppressed M1CCD cells, grown to confluency on semi-per- with BACE1 inhibitor. Thus, BACE1 inhibition increases GLUT4 meable supports. A detectable rise in ITE wasobservedinWT translocation supporting the notion that BACE1 reduction cells within 2h of aldosterone treatment, an effect which was enhances glucose uptake. However, at least part of this effect maximal after 24h. The induction of ITE by aldosterone was inhib- in these muscle cells appears insulin-independent. Raised 5’ ited in PKD1 suppressed cells. Using immunocytochemistry and AMP activated protein kinase (AMPK) activity increases GLUT4 laser scanning confocal microscopy, we observed a stark translocation independently of insulin (2). Treatment of C2C12 increase in ENaC alpha expression in WT cells treated with aldos- and L6 cells with BACE1 inhibitor increased AMPK phosphoryl- terone for 24h, an effect which was absent in the PKD1 sup- ation (1.66 ± 0.43 fold; P = 0.06, n = 8 and 1.72 ± 0.39 fold; P < pressed cells. Moreover, using a specific plasma membrane 0.05, n = 11 respectively) and phosphorylation of its substrate, marker, we observed an aldosterone-mediated induction of api- acetyl CoA carboxylase (ACC; 1.72 ± 0.17 fold; P < 0.01, n = 8 cal membrane insertion of the constitutively expressed ENaC and 1.33 ± 0.21 fold; P = 0.06, n = 11 respectively), indicative beta subunit in WT cells. Aldosterone treatment failed to affect of enhanced AMPK activity. Thus, we propose that skeletal mus- the subcellular localization of ENaC beta in PKD1 suppressed cle BACE1 levels modify glucose uptake in an insulin and AMPK cells. Overall, PKD1 plays a central role in the aldosterone-medi- dependent manner. ated regulation of ENaC activity, through both transcriptional Stachel et al. (2004) J. Med. Chem, 47, 6447-6450. control and subcellular trafficking. Nakano et al. (2006) Metabolism, 55, 300-8. 1. McEneaney, V., B.J. Harvey, and W. Thomas, Aldosterone rapidly activates protein kinase D via a mineralocorticoid receptor/EGFR Supported by GSK, BBSRC, Nuffield Foundation and the Well- trans-activation pathway in the M1 kidney CCD cell line. J Steroid come Trust. We acknowledge A. Klip for her kind donation of Biochem Mol Biol, 2007. 107(3-5): p. 180-90. the L6-GLUT4myc cells. 2. Liljedahl, M., et al., Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network. Where applicable, the authors confirm that the experiments Cell, 2001. 104(3): p. 409-20. described here conform with The Physiological Society ethical requirements. 3. Verrey, F., Transcriptional control of sodium transport in tight epithelial by adrenal steroids. J Membr Biol, 1995. 144(2): p. 93-110.

Where applicable, the authors confirm that the experiments C95 described here conform with The Physiological Society ethical requirements. Protein Kinase D Modulates Aldosterone-induced ENaC Activity in Renal Cortical Collecting Duct Cells through the Regulation of Subcellular Trafficking and MR-dependent Gene Expression C96 R. Dooley, V. McEneaney, B.J. Harvey and W. Thomas In-utero nutritional programming of kidney development Molecular Medicine, Royal College of Surgeons in Ireland, Dublin, and its long term impact on renal oxidative stress following Ireland juvenile obesity Aldosterone treatment stimulates the phosphorylation and H.P. Fainberg1, D.S. Gardner2, K.D. Sinclair3, H. Budge1 and activation of Protein Kinase D 1 (PKD1), in murine cortical col- M.E. Symonds1 lecting duct cells (M1CCD), through the transactivation of the 1Centre for Reproduction and Early Life, Institute of Clinical Science, epidermal growth factor receptor (EGFR) [1]. PKD1 belongs to School of Clinical Sciences, University of Nottingham, Nottingham, a family of serine/threonine kinases known to be important UK, 2School of Veterinary Medicine and Science, University of modulators of subcellular trafficking, through the regulation Nottingham, Nottingham, UK and 3School of Biosciences, University of vesicle fission from the Golgi [2]. The epithelial sodium chan- of Nottingham, Nottingham, UK nel (ENaC) is a major effector of aldosterone action in the kidney and plays a crucial role in the maintenance of whole body Maternal nutrient restriction, coincident with early fetal kidney sodium homeostasis. Active ENaC is believed to be a het- development, can increase glucocorticoid action in the new- erotrimer composed of one each of the alpha, beta and gamma born kidney1. Fetal adaptations in the kidney may also deter- subunits. ENaC activity is dynamically regulated by aldosterone mine the long term consequences following juvenile obesity through multiple mechanisms. The nuclear mineralocorticoid and the onset of hypertension. One important mechanism receptor (MR) in complex with aldosterone behaves as a ligand- implicated in both obesity and renal disease is enhanced oxida- dependent transcription factor which stimulates the tissue- tive stress, leading to glomerulosclerosis. Two key regulators specific upregulation of ENaC subunit expression. In the distal of this process are the angiogenic hypoxia inducible factor (HIF)- nephron, the ENaC alpha subunit is under the transcriptional 1α and the pro-apoptotic receptor Fas. The present study, there- control of ligand-bound MR, while beta and gamma are con- fore, examined how the early in-utero diet results in renal adap- stitutively expressed [3]. tations to oxidative stress induced by juvenile obesity. Using M1CCD cells where endogenous PKD1 was stably knocked Pregnant sheep (n=26) were randomly assigned to a normal (7 down, we examined the role PKD1 plays in the aldosterone- MJ/day) or nutrient restricted diet (NR, 3.5 MJ/day), from 30- mediated regulation of ENaC activity via transcription and/or 80 days of gestation (term = 147 days) and fed to requirements trafficking of pre-expressed ENaC subunits, over an extended at all other times. After weaning, the NR (NR-O, n=11) and obese period of aldosterone treatment. The amiloride-sensitive, trans- (O, n=7) offspring were reared in an obesogenic environment epithelial current (ITE) was measured in wild-type (WT) and PKD1 to promote fat deposition. The lean group (L, n=8) remained

55P Oral Communications out to pasture. At one year of age, all sheep underwent meas- diovascular response to stress, five regions of SBP/PI tracings urements of plasma cysteine concentration and were then were defined: baseline (BASELINE), air-jet stress (S), immedi- humanely euthanased with kidneys sampled for renal malon- ate recovery (IMMED. REC), and recovery period (REC). Con- dialdehyde (MDA) concentrations and real-time gene expres- comitant SBP increases and PI shortenings during REC were sion of the Fas and HIF-1α genes. analyzed separately (DEL RES). Obesity raised the total plasma cysteine (L 12.8±1.1; O 18.3±1; The sequence method revealed that in WR during S and IMMED. NR-O18±1.6(p<0.05))andintra-renal(i.e.MDA)oxidativestress REC, sBRR sensitivity was maintained, baroreflex effectiveness in both obese groups. In addition, although gene expression of (BEI) decreased, its operating range (OR) enlarged and the SBP HIF-1α also increased with obesity, this response was amplified set point was shifted towards higher values. In REC parameters in offspring born to NR mothers (L 1±0.285; O 1.964±0.552; NR- regained baseline values. DEL RES occurred associated with the O5.353±1.0252-(ΔCT)(p<0.05)),whereasmRNAabundancefor re-setting of sBRR (Baji ´c D et al., 2009). During S in BHR the Faswasreduced(NR-O1.89±1.56;O4.68±1.722-(ΔCT)(p<0.05)). sequence method revealed that sBRR sensitivity was preserved; Our study suggests that abnormal patterns of renal adaptation BEI showed the tendency to increase (0.72±0.08 vs. 0.79±0.09, are induced by juvenile obesity in NR offspring, with persistent paired t-test, p=0.16), the set point was shifted towards higher changesinmarkersofoxidation.Wehave,therefore,shown,for SBP values (139.7±11.3 mmHg vs. 141.3±12.0 mmHg, paired thefirsttimeinthismodeloffetalprogramming,thatincreased t-test, p=0.009), while the OR increased (523.8±340.4 vs. systemicandrenaloxidativestressaltersthegeneregulationof 1304.4±550.2, paired t-test, p=0.02). During IMMED. REC BEI angiogenic and pro-apoptotic genes such as HIF-1α and Fas. maintained the increasing tendency with respect to BASELINE These differential adaptations may delay the onset of glomeru- (0.72±0.08 vs. 0.78±0.1, paired t-test, p=0.19), SBP set point losclerosis2 without necessarily improving vascular function. decreased vs. S (139.7±11.3mmHg vs. 141.3±11.9mmHg, 1. Whorwood CB, et al. Endocrinology 2001;142(7):2854-64. respectively; paired t-test, p=0.008) and returned to the BASE- LINE level. In REC BEI and OR returned to the BASELINE values. 2. Williams PJ, et al. Kidney Int 2007;72(3):279-89. In DEL RES SBP set point and OR were at the BASELINE level. In WR air-jet stress modulates sBRR differentially during the time This study was supported by the British Heart Foundation course of stress and recovery affecting linear and nonlinear BP Where applicable, the authors confirm that the experiments and PI relationship. BHR manifested different cardiovascular described here conform with The Physiological Society ethical response to stress with respect to WR. It shows different barore- requirements. flex engagement, faster recovery of SBP set-point and less prominent form of DEL RES. The baroreflex dominance in stress response can be compared by dominant baroreflex regulatory response to acoustic stimulation (Silvani et al., 2001). Baji ´c D et al. (2009). Stress. The International Journal on the Biology C97 of Stress. In press. Silvani A et al. (2003). Sleep. 26(2):201-5. Cardiovascular Response to Air-jet Stress Differs in Borderline Hypertensive Rats with Respect to Wistar Rats-Sequence Where applicable, the authors confirm that the experiments Analysis of Spontaneous Baroreflex described here conform with The Physiological Society ethical requirements. T. Bojic1, S. Stojicic2, D. Bajic3, D. Murphy4, J.F.R. Paton4 and N. Japundzic-Zigon1

1Institute of Pharmacology, Clinical Pharmacology and Toxicology, C98 School of Medicine, Belgrade, Serbia, 2School of Dentistry, Belgrade, 3 4 Serbia, University of Novi Sad, Novi Sad, Serbia and University Role of VentroMedian Medullary Raphe neurons in of Bristol, Bristol, UK controlling heart rate variability in free behaving rats Cardiovascular morbidity and mortality can be triggered by M. Cerri, G. Zamboni, D. Tupone, D. Dentico, M. Luppi, emotional stress: it can cause development of arterial hyper- D. Martelli, E. Perez and R. Amici tension. Development of hypertension is also dependent on Department of Human and General Physiology, University of genetic background. Borderline hypertensive rats (BHR) with Bologna, Bologna, Italy respect to Wistar rats (WR) develop larger cardiovascular response to different stress models. Analysis of spontaneous A role of VentroMedian Medullary Raphe (VMMR) neurons in fluctuations of systolic blood pressure (SBP) and pulse interval cardiovascular (CV) control is suggested by studies on anaes- (PI) (spontaneous baroreceptor reflex, sBRR) permits an insight thetized animals. VMMR neuron disinhibition increased heart on baroreflex and central components of stress response. Effect rate, arterial pressure and sympathetic outflow to the heart (1). of air-jet stress on cardiovascular mechanisms was evaluated However, it is thought that VMMR neurons do not maintain the in freely moving WR (nw=6) and BHR (nbhr=6). Experiments in tonic sympathetic CV drive, due to the lack of major CV effects this study conformed to ECC Directive (86/609/EEC). Under after VMMR inhibition (2). Since general anaesthesia affects general anesthesia (halothane 4% induction, 2% via mask), rats sympathetic outflow and does not permit the measurement of were equipped by an arterial catheter and exposed to 2min heart rate variability (HRV), in the present study the role of stress. At the end of experiment rats were euthanized (thiopen- VMMR neurons in CV control has been assessed in free behav- tone sodium, 200mg/kg, i.p.). For a time course analysis of car- ing rats.

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activation of the autonomic nervous system to maintain car- Six male CD rats (Charles River), kept under a 12h:12h light- diac output. We reported (Flanagan et al., 2008) in anaes- dark cycle at 24±0.5∞C ambient temperature were used. Under thetised rats that an acute volume expansion decreased renal general anaesthesia (Diazepam, 5mg/Kg intramuscular; Keta- sympathetic nerve activity (RSNA) and that this reflex was mine-HCl, 100 mg/Kg, intraperitoneal), animals were implanted absent in rats with heart hypertrophy after isopre- with electrodes for EEG and EKG recording, a thermistor to naline/caffeine or thyroxine administration. At the kidney, measure hypothalamic temperature (Thy), a microinjection this raised level of RSNA would cause fluid retention exacer- guide cannula to target VMMR. One week after surgery, rats bating the load on the heart. This study examined whether were randomly microinjected, on different days, with 100nl of in conscious rats the ability to excrete a volume load was the following: i) GABA-A agonist muscimol (1mM); ii) GABA-A altered in any way in heart hypertrophy. A further objective antagonist bicuculline (1mM); iii) 0.9% saline. Preliminary analy- was to examine how these responses were altered following sis of data from muscimol injection was carried out during wake denervation of the kidneys. in two rats. HRV was analyzed within both the time (R-R mean; Two groups of Wistar rats (n=6) were used, one intact and the R-R standard deviation (STD-RR); root mean square succes- other subjected to a bilateral renal denervation 1 week previ- sive difference (RMSSD)) and frequency domains (High (HF, ously. The animals were anaesthetised (3% isoflurane in O ) and 0.6–2.4 Hz) and Low Frequency (LF, 0.06–0.6 Hz) bands). Effects 2 each kidney in turn was exposed via a flank incision, all fat and of injections were separated into six temporal blocks accord- connective tissue stripped from around the renal artery which ing to the dynamics of changes in core temperature following was then bathed with 10% phenol in ethanol for 1 min, rinsed VMMR inhibition (3). At the end of the experiment, animals with saline and then muscle and skin sutured. Tail vein blood were sacrificed and histological control was carried out. samples were obtained under brief isoflurane anaesthesia and Table 1 shows changes in HRV parameters and Thy (% of base- a 24h urine collection taken. On day 1 at 10.00 am, animals line levels, mean ± SEM) during the six temporal blocks. The LF received an oral gavage of tap water (2ml/100g body weight) band, expression of CV sympathetic drive, was clearly reduced and urine samples were collected every 2h for 6h. Thereafter, following VMMR neurons inhibition. Changes in HRV parame- the rats received isoprenaline (5mg/kg) every 72 h for 2 weeks ters appeared to be specifically due to VMMR neuron inhibition and drinking water containing caffeine (61.5mg/kg). At week and not to changes in Thy. In conclusion, VMMR neurons seem 1 and 2, tail vein sampling, oral gavage and urine collection was to participate in maintaining tonic sympathetic CV outflow. Table 1 repeated. Creatinine clearance (glomerular filtration rate, GFR), urine volume (UV) and absolute sodium excretion (UNaV) were determined. Data, means ± S.E.M. were taken as significant when P<0.05 (ANOVA) The intact rats had a basal GFR of 1.73±0.25ml/min/kg, UNaV of 0.52±0.1μmol/min/kg and UV of 22.8±3.08μl/min/kg which were unchanged at weeks 1 and 2. The water load in intact rats caused cumulative increases in UV, from 3.5±0.22ml at 2h to 5.3±0.21ml 6h, and in UNaV from 1.98±0.26μmol/min/kg at 1) Cao WH & Morrison SF. (2003). Disinhibition of rostral raphe pallidus ± μ neurons increases cardiac sympathetic nerve activity and heart rate. 2h to 16.48 3.87 mol/min/kg at 6h. At week 2 the magnitude In Brain Res, pp. 1-10. of the cumulative increases in UV and UNaV were reduced (both 2) Salo LM, Nalivaiko E, Anderson CR & McAllen RM. (2009). Control of P<0.05) by some 60%. In the rats subjected to bilateral renal cardiac rate, contractility, and atrioventricular conduction by medullary denervation, the water load resulted in a cumulative rise in UV, raphe neurons in anesthetized rats. Am J Physiol Heart Circ Physiol 296, from 3.26±0.30ml at 2h to 5.96±0.22ml at 6h, and UNaV from H318-324. 1.6±0.25μmol/min/kg over 2h to 7.11±0.74 μmol/min/kg at 3) Zaretsky DV, Zaretskaia MV & DiMicco JA. (2003). Stimulation and 6h, responses similar to those obtained in the intact rats. The blockade of GABA(A) receptors in the raphe pallidus: effects on body magnitude of the increases in UV and UNaV after the water load temperature, heart rate, and blood pressure in conscious rats. Am J were unchanged after the 2 weeks of isoprenaline/caffeine Physiol Regul Integr Comp Physiol 285, R110-116. treatment in the renally denervated group which was different Where applicable, the authors confirm that the experiments from the intact group (P<0.05). described here conform with The Physiological Society ethical These findings demonstrated that in the isoprenaline/caffeine requirements. model of heart hypertrophy there was a blunted ability to excrete a volume load, which was most likely due to a lack of suppression of RSNA as the excretory response was unchanged following renal denervation. Exactly how the cardiopulmonary C99 receptor sensitivity changes in heart hypertrophy remains to be explored. Renal excretory responses to a volume load in conscious Wistar rats with heart hypertrophy Flanagan ET, Buckley MM, Aherne CM, Lainis F, Sattar M & Johns EJ (2008). Exp Physiol 93, 1058-1064. M.M. Buckley, D.M. Brennan and E.J. Johns Department of Physiology, University College Cork, Cork, Ireland Where applicable, the authors confirm that the experiments Heart hypertrophy is considered to be an initial indicator of described here conform with The Physiological Society ethical a progression into heart failure which is accompanied by an requirements.

57P Oral Communications

Paxinos G & Watson S (1986) The rat brain in stereotaxic coordinates. C100 2nd Ed. Academic Press, New York Supported by a BBSRC Pfizer CASE studentship to ES Midbrain control of micturition in the rat

1 1 2 1 Where applicable, the authors confirm that the experiments E. Stone , J.H. Coote , J. Allard and T.A. Lovick described here conform with The Physiological Society ethical 1University of Birmingham, Birmingham, UK and 2Pfizer Ltd, requirements. Sandwich, UK

In most socialised animals voiding can be suppressed, even when the bladder is full, until the individual is in a safe and socially acceptable environment. In the rat, stimulation of blad- C101 der afferents during filling activates a spino-midbrain-spinal micturition reflex pathway that relays in the periaqueductal Changes in systolic blood pressure and the low frequency grey (PAG) (DeGroat, 2006). We have investigated whether the component of heart rate variability are positively correlated synaptic relay in the PAG could be a site of tonic inhibitory con- during periaqueductal grey matter stimulation in humans trol over micturition. J.A. Hyam1,2, C. Williams1, S. Wang1, D. Schlugman3, G. Lu1, Urethane anaesthetised (1.5g/Kg i.p.) male Sprague-Dawley J.F. Stein1, D.J. Paterson1, A.L. Green1,2 and T.Z. Aziz1,2 rats were instrumented to record femoral arterial pressure, heart rate and tracheal airflow. A cannula inserted through the 1Department of Physiology, Anatomy & Genetics, University of dome was used to measure intravesicular pressure and for infu- Oxford, Oxford, UK, 2Department of Neurosurgery, John Radcliffe sion of saline (6ml h-1) to induce periodic (0.6±0.05 min-1, Hospital, Oxford, UK and 3Department of Anaesthesia, John mean ± S.E.M.) increases in bladder pressure, accompanied by Radcliffe Hospital, Oxford, UK expulsion of urine through the urethra. The GABA agonist muscimol (250pmol in 50nl) microinjected Introduction: Deep brain stimulation applied to the dorsal peri- into the caudal ventrolateral PAG (vlPAG, P-8.8 Paxinos & Wat- aqueductal grey matter (PAG) in humans has been shown to son, 1986, n=3), but not at other sites in the PAG (n=16) com- increase arterial blood pressure during rest and resist postural pletely suppressed the periodic contractions. Microinjection blood pressure fall on standing whereas stimulation in the ven- of the GABAA antagonist bicuculline (1pmol in 50nl) into the tral PAG decreases BP during rest. The mechanism by which this caudal vlPAG (n=13) evoked an increase in the frequency (from effect is mediated is not clear. The low frequency (LF) compo- 0.7±0.1 to 2.3±0.4min-1, P < 0.05, Student’s paired t-test) and nent of heart rate variability (HRV) is generally accepted to rep- threshold (14.5±1.0 to 20.6±2.0 mmHg, P< 0.05) of contrac- resent sympathetic nervous system activity. The aim of this tion that lasted 1106±253s (range 263s-2911s). At 8/13 experiment was to measure cardiovascular responses during (68.5%) sites this effect was accompanied by a pressor PAG stimulation and correlate them to changes in sympathetic response (mean arterial pressure 86.9±4.6 to 123.9±8.5 nervous system activity. Methods: Five consecutive male mmHg, P< 0.01), which lasted 1680±253s (range 780s-2718s), patients of a mean age of 49.1 years underwent PAG stimula- tachypnoea (158.8±5.6 to 323.1±45.9 breaths min-1, P < tion for intractable pain syndromes. Intra-operative, continu- 0.05), exopthalmus and pupillary dilatation. At the remaining ous heart rate and invasive blood pressure recordings were sites (n=5) there were no accompanying cardio-respiratory made in patients who were awake (4 patients) or under gen- changes. eral anaesthesia (1 patient). Recordings were made for 100 sec- In contrast, bicuculline microinjected at more dorsal sites in onds before stimulation, 100 seconds during stimulation at one the caudal PAG (n=14) evoked a long lasting (>60min) irre- or two electrode locations within the PAG and for 100 seconds versible increase in bladder pressure (16.2±0.8 to after stimulation. This yielded recordings from a total of 7 PAG 25.9±1.9mm Hg, P < 0.01) on top of which frequent (3.1±0.5 electrode positions across patients. Results: PAG stimulation min-1) low amplitude contractions were superimposed. altered systolic blood pressure (SBP), diastolic blood pressure These changes in bladder activity were accompanied by a sus- and heart rate. During stimulation SBP increased in 3 cases by tained pressor response (88.1±3.1 to 139.1±5.6mmHg, P< 7.2-10.2mmHg, decreased in 2 cases by 3.1-11.5mmHg and 0.01), tachycardia (438.4±12.6 to 547.4±41.0 beats min-1, was unchanged in 2 cases. HRV also changed during stimula- P< 0.01), increases in respiratory amplitude (128.1±16.7 to tion. LF power increased when SBP increased and decreased 316.7±44.3, arbitrary units P < 0.01) and respiratory rate when SBP decreased. Furthermore changes in SBP during stim- (164.7±5.1 to 318.6±22.3 breaths min-1, P < 0.01) and pro- ulation correlated positively with changes in LF power (Pear- nounced signs of autonomic activation (exopthalmus and son’s r=0.82, p=0.024, n=7). Conclusions: PAG stimulation pro- pupillary dilatation). duced positively-correlated changes in SBP and LF power. This The results suggest i) the functional integrity of the caudal therefore suggests that PAG stimulation alters cardiovascular vlPAG is necessary for reflex micturition to occur in response performance via modulation of sympathetic nervous system to bladder filling and ii) the synaptic relays in this region are activity. under tonic GABAergic control. In addition, the dorsal PAG, whilst not essential for execution of the reflex, can facilitate Support grants are received from the UK Medical Research contractile activity of the bladder in concert with intense Council, UK Biomedical Research Collaborative of the National sympatho-activation. Institution for Health Research, Medtronic and the Norman Col- lisson Foundation. DeGroat WC (2006) Brit J Pharmacol 147, S25-S40. 58P Oral Communications

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

C102

Reduced synaptic inhibition underlies respiratory apneas in a mouse model of Rett syndrome (RTT)

A.P. Abdala2, M. Dutschmann3, J.M. Bissonnette1 and J.F.R. Paton2

1Obstetrics and Gynecology, Oregon Health & Science University, Portland, OR, USA, 2Physiology & Pharmacology, University of 3 Bristol, Bristol, UK and Membrane & Systems Biology, University Figure: Apnea in Mecp2+/- mouse. Traces are from above: integrated HN, of Leeds, Leeds, UK raw HN, integrated cVN, raw cVN, integrated phrenic and raw phrenic nerve activity. RTT is a neurodevelopmental disorder caused by mutations Chahrour, M & HY Zoghbi (2007) Neuron 56, 422-437. in the X-linked gene that encodes the transcription factor methyl-CpG-binding protein 2 (Mecp2) (1). Included in the Weese-Meyer DE et al (2006) Pediatr Res 60, 443-449 phenotype are respiratory disorders that include frequent Guy, J et al (2001) Nature Genet 27, 322-326. apneas and periodic breathing that are most prevalent in Paton, JFR (1996) J Neurosci Methods 65, 63-68. young females (2). Heterozygous female mice (Mecp2+/-) Stettner, GM et al (2007) J Physiol 579, 863-876. with deletion of the 3rd and 4th exons of Mecp2 mimic these respiratory disturbances but the central neuronal mecha- Support: March of Dimes Foundation #6-FY06-314 (JMB) nisms have not been fully determined. We have hypothe- International Rett Syndrome Foundation #0802 (JMB sized that insufficient GABAA synaptic inhibition underlies these respiratory disorders and augmenting GABA in awake & JFP) animals reduces their incidence. Studies were carried out National Institutes of Health NS057815 (JFP) in B6.129P2(C) tm1.1Bird (3) heterozygous females University of Bristol Benjamin Meaker Visiting (Mecp2+/-) and wild type littermates (Mecp2+/+) using an in situ preparation to record phrenic, central vagal (cVN), Professorship (JMB) hypoglossal (HN) and abdominal (ABD) nerve activity (4). Where applicable, the authors confirm that the experiments Animals were anaesthetized deeply with 5% halothane and described here conform with The Physiological Society ethical once they failed to respond to noxious pinching of a paw and requirements. the tail were decerebrated. Apneas (TE ≥ 1.0 s) were more frequent in Mecp2+/- (168.2±29.1/hr) (SEM) (n=9) than ± Mecp2+/+ mice (56.8 12.9/hr) (n=13) (p= 0.005, Student t C103 test). Apneas were characterized by prolonged postinspira- toryactivityincVN(asobservedpreviouslyinmaleMecp2- Knockdown of SERCA-2 in human airway smooth muscle /y mice;(5)) that terminated before the end of phrenic apnea. from healthy subjects recapitulates a phenotype associated In addition, during the apnea there was a hypoglossal nerve with asthma discharge that occurred at the time a phrenic burst was O.O. Ojo1, K. Mahn1, M.R. Holt3, S.J. Hirst2, T.H. Lee1 and anticipated from the respiratory cycle length of preceding J.P. Ward3 bursts. This continued throughout the duration of the apnea 1 (Fig). In some apneas the abdominal nerve exhibited a sus- MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, 2 tained burst. King’s College London, London, UK, Department of Physiology, 3 1-[2-[[(Diphenylmethylene)imino] oxy]ethyl]-1,2,5,6-tetrahy- Monash University, Melbourne, VIC, Australia and King’s College dro-3-pyridinecarboxylic acid hydrochloride (NO-711), 5 – London, London, UK 10μM in the perfusate was used to block GABA reuptake. Cytosolic [Ca2+] plays a critical role in the function of airway Apneas were reduced from 152.3±12.1 to 115.3±16.0/hr smooth muscle (ASM) and the sarco/endoplasmic reticulum (p=0.009, n=5) in Mecp2+/- mice. The apneas that remained [Ca2+] ATPase (SERCA) is an important mechanism for regu- after NO-711 were characterized by a shorter duration, lower lating cytosolic [Ca2+]. ASM from asthmatics is characterised activity in HN and absence of activity in ABD, consistent with by increased proliferative and migratory responses, and we reduced excitability of the network or increased synaptic inhi- have reported that ASM cells obtained from asthmatic volun- bition. The results suggest that apnea in this mouse model of teers exhibit abnormal Ca2+ homeostasis arising from reduced RTT results from, in part, insufficient GABA mediated synaptic SERCA-2 expression (Mahn et al, 2007). The aim of this study inhibition in the ponto-medullary respiratory network leading was to determine whether knockdown of SERCA-2 in ASM cells to a loss of rhythmic phrenic bursts. from healthy subjects could reproduce features of the pro-asth-

59P Oral Communications matic phenotype. METHODS: The study was approved by the astrocytes and this effect can be mimicked by the application local ethical committee and informed consent obtained. ASM of ATP. In the present study, we used an in vivo preparation to cells from subjects with and without mild asthma were cultured detect astrocytic calcium responses in ventral surface astro- from endobronchial biopsies (n=10). ASM cells from healthy cytes induced by respiratory acidosis. Methods: Six male donors were transfected with siRNA against SERCA-2 (sASM) Sprague Dawley rats were used in accordance with the UK Ani- or a scrambled siRNA (cASM). Knockdown efficiency was con- mals (Scientific Procedures) Act 1986 and associated guide- firmed by Western blot. Changes in cytosolic [Ca2+] were esti- lines. Animals were anaesthetised with a mixture of ketamine mated using Fura PE3; proliferation was assessed by 3[H]-thymi- (60 mg kg-1 I.M.) and medetomidine (250 μg kg-1 I.M.) and dine incorporation with migration determined by cell received bilateral microinjections into the rostroventrolateral spreading. RESULTS: sASM exhibited a 30% reduction in SERCA- medulla (2μl each side) of an adenoviral vector encoding a Ca2+ 2 protein expression, comparable with that found in ASM from sensitive protein Case12 (3) under control of an enhanced GFAP asthmatics. Bradykinin (1μM)-evoked [Ca2+] release was promoter. Anaesthesia was reversed with atipemazole (250 mg reduced by 47±24% (mean±SEM) in sASM compared with cASM kg-1 I.M.) and post-operative care was taken. Seven days after (p<0.05; n=4), consistent with reduced store capacity. Recov- injections, animals were anaesthetised with propofol (30 mg ery of cytosolic [Ca2+] to baseline, an index of SERCA-mediated kg-1 h-1, I.V.), paralysed with gallamine triethiodide (10 mg Ca2+ uptake, was increased from 109±27 sec in cASM to 274±78 kg-1, I.V.; then 1–2 mg kg-1 h-1, I.V.) and artificially ventilated. sec in sASM (p<0.05; n=4), similar again to that found with ASM The adequacy of anaesthesia during neuromuscular blockade from asthmatics (Mahn et al, 2007). SERCA-2 knockdown also was assessed by monitoring heart rate and blood pressure sta- increased 10% fetal bovine serum (FBS)-dependent prolifera- bility. The ventral surface of the medulla was exposed and con- tion from 285±10% (versus FBS-free medium) in cASM, to tinuously perfused with HBSS (pH 7.4) and phrenic nerve activ- 417±26% in sASM (p<0.02; n=3). The latter was comparable ity was recorded. GFP expression in the ventral surface of the with responses in ASM from asthmatics (450±90%; n=3). Cell medulla was visualized using a fluorescence microscope. Case12 2+ spreading, taken as a functional readout of cell motility, showed fluorescence was used as an indicator of [Ca ]i specifically in increased spreading or early laminapodia and filapodia forma- astroglia. At the end of recordings, animals were intracardially tion (expressed as pixels/3min) in sASM (387±118) compared perfused with 4% paraformaldehyde and the brainstems were with cASM (90±28; n=7; p<0.05). Conclusion: Knockdown of subsequently processed for immunohistochemical detection SERCA-2 in healthy ASM recapitulates key changes in cellular of Phox2B and GFAP. Results: Lowering ventral surface pH from function that have been previously observed in ASM from asth- 7.4 to 7.2 increased phrenic nerve activity and resulted in pro- 2+ matics. This suggests that reduced SERCA-2 expression, with found elevations in [Ca ]i in surface astrocytes. Immunohis- consequent changes in cytosolic Ca2+ homeostasis, may play tochemical analysis of the injected areas revealed strong GFP an important role in the development of ASM changes in air- expression in GFAP-expressing astrocytes located in the mar- way wall remodeling in asthma ginal area of the ventral medullary surface. These astrocytes Mahn K, Hirst SJ, Karner LC, Simcock DE, O’Connor BJ, Snetkov VA, Lee were often seen surrounding blood vessels and in close contact TH. Calcium buffering is delayed in asthmatic airway smooth muscle. with chemosensitive Phox2B-immunoreactive neurons of the Am J Respir Crit Care Med 2007;175:A523 retrotrapezoid nucleus (RTN). Conclusion: Medullary astrocytes Supported by the MRC, Asthma UK, the Dr Hadwen Trust for surrounding ventral penetrating arteries could play an impor- Humane Research, and the Guy’s and St Thomas’ NHS Foun- tant role in integrating chemosensory information by sensing dation Trust and King’s College London National Institute for physiological changes in arterial PCO2 and pH. We suggest that Health Research Biomedical Research Centre, these astrocytes shape the chemosensory responses of the RTN neurons and the central respiratory network as a whole. Where applicable, the authors confirm that the experiments Gourine AV et al. (2005). J Neurosci 25, 1211-1218. described here conform with The Physiological Society ethical Gourine AV et al. (2005). Nature 436, 108-111. requirements. Souslova EA et al. (2007). BMC Biotechnol 29, 37-46. Suppported by The Wellcome Trust and The British Heart C104 Foundation. Where applicable, the authors confirm that the experiments Chemosensitive astrocytes in the rostral ventrolateral described here conform with The Physiological Society ethical medulla requirements. N. Marina1, V. Kasimov1, M. Figuereido2, S. Kasparov2 and A. Gourine1 1 Neuroscience, Physiology and Pharmacology, University College C105 London, London, UK and 2Physiology and Pharmacology, University of Bristol, Bristol, UK Synaptic NMDA Receptor Subunit Composition and Plasticity ATP is released from the chemosensitive regions located on the in Principal Cells and Interneurons in Hippocampus ventral surface of the medulla oblongata in response to changes S.C. Harney and R. Anwyl in arterial PCO2 and pH, which results in the activation of the respiratory network (1, 2). In vitro studies using brainstem slices Physiology, Trinity College Dublin, Dublin, Ireland + have shown that increases in PCO2 or [H ] results in the gener- N-methyl-D-aspartate receptors (NMDARs) have a pivotal role 2+ ation of intracellular calcium waves ([Ca ]i) in ventral surface in long-term plasticity and NMDAR-mediated synaptic trans-

60P Oral Communications mission is subject to plasticity although the mechanisms in attractor states and the precise mechanisms involved remains involved are little understood. Subunit composition determines elusive. the properties of NMDARs and there is evidence for subtype Using a novel in-vitro model of spontaneous network bistabil- differences between receptors at different subcellular locations ity in acute rat brain slices we can for the first time examine the and between different neuronal cell types. NR2D-containing isolated effect of cholinergic modulation on spontaneous net- NMDARs are predominantly expressed at extrasynaptic loca- work bistability. tions and our previous data suggests that extrasynaptic NR2D- In whole cell patch clamp recordings from layer III entorhinal containing NMDARs relocate to the synapse during NMDAR- cortex principal cells we show that bath applied Acetylcholine LTP. Expression of the NR2D subunit has also been (ACh, 50-200 nM) leads to a concentration-dependent and demonstrated in inhibitory interneurons in hippocampus. Hip- reversible increase in UP state duration (control: 7.2 ± 3.5 s vs pocampal slices were prepared from male Wistar rats (3-5 200 nM ACh: 42.3 ± 23.2 s vs wash: 10.3 ± 4.2 s; n = 5; p < 0.001; weeks old) and whole-cell patch-clamp recordings were per- 2-way ANOVA) whilst DOWN states become shorter (control: formed to compare NMDAR-mediated synaptic transmission 43.6 ± 27.2 s vs 200 nM ACh: 13.5 ± 3.9 s vs wash 37.5 ± 13.7 in granule cells (GCs) and interneurons (INs) in dentate gyrus. s; n = 5; p < 0.001; 2-way ANOVA). Increasing cholinergic mod- NMDAR-EPSCs were evoked by stimulation of the medial per- ulation also leads to a reversible increase in cell firing (control: forant path, at a test frequency of 0.033 Hz, at 34 ∞C. Decay 1.24 ± 1.2 Hz vs 200 nM ACh: 3.25 ± 2.6 Hz vs wash: 2.79 ± 2.7 kinetics were significantly slower for NMDAR-EPSCs recorded Hz; n = 4; p < 0.001; 2-way ANOVA) (all data presented as mean τ ± in interneurons, with a weighted decay time constant ( W) of S.E.M.). Increasing ACh concentrations beyond 200 nM lead ± τ ± 85 8 ms (n = 21), compared to W of 32 1 ms in granule cells to a persistently active state, similar to those reported earlier (n = 17, p < 0.001). NMDAR-EPSC amplitude and 10-90% rise in layer III and V entorhinal cortex principal cells (Tahvildari et time was similar between granule cells and interneurons (ampli- al. 2007). tude 28 ± 3 pA and 24 ± 5 pA in GCs and INs, respectively, 10- Our results suggest that cholinergic tone alone may be suffi- 90% rise time 4.7 ± 0.3 ms and 5 ± 0.9 ms in GCs and INs, respec- cient to account for the switching between a bistable attractor tively). The NR2D-preferring antagonist UBP141, inhibited state for synchronous network activity, as observed during NMDAR-EPSCs in INs by 27 ± 4 % of control amplitude (n = 7) SWS, and a monostable regime of less synchronous network but had no effect on currents recorded in GCs (99 ± 3 % of con- activity, similar to that found in REM sleep or wakefulness. trol, n = 4). Ifenprodil had similar effects on NMDAR-EPSC ampli- Steriade M et al. (1993). J. Neurosci. 13, 3252-3265. ± tude in both cell types, inhibiting EPSCs by 28 6 % of control Steriade M et al. (2001). J Neurophysiol. 85, 1969-1985. in GCs (n = 5) and by 35 ± 9 % of control in INs ( n = 7). Our find- Tahvildari et al. (2007). Hippocampus. 17(4), 257-63. ings suggest that different NMDAR subtypes are expressed at perforant path synapses on principal cells and interneurons, MMKissupportedbyanOXIONWellcomeTrustPrizeStudentship. and this variation may be associated with differences in spike Where applicable, the authors confirm that the experiments timing and synaptic plasticity. described here conform with The Physiological Society ethical Supported by: Science Foundation Ireland. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. C107

Deletion of TASK1 and TASK3 channels disrupts firing but not glucose or pH responses in orexin neurones C106 A. Gonzalez and D. Burdakov Pharmacology, University of Cambridge, Cambridge, UK Cholinergic modulation of cortical network bistability in vitro Neurones in the hypothalamus that express the peptide M.M. Kohl, N. Rabinowitz and O. Paulsen hypocretin/orexin are essential for the regulation of sleep-wake transitions, appetite, and metabolism (Hara et al., 2001; Department of Physiology, Anatomy and Genetics, University of Adamantidis et al., 2007). They sense changes in extracellular Oxford, Oxford, Oxfordshire, UK glucose and pH that occur within the physiological range, such During slow-wave sleep (SWS) assemblies of cortical neurons that an increase in glucose is inhibitory and a decrease in pH is exhibit bistable behaviour in that they simultaneously switch excitatory (Burdakov et al., 2006; Williams et al., 2007). It has between more depolarised UP states and rather quiescent been demonstrated that both glucose and pH modulate the DOWN states (Steriade et al., 1993). In contrast, during rapid- excitability of orexin cells by regulating a leak K+ current, most eye-movement (REM) sleep or wakefulness the same neurons likely mediated by channels of the family of two-pore domain loose this synchronicity and are virtually constantly active and K+ channels. Specifically, pharmacological data suggested that thus monostable. Cholinergic stimulation from subcortical TASK1 and/or TASK3 channels were responsible for the glucose- structures has been linked to the abolition of the bistability that and pH-sensing properties of orexin neurones (Burdakov et al., occurs in the transition from slow-wave sleep to REM sleep or 2006; Williams et al., 2007). wakefulness (Steriade et al., 2001). However, whether Acetyl- To test this hypothesis, we performed whole-cell recordings choline (ACh) alone is capable of bringing about this change in vitro from identified GFP-labelled orexin cells from mice that lack TASK1 and TASK3 channels (KO mice). Animal procedures

61P Oral Communications were carried in accordance with the Animals (Scientific Proce- (Willoughby & Schwiening, 2002) with the fluorescent pH indi- dures) Act 1986 (UK). cator 8-hydroxypyryne-1,3,6-trisulphonic acid (HPTS, 125 μM) We found that intrinsic excitability was reduced in KO mice: and the calcium indicator Fura Red (62.5 μM) at pH 7.3. They compared to wild-type orexin cells, the relationship between were illuminated with alternating 405 nm and 488 nm light firing rate and current in KO neurones was significantly lower and pH-sensitive emission was collected at 505-550 nm and at high current levels (P < 0.01 for current ≥12 pA/pF). The action calcium-sensitive emission at 610-700 nm using a Leica SP5 potential threshold was more positive in KO cells (KO, -18.5 ± confocal microscope (40x 0.8 NA water immersion objective). 0.3 mV; wild-type, -21.4 ± 0.2 mV; P < 0.001, n > 150 spikes in The HPTS ratio was calibrated in vitro (Willoughby et al., 1998) each group), as was the after-hyperpolarization potential (KO, using a single-point normalization to the fluorescence of the -40.7 ± 0.3 mV; wild-type, -47.3 ± 0.3 mV; P < 0.001, n > 150 patch-pipette. Slices were maintained in HEPES-buffered spikes in each group). Spontaneous firing rates were not signi- Ringer’s solution. ficantly different between groups (KO, 9.4 ± 2.6 Hz; wild-type, The soma was always more acidic (pH 6.98±0.06 mean±SD) 13.8 ± 2.1 Hz; P = 0.21, n = 7 cells for each group). Moreover, than the dendrites (pH 7.27±0.14 mean±SD at ~120 μm from when the extracellular glucose concentration was increased the soma, n=7) or the patch-pipette (pH 7.3) ~3 mins after from 1 to 5 mM, all KO orexin cells tested (n = 10) were hyper- the last depolarization (1 s to ~ +20 mV). Further increasing the polarized in a manner indistinguishable from wild-type cells pH of the patch-pipette to pH 7.6 did not significantly alkalin- ± (change in Vm induced by glucose: KO, -21.8 2.3 mV; wild- ize the soma. This is not consistent with the pHi gradient result- type, -25.1 ± 3.7 mV, P = 0.46, n = 6 for each group). Post-synap- ing from simple dialysis with the patch-pipette. We considered tic currents modulated by glucose and pH in KO cells had the several other artefacts that might underlie the apparent pHi characteristics of leak-like K+ currents, i.e. a reversal potential gradient including non-linearities in the PMT gain, chromatic close to that of EK+ and a current-voltage rectification well- aberration and anoxia. To overcome chromatic aberration we described by the Goldman-Hodgkin-Katz equation. produced pH and calcium images from serial confocal sections Our results suggest that TASK1 and TASK3 channels are not using only in focus structures. These images showed little sys- essential for glucose- or pH-sensing in orexin cells. They do, tematic relationship between regional pH and either the depth however, enhance their membrane excitability and support in slice or Ca2+ level. However, regional pH and the rate of reg- their ability to generate high-frequency firing. ulation was related to distance from the soma. Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K & de Lecea L Our study suggests a heterogeneity of resting pH and evoked (2007). Neural substrates of awakening probed with optogenetic con- pH shifts in cerebellar Purkinje neurones. It is consistent with trol of hypocretin neurons. Nature 450, 420–424. most of the functional pH regulation occurring within the den- Burdakov D, Jensen LT, Alexopoulos H, Williams RH, Fearon IM, O’Kelly dritic tree rather than at the soma. This could result in the I et al. (2006). Tandem-pore K+ channels mediate inhibition of orexin uncoupling of excitability of soma and dendritic arbor and may neurons by glucose. Neuron 50, 711–722. explain the different reversal potentials reported for GABAA Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM, Sinton CM et receptors in different cellular regions. al. (2001). Genetic ablation of orexin neurons in mice results in nar- Xiong Z-Q et al. (2000) J Neurosci 20, 1290-1296. colepsy, hypophagia, and obesity. Neuron 30, 345–354. Willoughby D et al., (1998) Pflugers Arch 436, 615-622. Williams RH, Jensen LT, Verkhratsky A, Fugger L & Burdakov D (2007). Control of hypothalamic orexin neurons by acid and CO2 . Proc Natl Willoughby D & Schwiening CJ. (2002) J Physiol 544, 487-499. Acad Sci U S A 104, 10685–10690. We thank the MRC for financial support. Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

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Long-lasting pH gradients are induced by electrical activity Activity-dependent long-term potentiation produced by in rat cerebellar Purkinje neurons exogenous nitric oxide during NMDA receptor blockade in O. Larina and C.J. Schwiening hippocampal slices Physiological Laboratory, Department of Physiology, Development B.M. Pigott and J. Garthwaite and Neuroscience, University of Cambridge, Cambridge, UK The Wolfson Institute for Biomedical Research, University College London, London, UK Intracellular pH (pHi) is a powerful modulator of cell excitability (Xiong et al., 2000) yet there is no information on the rest- Nitric oxide (NO) is a freely diffusible intercellular signalling ing pHi in dendritic regions. Understanding how dendritic pHi molecule in most brain areas and has been widely implicated is regulated is important since epilepsy and some forms of in synaptic plasticity and other phenomena. NO generation is ataxia appear to involve pHi shifts. We have used fluorescence linked to NMDA receptor activation and physiological NO sig- confocal imaging to investigate regional resting pHi in Purkinje nal transduction is achieved through guanylyl cyclase activa- neurones. tion and cGMP accumulation (Garthwaite, 2008). In the hip- Purkinje neurones, in cerebellar slices, were whole-cell patch pocampus, NO has long been implicated as a retrograde clamped using pipettes containing a standard solution messenger in NMDA receptor-dependent long-term potentia-

62P Oral Communications tion (LTP), but its precise role remains unsettled. If NO were acting purely presynaptically and if presynaptic changes contribute C110 to LTP, it is predicted that exogenous NO should partially overcome the inhibitory effect of NMDA receptor blockade on LTP. Increased expression of transforming growth factor-b1 To test this hypothesis, NMDA receptor-dependent LTP was alters synaptic structure in vivo and regulates neuronal studied at Schaffer collateral-CA1 synapses using field poten- activity in vitro tial recording in hippocampal slices of male mice aged 6-8 J.J. Bae1,3, A. Martinez-Canabal2,4, P.W. Frankland2,4 and W. Lu1,3 weeks. LTP was induced using a 1-s, 100-Hz tetanus. Values of potentiation given below refer to mean field EPSP slopes 55-60 1Physiology, University of Toronto, Toronto, ON, Canada, 2Institute minutes after the tetanus, normalised to baseline responses of Medical Science, University of Toronto, Toronto, ON, Canada, (100 %) ± SEM. As expected, the NMDA antagonist, D-AP5 (50 3Clinical and Integrative Biology, Sunnybrook Health Sciences μM), reversibly blocked LTP (110 ± 6 % following tetanus dur- Centre, Toronto, ON, Canada and 4Neurosciences and Mental ing NMDA blockade; 169 ± 14 % following a second tetanus Health, Hospital for Sick Children, Toronto, ON, Canada after D-AP5 washout; n = 5). When present during tetanic stim- Transforming growth factor-beta 1 (TGF-β1) is a multifunctional ulation in the presence of D-AP5, the NO donor PAPA/NONOate injury-related cytokine that orchestrates key events of devel- ± produced a long-lasting potentiation (152 9 %; n = 5), as pre- opment, disease and repair (Gomes et al. 2005). Secreted by dicted by the retrograde messenger hypothesis. This NO- both neurons and glial cells in the central nervous system (CNS), induced potentiation was, however, slow to develop (20 min increased expression of TGF-β1 is associated with neurologi- to plateau) and, unexpectedly, it did not occlude subsequent cal diseases and brain trauma (Buckwalter et al. 2004). To date, ± LTP, which was, instead, substantially enhanced (266 9 %; n its role in the regulation of synaptic structures and transmis- = 5). Both the NO-induced potentiation and the subsequent sion in the mammalian CNS remains unclear. To investigate the enhancement of LTP were dependent on exogenous NO con- effects of chronic over-secretion of TGF-β1 on the structural μ centration (maximal at 3 M PAPA/NONOate) and on there and functional properties of neural cells, we used transgenic ± being a coincident tetanic stimulation (115 4 % during NMDA mice (T64) that over-express active form of TGF-β1inhip- ± blockade with exogenous NO but no tetanus; 190 11 % fol- pocampal and cortical astrocytes. Immunohistochemical analy- lowing a subsequent tetanus after washout of D-AP5 and sis demonstrated that in comparison with wild-type (WT) lit- PAPA/NONOate; n = 5). Both effects were also blocked by the termate controls, the number of cells in T64 mice expressing μ ± guanylyl cyclase antagonist, ODQ (10 M; 114 7 % following glial fibrillary acidic protein (GFAP, astrocyte marker), and tetanus during NMDA blockade and with exogenous NO; 159 CD11b (microglia marker) increased significantly in the hip- ± 10 % following subsequent tetanus; n = 5), implying that they pocampus and cortex. Notably, large immuno-clusters of both involve the generation of cGMP and so could be mecha- synaptophysin (presynaptic protein) and calbindin-D28K (CaBP, nistically linked. It is concluded that the apparent rescue of calcium binding protein) positive immunoreactivity of neurons LTP by exogenous NO paired with a tetanus when NMDA recep- were, respectively, decreased in the CA3 region and the den- tors are blocked cannot simply be explained by the NO com- tate gyrus through to the mossy fiber terminals. Decrease in pensating for a missing component of normal LTP. Instead, the CaBP and increase in GFAP protein levels in the T64 model were results show that exogenous NO, in an activity-dependent man- confirmed. Cultured primary astrocytes from T64 mice show ner, evokes a long-term enhancement of synaptic transmission higher rate in proliferation and more elongated cell bodies and of similar amplitude to that normally produced during LTP, but processes when compared with WT controls. To examine the that is additive to normal LTP. effect of TGF-β1 alone in the regulation of neuronal functions, Garthwaite J (2008). Eur J Neurosci 27(11), 2783-2802. we cultured hippocampal neurons from WT rat embryos in the β Supported by the BBSRC. presence or absence of TGF- 1 (4.0 ng/ml, 7-12 days). Voltage- clamp recordings in these cultured neurons revealed that TGF- Where applicable, the authors confirm that the experiments β1 significantly increased the amplitudes of voltage-gated K+ described here conform with The Physiological Society ethical currents, Na+ currents and glutamate-induced currents requirements. (p<0.05, Student’s t-test) in treated cells. Taken together, our results suggest that chronically increased expression of TGF- β1 may activate glia and induce alterations in synaptic structures of surrounding neuronal populations. Further, TGF-β1 alone appears, at least in culture condition, to increase neuronal activity. Buckwalter M & Wyss-Coray T (2004). J Neuroinflammation 1, 10 Gomes FC, Sousa Vde O, Romão L (2005). Int J Dev Neurosci 5, 413-424

This study was funded by the Canadian Institute for Health Research. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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C112 C111 The effect of TNF-a treatment on skeletal muscle myokine Motor Unit Size-Dependent Intrinsic Withdrawal of production Neuromuscular Synapses During Postnatal Development of Mouse Muscle A.P. Lightfoot, D.C. Harrison, A.C. Kayani, F. McArdle, A. Teriakidis1, D.J. Willshaw2 and R.R. Ribchester3 R.D. Griffiths and A. McArdle 1Neuroinformatics DTC, University of Edinburgh, Edinburgh, UK, PathophysiologyResearchUnit,UniversityofLiverpool,Liverpool,UK 2 Institute for Adaptive Neural Computation, University of The role of skeletal muscle in locomotive function is well 3 Edinburgh, Edinburgh, UK and Euan MacDonald Centre for MND defined; however recent research has indicated a role as an Research, University of Edinburgh, Edinburgh, UK immunogenic organ. During systemic inflammation, mus- At birth every muscle fibre has functional synaptic inputs from cle is exposed to pro-inflammatory factors and Tumor necro- α multiple motor neurons, all but one of which are eliminated. sis factor alpha (TNF- ) has been implicated as a key initia- Synapseeliminationisdrivenbycompetitionbetweensynapses tor of the early inflammatory response. Studies have co-innervating a muscle fibre (Betz et al., 1980). However, an indicated that muscle responds rapidly to stress by the unresolved question is whether synapse elimination can occur increased production of myokines and stress or Heat Shock in the absence of competition. Some evidence suggests that Proteins (HSPs) and data suggest that that these proteins ‘intrinsic withdrawal’ of synapses continues at some neuro- may be released as signalling proteins to other cells (1, 2, α muscular junctions (NMJs) after partial denervation at birth 3). We hypothesise that elevated levels of TNF- initiates (FladbyandJansen;1988),leavingsomeendplatesuninnervated. the production and release of myokines and HSPs from We have re-examined the question of ‘intrinsic withdrawal’ by skeletal muscle. directly measuring motor unit (MU) size in transgenic mice C2C12 myotubes were cultured & differentiated in vitro (4). α expressing yellow fluorescent protein in motor neurons Myotubes were treated with TNF- in culture media (25ng/ml (thy1.2:YFP). We used confocal microscopy to determine MU media) for 3 or 6 hours; culture media and cell lysates were har- sizes in the 4th deep lumbrical (4DL) muscle of unoperated con- vested and analysed for the presence of cytokines using trol mice, adult mice whose muscles had been partially dener- Luminex multiplex (20-plex) bead analysis. RNA was isolated vated at birth by bilateral tibial nerve cuts (neonates anaes- using TRI reagent/Rneasy clean up extraction method and thetised by chilling) leaving a single innervating axon through reverse transcribed to single strand cDNA. Gene expression was the sural nerve (AwPD) and neonates. We found that the aver- analysed using a murine cytokine qPCRarray assay. Western age MU size was significantly smaller in control mice (54 ± 11, blotting was used to quantify levels of HSPs in the lysate and α n = 29, mean ± SD) than in neonates (137 ± 63, n = 10, p<0.001, media following TNF- exposure (10, 25 or 50ng/ml). Cell via- Tukey HSD) and in AwPD (103 ± 29, n = 10, p<0.001, Tukey HSD) bility was assessed by trypan blue exclusion and LIVE/DEAD though the difference between neonates and AwPD only staining. approached significance (p=0.058, Tukey HSD). While the small- qPCR array analysis of cell lysates showed greater than four-fold est MU size in those two groups was approximately the same, up-regulation of complement component 3, MCP-1, CCL5, there were several MUs that were larger in the neonates than CXCL1, CXCL5, CXCL9 and CXCL10. Luminex multibead analy- ≤ the maximum MU size in the AwPD, suggesting that only motor sis showed significant (ANOVA, P-value 0.05, n=6) levels of ± units above a certain size may lose synapses in the absence of IL-6 and RANTES located in the media at 3 hours (288pg/ml ± competition. 70.30; 7200pg/ml 626.64 respectively) and 6 hours ± ± α The present data suggest that there is an intrinsic limit to the (553pg/ml 147.63; 2122pg/ml 307) following TNF- expo- number of synapses a motor neuron can sustain and that this sure. Western blot analysis, showed significant up-regulation number may decline as axons and muscle fibres grow. We have of intracellular levels of HSP60 and HSP70, which peaked at 3 been modifying a computational model of synapse elimination hours following treatment and significant levels of HSP60 were ≤ to examine the effect of growth on MU size (Rasmussen & Will- detected in the culture media (ANOVA, P-value 0.05, n=6). shaw, 1993). Furthermore there was no significant loss of cell viability for up to 8 hours following TNF-α exposure. WJ Betz, JH Caldwell, RR Ribchester (1980). The effects of partial denervation at birth on the development of muscle fibres and motor units C2C12 myotubes showed significant up-regulation of myokine in rat lumbrical muscle. J Physiol, Vol. 303, pp. 265-279. expression in response to TNF-α treatment and increased release of myokines. These data further support the theory that T Fladby, JK Jansen (1987). Postnatal loss of synaptic terminals in the partially denervated mouse soleus muscle. Acta Physiol Scand, Vol. muscle can release cytokines in response to stress, thus act- 129, No. 2., pp. 239-246. ing as an immunogenic organ. The specific release of HSP60 supports the suggestion that heat shock proteins can be CE Rasmussen & DJ Willshaw (1993). Presynaptic and postsynaptic competition in models for the development of neuromuscular connections. released into the extracellular environment in response to Biological Cybernetics 68(5):409-19 stress, and we hypothesise that they may function as danger signals for the immune system(5). This work was funded by the EPSRC/MRC 1. Pedersen BK, Akerstrom TC, Nielsen AR, Fischer CP. Role of myokines Where applicable, the authors confirm that the experiments in exercise and metabolism.(2007). J Appl Physiol.3:1093-8. described here conform with The Physiological Society ethical 2. Nielsen S, Pedersen BK. Skeletal muscle as an immunogenic requirements. organ.(2008). Curr Opin Pharmacol. 3:346-51.

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3. Asea A. Hsp70: a chaperokine.(2008). Novartis Found Symp. the rates in wild type and transgenic mice. Fibres from nNOS 291:173-9; overexpressors showed a decrease in ethidium fluorescence 4. Maglara AA, Vasilaki A, Jackson MJ, McArdle A. Damage to develop- compared with the fibres from control mice at rest (p<0.05). ing mouse skeletal muscle myotubes in culture: protective effect of Following DCFH loading, no differences were seen in DCF flu- heat shock proteins.(2003). Journal of Physiology.548(Pt 3):837-46. orescence between the groups. Discussion: Contractile activity increased ROS and RNS in fibres from both wild type and 5. Calderwood SK, Mambula SS, Gray PJ, Jr. Extracellular heat shock pro- teinsincellsignalingandimmunity.(2007).AnnNYAcadSci.1113:28-39. nNOS overexpressing mice. The mice overexpressing nNOS appeared to generate more NO. at rest compared with those The authors would like to thank MRC and the University of Liv- from control mice. However following contractile activity the erpool for funding this study. fluorescence from fibres did not differ between the groups. These data indicate that overexpression of nNOS can increase Where applicable, the authors confirm that the experiments NO. bioavailability in skeletal muscle fibres and reduce the avail- −. described here conform with The Physiological Society ethical ability of O2 . requirements. Powers SK & Jackson MJ (2008). Physiol Rev. 88, 1243-1276 Nguyen HX & Tidball JG (2003). J Physiol. 550, 347–356 C113 Palomero J et al. (2008). Antioxid Redox Signal. 10, 1463–1474 Pye D et al. (2008). J Physiol. 581, 309–318 Real-time measurement of intracellular reactive oxygen and nitrogen species in single isolated mature skeletal muscle The authors thank the Wellcome Trust, UK and the Alexander fibres in mice overexpressing nNOS S. Onassis Public Benefit Foundation, Greece for generous financial support. G.K. Sakellariou1, J. Palomero1, H. Van Remmen2 and M.J. Jackson1 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical 1Pathophysiology Research Unit, School of Clinical Sciences, requirements. University of Liverpool, Liverpool, UK and 2University of Texas Health Center at San Antonio and Barshop Institute for Longevity and Aging Studies, San Antonio, TX, USA C114 Introduction: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are continuously produced by skeletal mus- Effects of genetic variation on activity of citrate synthase cle. Their generation is augmented during contractile activity and levels of signalling proteins in skeletal muscles of mice and may play an important role in signaling adaptive responses and/or mediate some degenerative processes [1]. Purpose: To A. Ratkevicius1,2, A. Carrol1, T. Venskunas2, A. Kilikevicius2, elucidate the roles of ROS and RNS in detail, we examined their S. Gray1, H. Wackerhage1 and A. Lionikas1,2 intracellular activities using fluorescence microscopy in single 1School of Medical Sciences, University of Aberdeen, Aberdeen, isolated mature skeletal muscle fibres from mice overexpress- UK and 2Department of Applied Physiology and Kinesiotherapy, ing neuronal nitric oxide (nNOS) and wild type control mice Lithuanian Academy of Physical Education, Kaunas, Lithuania both at rest and following a period of contractile activity. Meth- ods: Genetically modified mice overexpressing nNOS (type I Indentifying genetic factors modulating skeletal muscle char- NOS) [2] and wild type (C57Bl/6) mice were used in this study. acteristics can provide an insight into health and disease. A Each group consisted of 9 mice aged 22 ± 1 months. Mice were study of inbred strains of mice is a starting point of such an killed by cervical dislocation and the flexor digitorum brevis mus- effort. Genetic variations among strains often result in differ- cles were dissected. Muscles were incubated in collagenase to ences in muscle size2 and exercise performance3, but the cel- isolate single muscle fibres. Fibres were then plated on culture lular mechanisms behind these differences are not clear. The dishes which had been previously coated with collagen and cul- aim of the study was to investigate activity of citrate synthase tured for 24 h [3, 4]. Contractile activity was induced by elec- (CS), a marker for mitochondrial oxidative capacity4, and lev- trical stimulation [3, 4]. Fibres were loaded with 4-Amino-5- els of protein kinase B (PKB) and extracellular signal-regulated Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM DA), kinase 1 and 2 (MAPK(erk1/2)), as two growth regulators1, in Dihydroethidium (DHE) or 2’,7’-Dichlorofluorescein Diacetate quadriceps muscle of the A/J, BALB/cByJ, DBA/2J, C3H/HeJ, (DCFH DA), fluorescent probes for the assessment of Nitric C57BL/6J and PWD/PhJ mice. The quadriceps muscle of 14 week . −. Oxide (NO ), Superoxide (O2 ) and a general probe for ROS old male mice was dissected after sacrifice and snap frozen. 40- respectively. Unpaired Student’s t test for single comparisons, 70 mg of the muscle were homogenised and centrifugated. The significance p<0.05. Results: At rest the nNOS mice had a higher supernatants were taken and the protein concentration was rate of increase in DAF-FM fluorescence compared with the con- measured using the Bradford assay. The CS activity was assessed trol group implying a higher NO. generation (p<0.05). How- applying a standard spectrophometric assay. The analysis of ever, no significant differences were observed between the the specific proteins was performed by the Western blotting. stimulated fibres from the two groups. Ethidium fluorescence One way ANOVA showed that CS activity (n=9 per strain) was from DHE-loaded fibres increased in both groups indicating a strain dependent (P<0.0001) with A/J mice showing lower val- −. potential increase in the rate of O2 generation in the sar- ues (P<0.01, Tukey’s test) than the other strains. The difference coplasm after contractions (p<0.05) with no difference between between the A/J and PWD/PhJ mice was particular marked

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(281±60 versus 731±164 μmol (g protein)-1min-1, mean±S.D., of E2 and IGF-I. Both hormones influenced the three muscle dif- respectively). The ANOVA also showed that the Strain factor ferentiation markers considered, even if the E2 efficacy was less had a significant effect on the level of PKB (n=5 per strain, than IGF-I. In addition, E2 effects were completely prevented ANOVA, P<0.01). Muscles from mice of the PWD/PhJ strain had by the pure ER inhibitor. The contribute of both nuclear and lower levels of PKB than muscles in the A/J, BALB/cByJ or DBA/2J extra-nuclear action mechanisms on E2-induced modification mice (P<0.01, Tukey’s test). There were no significant differ- of differentiation markers was thus evaluated by pre-trating ences in MAPK(Erk1/2) between the strains. Results of the study cells with cycloheximide, actinomycin, and the palmitoylation show that the genetic variations influence CS activity and lev- inhibitor 2-Br, that prevents the membrane starting signals of els of some growth regulators in muscles of mice. This might both receptor isoforms. These data indicate that both E2- have a direct effect on muscle morphology and function. Thus dependent rapid signals and nuclear action are required for in studies of inbred mice can improve our understanding of the E2-induced L6 differentiation. In particular, ERα-dependent AKT underlying mechanisms of variation in muscular function. activation is necessary to control the first step of myogenic dif- Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ and Wackerhage H. ferentiation. Moreover, both receptors mediate the E2-induced Selective activation of AMPK-PGC-1a or PKB-TSC2-mTOR signaling can activation of p38 which, in turn, affects the expression of myo- explain specific adaptive responses to endurance or resistance train- genin and MHC. The contribute of ERα in activating the previ- ing-like electrical muscle stimulation. FASEB J 2005; 19: 786-8. ously identified E2-dependent effects has been evaluated by Brockmann GA, Kratzsch J, Haley CS, Renne U, Schwerin M, Karle S. Sin- using both ERα and ERβ selective agonists (PPT and DPN, gle QTL effects, epistasis, and pleiotropy account for two-thirds of the respectively) and by reducing ERα with specific siRNAs. As phenotypic F(2) variance of growth and obesity in DU6i x DBA/2 mice. expected, E2 was unable to induce AKT phosphorylation, Genome Res 2000; 10:1941-57. whereas the E2-dependent activation of p38 was still present, LightfootJT,TurnerMJ,PompD,KleebergerSR,LeamyLJ.Quantitativetrait in ERα-silenced cells. Intriguingly, E2 was not able any more to loci for physical activity traits in mice. Physiol Genomics 2008; 32:401-8. induce the Glut-4 translocation and the increase of myogenin α Spina RJ, Chi MM, Hopkins MG, Nemeth PM, Lowry OH, Holloszy JO. and MHC level in ER -depleted cells, thus confirming the piv- Mitochondrial enzymes increase in muscle in response to 7-10 days of otal role of ERα in the L6 differentiation. All together these data cycle exercise. J Appl Physiol 1996, 80:2250-4. indicate that E2, like other extra-cellular growth factors, modulates specific cell signals affecting the skeletal muscle devel- We thank Mrs Rita Zelniene for technical assistance. opment providing the basis of gender-related physiological differences in skeletal muscle recovery after damage. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

C115 C117 ERa-mediated rapid signals are requested for estradiol- induced skeletal muscle differentiation Effects of exogenously induced fever and hyperthermia on endocrine functions and behavior in the pre-pubertal rat P. Galluzzo, P. Bulzomi, F. Acconcia, V. Pallottini and M. Marino F. Mete1, E. Kilic2, A. Somay3 and B. Yilmaz2 University Roma Tre, Rome, Italy 1Pediatrics, Vakif Gureba Education and Research Hospital, Istanbul, β 17 -estradiol (E2) mediates a wide variety of complex biolog- Turkey, 2Physiology, Yeditepe University, Faculty of Medicine, ical processes. While E2 effects on the reproductive system are Istanbul, Turkey and 3Pathology, Vakif Gureba Education and quite well established, less is known about how it affects the Research Hospital, Istanbul, Turkey physiology of other tissues. Skeletal muscle expresses both estrogen receptor isoforms (ERα and ERβ). In addition, female Hyperthermia may cause pathological changes in various rat muscles show fewer histopathological changes after organs including the endocrine system and brain. In this study, repeated eccentric contractions than male muscles; ovariec- we examined effects of exogenously induced fever and hyper- tomized female rats exhibit higher indexes of exercise-induced thermia on adrenal and thyroid functions and behaviors in pre- muscle membrane damage which disappear in after estradiol pubertal male Sprague-Dawley rats. Three groups of 30-day treatment. the mechanisms underlying the role played by E2 old rats were used. Body temperature was increased to 39∞C and remain still elusive. In actively proliferating rat myoblast (fever; Group I) and 41∞C (hyperthermia; Group II) in a hyper- cells (L6) (grown in 10% serum), E2 rapidly increased the glu- thermia induction chamber for 30 min. The rats in the Group cose transporter type 4 (Glut-4) translocation at the cell mem- III served as control (36 ∞C). Temperature of the laboratory was branes; successively, the increase of well known differentiation 21∞C. Core temperature of the animals was monitored by using markers of myogenesis (i.e., myogenin and myosin heavy chain, a rectal probe throughout the experiments. All animals received MHC) was evidenced by Western blot analysis at 6 and 24 h, saline (0.2 ml x 4 times, ip) and were decapitated 48 h after the respectively, after E2 stimulation. Seven days after E2 treat- experiments (day 32). Blood samples were collected. Serum ment the alignment and fusion of L6 myoblasts into multinu- cortisol, fT3, fT4 and dehydroepiandrosterone (DHEA) levels cleated myotubes were visible. Next we compared the effect were determined by chemiluminescence assay. Pituitary and

66P Oral Communications adrenal glands were dissected out and processed for thetized with pentobarbital (70 mg/kg body weight)and brains histopathological examination. To assess the activity and anx- were quickly removed. The arcuate nucleus and the paraven- iety of the animals, the open field test and elevated-0-maze tricular nucleus (PVN) were extracted from 1mm slices with a test, respectively, were used in all groups 24h before (day 29) punch needle. Activity of the AMPK pathway was assessed by and after (day 31) hyperthermia induction. Experiments were measuring the phosphorylation levels of both AMPK and acetyl- approved by the local ethics committee. Results were statisti- CoA-carboxylase (ACC). Activity of the mTOR pathway was cally analyzed by using One-Way Analysis of Variance followed examined by the phosphorylation level of p70 S6 kinase (p70 by LSD test. S6K) and the S6 ribosomal protein. Serum cortisol levels (3.22±1.3) were significantly reduced Results: The AMPK pathway in the arcuate nucleus was proin the fever (1.3±0.9) and hyperthermia (1.09±0.7) induced gressively affected during hypoxia, as a result of diminished groups (p<0.01). Serum levels of thyroid hormones did not levels of both AMPK and ACC. This confirmed what we pre- significantly differ among the groups. DHEA values were viously reported on the whole hypothalamus after 6h of below the limit of detection in all groups. Histopathological hypoxia. Conversely, in the arcuate nucleus, the activity of examination revealed that fever caused hyperemia in the pitu- the p70 S6K and of it’s target S6 ribosomal protein were itary and adrenal glands. However, mild degeneration was markedly enhanced after exposure to hypoxia affording for observed in both glands in the hyperthermia group. Pro- an increased activity of the mTOR pathway. A cross-regula- gression time in the open field test was significantly tion between AMPK and mTOR seemed to proceed to con- decreased and anxiety test scores increased in animals trol food intake in the arcuate nucleus, which is consistent exposed to 39∞C compared to the control values (p<0.01). with anorexigenic signals led by ambient hypoxia. Such These parameters were more pronounced in the hyperther- modifications were not observed in the PVN, suggesting a mia group (p<0.01). In conclusion, hyperthermia induced specific response of the AMPK and mTOR pathways within stress may cause delayed reduction in serum cortisol levels the hypothalamus during environmental hypoxia. Icv injec- which may be associated with behavioral abnormalities in tions of the AMPK activator AICAR and of an inhibitor of pre-pubertal male rats. mTOR (rapamycin) will provide further knowledge of the molecular mechanisms that drive this hypoxia-induced Where applicable, the authors confirm that the experiments anorexia. described here conform with The Physiological Society ethical Conclusion: These findings provide support for the hypothesis requirements. that AMPK and mTOR interact in the arcuate nucleus to regulate feeding during acute environmental hypoxia. Simler N et al. (2007). J Appl Physiol 102, 2135-2141.

C118 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Involvement of both AMPK and mTOR pathways in the requirements. arcuate nucleus of rat hypothalamus during ambient hypoxia

N. Simler, T. Chaillou and X. Bigard

CRSSA, La Tronche, France C119 Acute environmental hypoxia is known to promote reduction of body weight and marked decrease in food intake, as a result Growth Hormone is present in Human Retinal Ganglion Cells of hypoxia per se. This alteration in feeding behavior is known and Correlates with Cell Survival as altitude–induced anorexia but the molecular mechanisms underlying such alterations remain unclear. AMP-activated pro- E.J. Sanders, E. Parker and S. Harvey tein kinase (AMPK) functions as a major regulator of cellular Physiology, University of Alberta, Edmonton, AB, Canada metabolism and recent data demonstrated that together with the mammalian target of rapamycin (mTOR), AMPK plays a crit- Locally synthesized growth hormone (GH) may act as a survival ical role in regulating food intake. Decreased AMPK activity in factor in a number of tissues (Sanders & Harvey, 2008). Exper- the hypothalamus reduces food intake and body weight, imental studies with chick retinal ganglion cells (RGCs) suggest whereas mTOR activation is required for the appetite-sup- that GH, synthesized within the developing retina, may have pressing response to a variety of anorexigenic signals. We pre- autocrine/paracrine roles in the regulation of the waves of cell viously reported that hypothalamic AMPK activity was reduced death characteristic of RGC differentiation (Sanders et al. 2008). by hypoxic stimulus concomitant to hypophagia in rats, There is also evidence that endogenous GH may have a similar response which could be linked to the transient hyperglycemia neuroprotective function in the adult rat retina, however, there observed at the same time (Simler et al). Here we determine is no information concerning the possible presence or action whether this reduction occurs specifically in the arcuate nucleus of GH in the human retina. In this study we show, for the first of the hypothalamus and if the mTOR signaling could contribute time, that GH is present in the human retina and that the local to the mechanisms of altitude-induced anorexia. expression of retinal GH correlates with RGC cell survival. GH- Adult male wistar rats were either submitted to normobaric immunoreactivity, identical in size to that in the pituitary gland hypoxia (10% O2) during 2 or 6 h, or maintained in normoxia. (22kDa), was detected by Western blotting as a single band in At the end of experimental conditioning, animals were anes- extracts of cadaver retinas (obtained with ethical approval).

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Using tissue sections from eyes collected post-mortem (with to form HI (10.39 to 8.00 ln ms2) and BELOW (7.79 to 5.84 ln ethical approval), this immunoreactivity was largely confined ms2) groups. Independent sample t-tests were used between to large, rounded cells in the GLC (ganglion cell layer) of the groups, with repeated measures ANOVA to identify changes neural retina and co-localized in RGCs, labeled by a synuclein from baseline within groups. Mean R-R interval was not signi- antibody. GH receptor (GHR) immunoreactivity was similarly ficantly different between the groups at any time point indi- located in the GCL and 35% of these cells were both GH- and cating that mean R-R interval values are not wholly dependent GHR- positive. None of the cells that had GH immunoreactiv- on vagal activity. Meanwhile, vagal activity (lnHF and RMSSD) ity were apoptotic, as determined by terminal deoxynucleotidyl was reduced in both groups 5 min post-exercise (p<0.01). transferase dUTP nick end labeling (TUNEL). In summary, these Thereafter, HI and BELOW reacted differently with BELOW results demonstrate the presence of GH and its receptor in RGCs returning to, then overshooting baseline values more rapidly of the human retina in which GH expression correlates with cell than HI in both vagal measures. Interestingly those with lower survival. These results are consistent with our earlier in vivo and vagal activity return to baseline quicker and even overshoot in vitro experimental studies in chick embryos that show baseline. This may indicate that individuals with vagal activity autocrine or paracrine actions of GH in RGCs that promote cell below a certain threshold but above another level (as these are survival during development and suggest that GH is a novel young, healthy subjects with relatively high basal vagal activ- neurotrophic factor for human RGCs. ity), may gain cardioprotection following hard exercise whereas Sanders EJ & Harvey S (2008) Peptide hormones as growth and differ- the HI group may already have a basal vagal tone above the entiation factors during development. Developmental Dynamics 237: threshold to elicit cardioprotection. 1337-1352 Sanders EJ, Parker E & Harvey S (2008) Growth hormone-mediated survival of embryonic retinal ganglion cells: signaling mechanisms. Gen- eral and Comparative Endocrinology 156: 613-621

Supported by the Natural Sciences and Engineering Research Council of Canada

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

Figure 1: lnHF (ln ms2) during recovery from exercise in HI and LOW groups. * shows significant differences from baseline (p≤0.01); + shows differences between groups (p≤0.05). C120 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Recovery from exercise in humans: dependence on vagal requirements. activity

V. Gladwell1, G. Sandercock1 and S. Dawson2 C121 1BiologicalSciences,UniversityofEssex,Colchester,UKand 2Biomolecular and Sports Science, University of Coventry, An evaluation of autonomic nervous system modulation in Coventry, UK humans during written exam stress Arrhythmic event risk is augmented with low vagal activity. I. Denna, G. Sandercock and V. Gladwell Exercise training can reduce this risk by increasing vagal activity. The reduction in vagal activity immediately following exer- Biological Sciences, University of Essex, Colchester, UK cise may increase the risk of cardiac arrhythmias and sudden Cognitive and emotional stress induce measurable cardiovas- cardiac death. To date the effect of baseline vagal activity on cular responses including increases in heart rate (HR) and blood the recovery of cardiac autonomic activity up to 1 hour post- pressure. Revision, in preparation for exams, is a highly cogni- exercise has not been examined. 28 healthy, untrained volun- tively demanding process but it is less emotionally stressful than teers (10 females, 21.0±2.6 years) performed a 20 min hard the taking of a written exam were performance on the day is intensity (3mmol.l-1 blood lactate concentration) constant load of consequence. This study examined the cardiac autonomic test. ECG data were collected for 5 min at baseline and then nervous system (ANS) modulation in response to the stress for 5 min epochs at: 5, 15, 30, 45 and 65 min post-exercise. induced by a scheduled University written exam compared to Heart rate variability (HRV) analysis to assess cardiac autonomic a revision session at home using non invasive heart rate vari- activity was performed to obtain: mean R-R interval, root mean ability (HRV). Following ethical approval, eight (2 female) squared of successive differences (RMSSD) and frequency analy- healthy students undertaking a Masters Degree in Sports Sci- sis of high frequency spectral power (HF), both representing ence (age: 25.3.3±3.1years, height: 171.8±6.46cm, weight vagal activity (HF was naturally log transformed prior to analy- 68.8±14.5kg) gave informed consent to participate. At the first sis). Volunteers were divided according to basal HF by finding visit all the steps of the protocol were explained and partici- the median lnHF value of all volunteers at rest (7.89 ln ms2) and pants were shown how to attach a Polar S810i HR monitor (Polar then grouping the volunteers above or below this threshold Electro OY, Kempele, Finland) for RR-interval recording. Par-

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whilst statistical analysis was performed using 2-factor repeated ticipants were instructed to make two separate RR-interval measures ANOVA with Bonferroni post tests, P<0.05 was con- recordings; at the start of a revision session at home and at sidered significant. During control, VNS decreased heart rate the start of the written exam. The revision session was recorded from 147.8±6.5 to 88.9±6.4 bpm (P<0.001) whilst significantly at the same time of day as the scheduled exam (MSc in Sports increasing both ERP and VFT from 130.0±6.7 to 147.8±6.7ms Science). The HR data was analysed post hoc using Kubios HRV (P<0.001) and from 2.2±0.3 to 5.3±0.5mA (P<0.001) respec- analysis software, (University of Kupio, Finland). Following the tively. During perfusion with atropine, the effects of VNS on removal of any artefacts, the data were divided into 12, five heart rate (143.1±9.8 to 142.2±9.3 bpm, P>0.05) and ERP min epochs. Results from all epochs were averaged. Three (137.8±5.7 to 133.3±4.1 ms, P>0.05) were lost whilst the indices of vagal activity were calculated: the root mean square increase in VFT was preserved where VNS increased VFT from of successive differences (RMSSD), natural logarithm of high 2.0±0.3 to 4.7±0.7mA (P<0.001). These data suggest an acetyl- frequency power (frequency domain) (lnHF), and standard devi- choline / muscarinic receptor independent mechanism under- ation of instantaneous beat to beat variability (SD1). Paired lying the vagal protection of the heart against VF. The context sample t-tests were used to compare the differences between of these findings needs further exploration in relation to the the days, level of significance (p≤0.05). HR during exam was possibility of alternative neurotransmitters and the vagus-NO 9.7 beats per minute (bpm) higher compared with revision pathway. (82.6±9.0 bpm Vs 72.9±9.8 bpm) (p value: 0.002). There was Brack KE, Patel VH, Coote JH Ng GA 2007. J Physiol;583.2:695-704. a significant decrease in all three indices of vagal activity during exam: RMSSD, lnHF, SD1 (with p values: 0.01; 0.03; 0.017, Ng GA, Brack KE, Coote JH 2001. Exp Physiol;86.3:319-329. respectively). These results suggest that there is a signficant Ng GA, Brack KE, Patel VH, Coote JH 2007. Cardiovascular reduction in cardiac vagal activity under conditions of emo- Research;73:750-760. tional stress and cognitive challenge compared with the response to cognitive challenge alone. Moreover, this suggest We would like to acknowledge the BHF and the Garfield Weston that HRV can be used to assess changes in the ANS with men- Trust for financial support tal stress. Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

C122 C123 The protective effect of vagus nerve stimulation against ventricular fibrillation is preserved during muscarinic Action potential, sodium and gap junction channels in rat receptor blockade pulmonary vein myocytes K.E. Brack1, V.H. Patel1, J.H. Coote2 and G. Ng1 Y. Song1, G. Hao1, M. Boyett1, X. Yang2, Y. Du2 and Z. Shui1,2 1 Cardiovascular Sciences, University of Leicester, Leicester, UK and 1Cardiovascular Research Group, University of Manchester, 2 Pharmacology, University of Birmingham, Birmingham, UK Manchester M13 9NT, UK and 2Departments of Cardiovascular Classically the effect of vagus nerve stimulation is considered Surgery and Cardiology, Union Hospital, Wuhan, China to be entirely mediated via muscarinic receptor activation. We The pulmonary veins (PV) are important in the initiation and have previously shown that direct vagus nerve stimulation maintenance of atrial fibrillation, which may be related to slow (VNS) reduces the slope of action potential duration restitution excitation-conduction in the PV (Nattel, 2002; Aldhoon et al. whilst simultaneously protecting the heart against ventricular 2009). In this study, the essential factors for action potential fibrillation (VF) initiation in the absence of background sym- conduction in the PV have been investigated with single pathetic activity (Ng et al, 2007). We have extended this find- myocytes isolated from rat left atrium (LA) and four PVs (left ing to show that this effect is mediated via nitric oxide (NO) superior LSPV, left inferior LIPV, right superior RSPV and right (Brack et al, 2007). In this study our aim was to determine if the inferior RIPV). Whole cell current clamp and voltage clamp were protective effects of VNS are dependent on muscarinic recep- used to record action potential (AP) and the fast Na+ current tor activation. Adult male New Zealand rabbits (2.9±0.1Kg, (I ). Single cell real-time PCR was employed to measure the n=9) were used. The isolated heart preparation with intact auto- Na abundance of mRNAs for Na 1.5 and gap junction channel nomic nerves was obtained under propofol anesthesia (1 v (Cx43), and the expression of Cx43 protein on cell membrane mg/kg, i.v.) as previously described (Ng et al, 2001). The cer- was located by immunofluorescence. The results showed that: vical vagus nerves were stimulated at 9.6±1.6Hz, 7.2±1.1V. [i] the maximum velocity of AP phase 0 upstroke was signifi- Ventricular effective refractory period (ERP) was measured with cantly faster (62±10.5 V/s) in LA myocytes than in all PV the single extrastimulus method following a 20-beat drive train myocytes (Fig. 1 A and D); [ii] INa density was remarkably higher (300ms cycle length) whilst ventricular fibrillation threshold (16.4±2.9 pA/pF) in all PV myocytes (apart from RSPV (VFT) was determined as the minimum current required to myocytes) than in LA myocytes (Fig. 1 B, C and E); [iii] the rel- induce sustained VF with rapid pacing (30 stimuli x 30ms). ERP ative abundance of Na 1.5 mRNA in the LSPV and LIPV myocytes and VFT were studied at baseline and during VNS, before and v was 1.7 times higher than in LA myocytes (Fig. 1F), whereas during perfusion with 0.1μM atropine. Data are mean±SEM,

69P Oral Communications no difference in the Cx43 mRNA abundance between LA and PV myocytes was observed; and [iv] the majority of Cx43 C124 expression in LA myocytes was located at the cell-ends in the intercalated disc, whereas the Cx43 expression in PV myocytes Role of AV node functional properties in the generation of was distributed at both cell-ends and in the lateral cell mem- ventricular rhythms during atrial tachyarrhythmias brane. It is surprising and interesting that I density and the Na M. Masè1, L. Glass2 and F. Ravelli1,3 abundance of Nav1.5 mRNA in PV myocytes are inconsistent with the depolarization velocity of AP as compared with those 1Department of Physics, University of Trento, Povo, Trento, Italy, in LA myocytes. In general, the conduction velocity is mainly 2Department of Physiology, Centre for Nonlinear Dynamics in associated with Na+ and gap junction channels as well as their Physiology and Medicine, McGill University, Montreal, QC, Canada 3 functional balance, and INa is the large current that has suffi- and Fondazione Bruno Kessler FBK, Povo, Trento, Italy cient reserve or redundancy for the fast depolarization. There- The atrioventricular (AV) node is the specialized conduction fore, it is concluded that the difference in the cellular distribu- system, which provides electrical coupling between the upper tion of Cx43 protein in PV myocytes is predominant to slow and lower chambers of the heart. Thanks to its peculiar anatom- excitation-conduction as a result of anisotropic properties of ical and functional properties, the AV node plays a key role in the tissue more susceptible to atrial fibrillation. both physiological and pathological conditions. In particular, at high atrial rates, it protects the heart from life-threatening ventricular arrhythmias. Despite the impressive amount of information concerning AV nodal conduction, the actual role played by AV node functional properties (as nodal recovery (1;2) and concealed conduction (3)) in the generation of ventricular rhythm during spontaneous atrial tachyarrhythmias, as atrial flutter (AFL) and fibrillation (AF), is still incompletely understood. To clarify these aspects, AV synchrograms (4), displaying the time course of the phase of atrial activation in ventricular cycle, were constructed and quantified in 12 patients (62.7+/-16.4 years) with AFL and AF. Atrial electrograms and ECGs were recorded during an electrophysiological study after light sedation with diazepam (10 mg i.m.). The occurrence (i.e. percentage of synchronized beats, SB) and order (i.e. locking ratio of ventricular beats over atrial beats, LR) of synchronization between atrial and ventricular electrical activities were Figure 1. Action potential, I and Na 1.5 mRNA in LA and PV myocytes Na v analyzed in relation with atrial rate changes and arrhythmia A, recordings of action potentials from LA and PV myocytes. B and C, INa irregularity. Synchronized epochs were identified in all patients, traces from a LA and a LIPV myocytes during voltage clamp. D, maximum demonstrating the non-randomness of ventricular rhythm in velocity of phase 0 upstroke of AP in the various myocytes (n=10~19). E, AF. The occurrence of synchronization decreased at decreas- peak density of INa (n=11~15). F, relative abundance of Nav1.5 mRNA (n=7). Significantly different * from LA myocytes and # from LSPV myocytes (P < ing atrial cycle length, ACL, (SB=71.2+/-28.0% at ACL=228.6+/- 0.05, one-way ANOVA). 33.0ms to SB=47.1+/-24.2% at ACL=174.7+/-16.4 ms, p<0.05) Nattel S (2002). Nature 415, 219-226. and at increasing atrial cycle length variability (SB=70.1+/-31.0% Aldhoon B et al. (2009). Physiol Res. (Epub ahead of print). at stdACL=4.5+/-1.7ms in AFL vs SB=48.2+/-21.4% at stdACL=24.4+/-12.9ms in AF, p<0.05). The decrease in atrial This work is supported by the British Heart Foundation and the cycle length involved a global decrease in locking ratio from National Natural Science Foundation of China. LR=0.36+/-0.11 to LR=0.28+/-0.09 (p<0.005). More specifically, AV synchrograms showed that locking orders organized accord- Where applicable, the authors confirm that the experiments ing to a Farey sequence at increasing atrial frequency (5). The described here conform with The Physiological Society ethical decrease of synchronization and concurrent decrease in lock- requirements. ing ratio (corresponding to an increase in the number of blocked atrial beats) at increasing atrial frequencies is consistent with an increased contribution of concealed conduction effects (3), while the well-defined sequence of locking orders at changing atrial frequency is a clear marker of the nonlinear recovery of excitability in the nodal tissue (2;5). Thus these results provide evidence for a crucial role of AV node functional properties, as nodal recovery and concealed conduction, in the deter-

70P Oral Communications mination of ventricular rhythm during atrial tachyarrhythmias. fluorescence (3) and cellular fractionation (1). Three inde- Since both properties progressively hinder AV conduction in pendent experiments were performed for each condition presence of high atrial rates, they contribute to protect the tested. Our results show that both endogenous MR in the left heart from dangerous high frequency ventricular rhythms. ventricle and transfected MR in HL-1 cells are detected exclu- Billette J & Amellal F, in Atrial-AV Nodal Electrophysiology: A View from the Millennium, edited by Mazgalev T & Tchou P (Futura Publishing, sively as nuclear chromatin-bound factors. Depletion of corti- Armonk, NY, 2000), pp. 155-174. costeroids by ADX in mice or by culturing HL-1 cells with charcoal-striped serum or serum-free medium did not affect MR Shrier A et al. (1987). Circulation 76, 1196-1205. chromatin binding. Deletion of nuclear localization signal 0 Langendorf R (1948). Am Heart J 35, 542-552. (NLS0) (1) rendered mutants exclusively cytoplasmic, while Masè M et al. (2006). Comp Cardiol 33, 761-764. mutations in NLS1 partially redistributed MR to the cytoplasm. Chialvo DR & Jalife J (1987). Nature 330, 749-752. Taken together, our results suggest that MR is constitutively nuclear in mouse cardiac myocytes, independently of corti- Supported by PAT (Health Research Grant 2007). costeroids. The subcellular localization of the naïve receptor is Where applicable, the authors confirm that the experiments mainly determined by NLS0. This stands in sharp contrast to described here conform with The Physiological Society ethical the behaviour of MR in other cell types, where it appears evenly requirements. distributed over the nucleus and the cytoplasm. The differential behaviour of MR in cardiac myocytes might have important consequences for its function, both in genomic and non- genomic pathways. C126 (1) Mendez J & Stillman B (2002). Mol Cell Biol 20, 8602-8612.

Corticosteroid-independent nuclear localization of (2) Claycomb et al. (1998). Proc Natl Acad Sci USA 95, 2979-2984. mineralocorticoid receptor in mouse cardiac myocytes (3) Walther RF et al. (2005). J Biol Chem 280, 17549-17561. I. Hernandez-Diaz1,4, T. Giraldez3, M. Arnau2, V. Smits1,4 and D. Alvarez de la Rosa1,4 This work was funded by the Spanish Ministry of Science (grant BFU2007-61148). 1Pharmacology, University of La Laguna, La Laguna, Santa Cruz de Tenerife, Spain, 2Animal Care Services, University of La Laguna, La Where applicable, the authors confirm that the experiments Laguna, Santa Cruz de Tenerife, Spain, 3Research Unit, Hospital described here conform with The Physiological Society ethical Universitario Ntra. Sra. de Candelaria, Santa Cruz de Tenerife, Spain requirements. and 4Institute of Biomedical Technologies, University of La Laguna, La Laguna, Santa Cruz de Tenerife, Spain C127 Aldosterone plays a key role in extracellular volume homeostasis by promoting sodium reabsorption in the kidney distal tubule. Volumetric analysis in Xenopus laevis oocytes expressing the The accepted model for aldosterone action involves the bind- rat g-aminobutyric acid transporter GAT1 and the not ing of the hormone to the mineralocorticoid receptor (MR) in functional mutant Q291N the cytoplasm and subsequent translocation of the aldosterone-MR complex to the nucleus, where it acts as a tran- M. Santacroce, M. Castagna and V.F. Sacchi scription factor. MR is also expressed in non-epithelial tissues Department of Molecular Sciences Applied to Biosystems, Università such as the heart, where its physiological role is unclear. How- degli Studi of Milan, Milan, Italy ever, inappropriate MR activation leads to the development of cardiac fibrosis and renovascular disease. To improve our under- Water transport across membranes is a fundamental process standing of MR functional roles in the heart, we investigated for cells. Currently accepted water transport pathways are the the subcellular localization of the receptor in mouse left ven- lipid bilayer itself and specialized proteins, the aquaporins. tricle and in immortalized cardiac myocytes in the absence or Recently it has been shown that several cotransporters, such as presence of corticosteroids. Tissues were obtained from adre- SLGT1 and GAT1 mediate a water influx across membranes (Loo nal-intact or adrenalectomized (ADX) 8-week-old C57BL/6 et al., 1999; Lapointe et al., 2002). Some observations support mice. Preparative surgery was performed on mice anesthetized the idea that local osmotic gradients built up immediately after by intraperitoneal administration of 50 mg/kg ketamine and 1 cotransport activity are fully responsible for the cell swelling mg/kg medetomidine. After the procedure, anaesthesia was (Lapointe et al., 2002). An alternative hypothesis suggests a reversed with 1 mg/kg atipamezole. Mice were kept for four direct coupling of water, ion and solute (Zeuthen et al., 1996). days after ADX with free access to saline prior to harvesting the γ-Aminobutyric acid (GABA) is the major inhibitory neuro- tissue. The procedures were approved by the local ethics com- transmitter in mammalian brain and the GABA transporter GAT- mittee and accorded with current national legislation. Three 1, belonging to the neurotransmitter sodium symporter fam- independent experiments with 3 mice in each group were per- ily (NSS), is an integral membrane protein responsible for the formed. MR localization in the left ventricle was studied by reuptake of GABA from the synaptic cleft. cellular fractionation (1). MR subcellular localization was fur- Glutamine 291 is a strictly conserved residue in all members of ther tested by transient transfection of wild type or mutant MR the NSS family. It has been previously published that Q291 in HL-1 cells derived from adult mouse cardiac myocytes (2). mutants cannot transport GABA or give rise to currents even MR localization in HL-1 cells was studied by indirect immuno- though they are targeted to the plasma membrane (Mari et al., 2006).

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In order to better understand water transport in cotransporters name of the first proteins discovered PSD95, DLG and ZO1). we verified whether the not functional mutant GAT1 Q291N is These proteins contain multiple protein-protein interaction able to mediate water transport. domains, that bring together and localize transporters, chan- To this aim, we performed six independent volumetric analy- nels, enzymes and receptors in specific plasma membrane sis experiments in hypoosmotic conditions (ΔmOsm = 167) domains [2]. using Xenopus laevis oocytes expressing the wild type (wt) and In the C-terminal of EAAC1 there is a class I PDZ proteins target the mutated protein (Dorr et al., 2007). sequence (-SQF), whose removal induced internalization of the The expression of GAT1 on oocyte surface was confirmed by transporter in endocytotic compartments (Fig.1). Therefore, radiolabelled aminoacid transport experiments. PDZ proteins play a key role in regulating EAAC1 expression in In each experiment volumetric variations were measured in the plasma membrane, but none of the EAAC1 interacting part- three groups of 7-14 oocytes (GAT1, GAT1 Q291N and control ners have been so far identified. Aim of this work was to iden- oocytes) and the mean permeability factors (Pf) of each group tify the PDZ proteins interacting with EAAC1 and to verify were calculated. The Pf of GAT1 expressing oocytes ranged whether they play a role in the EAAC1 localization at plasma from 1.41×10-3 ± 1.93×10-4 cm/s to 1.99×10-3 ± 7.74×10-5 cm/s membrane. (mean ± S.E.); the Pf of Q291N ranged from 1.33×10-3 ± In epithelia, possible candidate are members of the NHERF ( 6.44×10-5 cm/s to 1.75×10-3 ± 7.27×10-5 cm/s (mean ± S.E.); Na+/H+ Exchanger Regulatory Factor) family, PDZ proteins that the Pf of control oocytes ranged from 7.30×10-4 ± 4.57×10-5 organize the apical domain of the plasma membrane [3]. cm/s to 1.46×10-3 ± 5.63×10-5 cm/s (mean ± S.E.). The interaction between PDZK1/NHERF3 and EAAC1 was As expected, in five experiments out of six the Pf of GAT1 was proved by means of several experiments. By yeast two hybrid higher than the Pf of control oocytes (p<0.05 Student’s t-test). system assay [4], we found that the C-terminal tail of EAAC1 Interestingly, in five experiments out of six also the Pf of GAT1 (last 40 or 26 aminoacids) directly interacted with PDZK1, and Q291N was significantly higher than control Pf value (p<0.05). the interaction required the first and second PDZK1 PDZ Our experiments thus showed that both wt protein and mutant domain (n=3). Affinity chromatography [5] confirmed that mediate a water influx across Xenopus oocyte membrane. PDZK1 is retained by the C-terminal tail of wild type EAAC1 In conclusion though at the moment we cannot choose for one (WT) (n=4). of the above described hypothesis regarding water transport Co-immunoprecipitation experiments demonstrated the bind- through cotransporters, our results indicate that, under par- ing of PDZK1 to wild type transporter (WT GFP-EAAC1), but ticular conditions, a water influx occurs both when the cotrans- not to a mutant transporter lacking the PDZ target sequence port mechanism can take place (GAT1 wt) and when it cannot (ΔTSQF GFP-EAAC1) (n=3) (Fig.2). Similar experiments per- (GAT1 Q291N). formed in intestinal tissues confirmed the EAAC1/PDZK1 inter- Dorr R, Ozua M & Parisi M (2007). J Neurosci Meth 161, 301–305. action in vivo (n=3). Lapointe J-Y, Gagnon MP, Gagnon DG & Bissonnette P (2002). Biochem. In agreement with these results we found co-localization of Cell Biol. 80, 525–533. PDZK1 with WT EAAC1 in over-expressing system (MDCK) as Loo DDF, Hirayama BA, Meinild A-K, Chandy G, Zeuthen T & Wright well as in native tissues (n=3). EM (1999). J Physiol 518, 195—202. PDZK1 not only co-localized but also functionally interacted with the transporter, because the presence of mycPDZK1 Mari SA,Soragna A, Castagna M, Santacroce M, Perego C, Bossi E, Peres ± ≤ A & Sacchi VF (2006). Cell. Mol. Life Sci. 63, 0100–0111. caused a 1.34 0.13 fold increase (T-test, p 0.05) in the WT EAAC1 surface activity, without affecting ΔTSQF activity. Zeuthen T, Hamann S & La Cour M (1996). J Physiol 497, 3-17. Thus, PDZK1 interacts with EAAC1 in vitro e in vivo, regulating Where applicable, the authors confirm that the experiments its localization and function at plasma membrane. Further stud- described here conform with The Physiological Society ethical ies are needed to clarify the molecular mechanism by which requirements. PDZK1 controls the membrane localization of EAAC1.

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The adaptor protein PDZK1 interacts with the glutamate transporter EAAC1 and regulates its surface expression E.S. Di Cairano, G. Fantin, A. D’Amico, A. Soragna, V.F. Sacchi and C. Perego Dep. of Molecular Science applied to Biosystem, University of Milan, Milan, Italy The glutamate transporter EAAC1/EAAT3 is expressed in the central nervous system (CNS), where it mediates the uptake of the neurotransmitter from the synaptic cleft. It is also expressed in non neuronal tissues, in particular intestine and kidney, where it represents the main pathway of amminoacid reabsorption [1]. Its surface expression is strictly regulated by interaction with accessory protein, e.g. PDZ proteins (from the

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Navarro-Lérida I, Martínez-Moreno M, Ventoso I, Alvarez-Barrientos A, Rodriguez-Crespo I. Binding of CAP70 to inducible nitric oxide synthase and implications for the vectorial release of nitric oxide in polarized cells. Mol Biol Cell., 2007 18: 2768-2777.

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

C129

The Importance of Flavonone B-Ring Catechol in Suppression of Intracellular Fenton Reactions R.J. Naftalin1, E. Vlachodimitropoulou1 and P. Sharp2 Immunofluorescence. MDCK cells were stably transfected with the indicated GFP-EAAC1 mutant transporters, fixed and analyzed by confocal 1Physiology, King’s College London, London, UK and 2Nutrition, microscopy. WT EAAC1 is mainly expressed at the plasma membrane, King’s College London, London, UK whereas ΔTSQF mutant is expressed in an endocytotic compartment. Scale bar, 10 μm Quercetin, like dehydroascorbate (DHA) is transported into cells via the passive glucose transporters, GLUTs 1 and 4(1). We have demonstrated that quercetin, like ascorbate, acts as an electron donor to the erythrocyte transmembrane oxidoreductase, 2+ 3+ DcytB(3;4). Ascorbate also interacts with Fe /Fe and H2O2 to produce very reactive OH. radicals. In contrast to ascorbate, quercetin is a known suppressant of the Fenton reaction (2). We have compared the iron chelating efficacy of several flavonones to determine which hydroxyl groups suppress intracellular free OH. radical formation. Fol- lowing overnight loading of Madin Darby canine kidney cells with 20μM ferrous sulphate, the cells were washed free of adherent extracellular Fe2+ with 300μM desferrioximine, an impermeant iron chelator which suppresses extracellular Fen- ton reactions, then exposed to varying concentrations of per- μ . meable polyphenols, 100 M H2O2 and the luminescent OH radical sensor L-012. The intracellular luminescence was assayed in a 96 well luminescence plate reader. The I.C.50 of quercetin and luteolin-induced inhibitions of intracellular iron-dependent luminescence was 3.4±0.9 and 3.7±0.8 μM respectively and with 3,5-dihydroxyflavone and chrysin was 9.4±1.5 μM and 18.1±14 μM (S.E.M, 2 experiments, with each compound, 6 concentrations and 3 replicates). The differences between the efficacies of the flavonone suppression of the Fenton reaction shows the key importance of Co-immunoprecipitation. Lysates of MDCK cells stably expressing EAAC1 the 3’-4’ dihydroxylated B ring as an Fe2+ chelator, present in wild type or ΔTSQF were immunoprecipitated with an anti-PDZK1 antibody; the immunocomplexes were resolved by 9% SDS-PAGE and immunostained both quercetin and luteolin. with an anti-GFP antibody. 10% of the cell lysate used in the immunopre- Since human ingestion of the natural product quercetin, even cipitation assay was probed (Lysate). PDZK1 antibody was able to co- in large quantities, is non-toxic (5), it may be a useful thera- immunoprecipitate only WT EAAC1 from cell lysates. peutic agent against oxidative stress induced by iron overload, Danbolt NC. Glutamate Uptake. Prog Neurobiol., 2001 65(1): 1-105 as in thallasemia and pre-eclampsia. 1. Cunningham, P et al (2006) J Biol Chem 281. 5797-5803. Hernando N, Wagner CA, Gisler SM, Biber J, Murer H. PDZ proteins and proximal ion transport. Curr Opin Nephrol Hypertens., 2004 13: 2. Yu, F et al (2008)J Agric Food Chem 56.730-735 569-574. 3. McKie AT et al (2001)Science 291.1755-1759. Brône B, and Eggerment J. PDZ proteins retain and regulate membrane 4. Vlachodimitropoulou E et al (2008)J Physiol Biochem 64.326. transporters in polarized epithelial cell membranes. Am J Phiysiol, 2005 288: 20-29. 5. Harwood M, et al (2007)Food Chem Toxicol 45. 2179-205. Anzai N, Miyazaki H, Noshiro R, Khamdang S, Chairoungdua A, Shin HJ, Where applicable, the authors confirm that the experiments Enomoto A, Sakamoto S, Hirata T, Tomita K, Kanai Y, Endou H. The mul- tivalent PDZ domain-containing protein PDZK1 regulates transport described here conform with The Physiological Society ethical activity of renal urate-anion exchanger URAT1 via its C-terminus. J. Biol. requirements. Chem., 2004 279: 564-573.

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Where applicable, the authors confirm that the experiments C130 described here conform with The Physiological Society ethical requirements. Transport of the photodynamic therapy agent 5- aminolevulinic acid by the amino acid transporter PAT1 (SLC36A1) and the dipeptide transporter PepT1 (SLC15A1) C131 C. Anderson1, M. Jevons1, T. Muthusamy2, S. Woods1, N.J. Conlon1, V. Ganapathy2 and D.T. Thwaites1 Megalin binds to NHERF1 and NHERF2 scaffold proteins 1Epithelial Research Group, Institute for Cell & Molecular Biosciences, D. Hryciw1, C. Slattery2, K. Jenkin1 and P. Poronnik3 2 Newcastle University, Newcastle upon Tyne, UK and Dept. of 1School of Biomedical and Health Sciences, Victoria University, Biochemistry and Molecular Biology, Medical College of Georgia, Melbourne, VIC, Australia, 2UCD School of Biomolecular and Augusta, GA, USA Biomedical Science, UCD Conway Institute, Dublin, Ireland and 3 5-Aminolevulinic acid (ALA) is a pro-drug used in photodynamic School of Medical Sciences, Royal Melbourne Institute of therapy, fluorescent diagnosis and fluorescent-guided resec- Technology, Bundoora, VIC, Australia tion which produces selective accumulation of protoporphyrin Albumin endocytosis in the renal proximal tubule is regulated IX in tumour tissue (1). Relatively high oral doses of ALA are by a number of transmembrane and accessory proteins includ- used to obtain protoporphyrin IX accumulation in colonic ing the scavenger receptor megalin. Previously we have demon- tumours as there is also accumulation in normal gastrointesti- strated an essential role for the scaffold proteins NHERF1 and nal mucosa. Due to the structural similarity of ALA and GABA, NHERF2 in albumin uptake. NHERF1 and NHERF2 are PDZ + a substrate for the H -coupled amino acid transporter PAT1 domain containing proteins that interact with specific (SLC36A1), we investigated the role of PAT1 in luminal ALA sequences that form a PDZ binding domain (S/TXXΦ) in the C- 3 3 μ uptake. [ H]ALA and [ H]amino acid uptake (10-100 M, 0.5- terminus of proteins. Interestingly, megalin contains a func- μ -1 + 5 Ci.ml , Na free, pH 5.5 buffer) were measured across the tional PDZ binding domain (SDV), however the interaction with apical membrane of human intestinal Caco-2 cell monolayers the scaffold proteins NHERF1 and NHERF2 has not been inves- ∞ (5min, 37 C) grown on permeable filters and in PAT1- or PepT1- tigated. ∞ expressing X. laevis oocytes (40min, 22 C), as described pre- In this study we investigated if there is an interaction between ± viously (2). Data are mean SEM (n) with ANOVA plus Tukey megalin and NHERF1 and NHERF2, and then characterized the post-test. In Caco-2 cell monolayers, ALA (10mM) inhibited api- specific domains required for this interaction. Firstly, we inves- cal uptake of several PAT1 substrates (e.g. GABA) but not that tigated if the proteins co-localize in a proximal tubule cell model, of other amino acids (e.g. methionine). In PAT1-expressing the opossum kidney (OK) cell line. Confocal analysis of OK cells 3 oocytes, [ H]ALA uptake was significantly greater than that in demonstrated that the distribution of megalin was predomi- water-injected oocytes at pH 5.5 (p<0.001) and pH 6.5 (p<0.01) nantly apical with some cytosolic localization. Importantly, 3 but not at pH 7.4 (p>0.05). PAT1-mediated [ H]ALA uptake was NHERF1 had a strong apical localization which overlapped with ± ± reduced (p<0.001) by 91.6 2.7% (n=20) and 96.4 1.7% (n=20) megalin. Further, as previously described (Hryciw et al, 2006) by GABA and 5-hydroxy-tryptophan [OH-Trp, a PAT1 inhibitor NHERF2 was predominantly cytosolic, and this protein co-local- (3)], respectively (both 20mM). ALA is known to be a PepT1 ized with megalin in this region. This indicated that the pro- 3 substrate (4). In PepT1-expressing oocytes, [ H]ALA uptake teins had an overlap in distribution in proximal tubule cells. Fur- was inhibited by the PepT1 inhibitor 4-aminomethylbenzoic ther, immunoprecipitation experiments were performed using ± acid [AMBA, 30mM (5)] by 81.8 3.9% (n=20). Rheogenic trans- anti-megalin, anti-NHERF1 and anti-NHERF2 antibodies that port was measured by two-electrode voltage-clamp in PAT1- were incubated with rat kidney lysate. The immunoprecipitates + or PepT1-expressing oocytes (-60mV, Na free, pH 5.5). ALA were analysed by Western blot analysis using the anti-NHERF1 + induced current consistent with H /ALA symport with a Km of and anti-NHERF2 and anti-megalin antibodies, respectively. ± ± 1.6 0.9 mM for PepT1 (n=4) and 10.4 5.6 mM for PAT1 (n=4). These studies clearly indicated that megalin bound to NHERF1 In Caco-2 cells, OH-Trp and AMBA significantly (p<0.001) inhib- and NHERF2 in vivo. To determine which domains in NHERF1 ited apical ALA uptake, in an additive manner. The relative and NHERF2 were required for this interaction, GST fusion pro- expression of PAT1 and PepT1 will determine ALA uptake in nor- teins were generated as described previously (Hryciw et al. mal mucosa and gastrointestinal tumours and, in turn, influ- 2006; Lee et al 2007). These fusion proteins included the full ence both ALA bioavailability and tumour-specific accumula- length NHERF proteins as well as their 2 PDZ domains (PDZ1 tion of protoporphyrin IX. and PDZ2) and C-terminal ezrin binding domain. Incubation 1. Fukuda H et al. (2005) Int J Biochem Cell Biol 37, 272-276 with rat kidney lysate and analysis by Western blot analysis indi- 2. Anderson CMH et al. (2009) J Physiol 587, 731-744 cated that megalin bound to PDZ2 of NHERF1 and PDZ2 and 3. Metzner L et al. (2005) FASEB J 19, 1468-1473 the C-terminal ezrin binding domain of NHERF2. Finally, we used fusion proteins to determine if the C-terminus of megalin was 4. Doring F et al. (1998) J Clin Invest 101, 2761-2767 the site of interaction with NHERF1 and NHERF2. GST-pull down 5. Meredith D et al. (1998) J Physiol 512, 629-634 experiments and rat kidney lysate supported this. Funded by the Wellcome Trust: 078640/Z/05/Z. Therefore, we have described for the first time an interaction between megalin and the scaffold proteins NHERF1 and NHERF2. As the NHERF proteins have been shown to be required for the formation of macromolecular complexes in other cell

74P Oral Communications systems, as well as binding to NHE3 and ClC-5 that are essen- ATPase pump and the NKCC1 cotransporter. CONCLUSIONS: tial transmembrane proteins required for endocytosis, further These studies demonstrate a novel role for PHDs in regulating investigation should determine if the a complex is required for intestinal epithelial secretory function. Our data suggest that albumin endocytosis in proximal tubule cells. by virtue of their ability to modulate transport protein expres- 1. Hryciw DH, Ekberg J, Ferguson C, Lee A, Wang D, Parton RG, Pollock sion, PHDs are likely to be important regulators of intestinal CA, Yun CC, Poronnik P. (2006) Journal of Biological Chemistry. fluid and electrolyte transport in hypoxic conditions. 281(23):16068-77. The authors would like to acknowledge Science Foundation Ire- 2. Lee A, Rayfield A, Hryciw DH, Ma TA, Wang D, Pow D, Broer S, Yun land for financial support. C, Poronnik P. (2007). Glia. 55(2):119-29. Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

C133 C132 Farnesoid X receptor activation attenuates colonic epithelial Prolyl Hydroxylase Inhibition Attenuates Colonic Epithelial secretory function Secretory Function N. Keating and S.J. Keely J.B. Ward1, K. Lawler1, C.T. Taylor2 and S.J. Keely1 Molecular Medicine, Royal College of Surgeons, Ireland, Dublin, 1Molecular Medicine, Royal College of Surgeons in Ireland, Ireland Dublin 9, Ireland and 2UCD Conway Institute, UCD, Dublin, At pathophysiological (mM) concentrations, bile acids acutely Ireland stimulate chloride and fluid secretion across colonic epithelial BACKGROUND: Inflammation of the gastrointestinal tract due cells, leading to the onset of diarrhoea. However, our previous to inflammatory bowel diseases and ischemic colitis is com- studies show that at physiological (μM) concentrations, the monly associated with hypoxia and altered epithelial transport. predominant colonic bile acid, deoxycholic acid (DCA) chron- - In hypoxia, key O2 sensors called prolyl hydroxylases (PHDs) are ically inhibits colonic Cl secretion. However, the underlying inactivated, thereby allowing transcription factors such as mechanisms remain unknown. hypoxia inducible factor (HIF) and nuclear factor κB (NFκB) to The Farnesoid X receptor (FXR) is a nuclear hormone receptor be activated. While recent studies have shown that inhibition that is activated by bile acids. FXR is involved in the regulation of PHDs is protective in murine colitis, little is known of their of bile-acid biosynthesis and FXR agonists have received much role in regulating intestinal epithelial transport function. AIM: research interest in treatment of diseases associated with bile To investigate the role of PHDs in regulation of epithelial secre- acid malabsorption. However, to date a role for the FXR in reg- tory function. METHODS: The pan specific inhibitor dimethy- ulating epithelial transport processes has not been reported. loxalylglycine (DMOG) was used to inhibit hydroxylase activ- To investigate the role of FXR in regulating colonic epithelial Cl - ity. Transepithelial Cl- ion secretion, the primary driving force secretion. for fluid secretion in the intestine, was measured as changes Cl - secretory responses to the Ca2+-dependent agonist, car- μ in short circuit current (Isc) across voltage clamped monolay- bachol (CCh; 100 M) or the cAMP-dependent agonist, forskolin μ ers of T84 cells grown on permeable supports. Results are (FSK; 10 M), were measured as changes in short circuit cur- ± expressed as mean standard error of the mean for a series of rent (ISC) across voltage-clamped monolayers of T84 colonic n experiments. Statistical analyses were made by one way epithelial cells. Immunocytochemistry and laser scanning con- ANOVA using Tukey multiple comparisons test. p values ≤ 0.05 focal microscopy were used to examine the subcellular local- were considered to be significant. RESULTS: Pre-treatment of ization of FXR. Results are expressed as mean ± SEM and sta- T84 cells with DMOG (1mM, 24 hrs) significantly attenuated tistical analyses were made by one way ANOVA and Student Cl- secretion in response to the cAMP and Ca2+ dependent sec- Newman Keul’s post-hoc test retagogues, forskolin (FSK) and carbachol (CCh), respectively. FXR expression was confirmed in T84 cells by western blotting. Responses to CCh and FSK were 20.2% ± 2.6% (n = 16; p < 0.001) Treatment with the FXR agonist, GW4064 (2μM for 24 hrs) and 38.6% ± 6.7% (n = 11; p < 0.001) of those in control cells, induced FXR translocation from the cytosol to the nucleus. respectively. Transepithelial resistance was not altered by GW4064 pretreatment attenuated subsequent secretory DMOG treatment indicating that it did not exert toxic effects. responses to CCh and FSK to 54.8 ± 4% and 72.2 ± 3% of those The effects of DMOG on secretory responses were apparent in control cells, respectively (p<0.001, n=14). The effects of after 3 hours and maximal at 18 hours, and this was concurrent GW4064 were concentration-dependent, with antisecretory with the cellular accumulation of HIF, which was apparent at 3 effects apparent at 0.1 μM. As previously reported, pretreat- hours and sustained up to 24 hours. Secretagogue-induced ment of T84 cells with DCA (50μM for 24 hrs) also attenuated basolateral K+ and apical Cl- conductances were not altered by secretory responses to CCh and FSK. However, the antisecre- DMOG. In contrast, Na+/K+ ATPase activity was significantly tory effects of GW4064 and DCA were additive and effects of reduced to 27.6% ± 8.1% (n = 7; p ≤ 0.01) by DMOG. Western GW4064, but not DCA, were inhibited by an FXR antagonist, blot analysis of transport proteins revealed that DMOG also guggulsterone (5μM), suggesting independent mechanisms decreased expression of both the catalytic subunit of the Na+/K+ of action. Furthermore, CCh-stimulated Na+/K+ ATPase activ-

75P Oral Communications ity was not altered by DCA but was inhibited 31.3 ± 6% by (n=28) at 30 min, and 7.86±0.92 μm (n=10) at 45 minutes and GW4064 compared to untreated controls (p<0.01; n=7). appears to be maintained at 48 hours of stimulation in CuFi-1 These studies demonstrate a novel role for the FXR in down- (n=9). LXA4 effect on ASL height is abolished using the lipoxin regulating colonic epithelial secretory capacity. This antise- receptor antagonist, boc2 (10nM) on a 15 and 30 minutes sub- cretory effect of FXR activation appears to be distinct from that sequent exposure of LXA4 (1nM, n=27). Discussion: LXA4 treat- elicited by DCA and is mediated by inhibition of Na+/K+ ATPase ment resulted in an increase of the ASL volume and may pro- activity. By virtue of their ability to downregulate epithelial vide a novel avenue in complementing existing therapy secretory function, our data suggest that FXR agonists may inflammatory in CF. Future Perspectives: The role of the ENaC have therapeutic potential as anti-diarrhoeal agents. channel in modulating the ASL via LXA4 will be investigated in addition to its downstream cell signalling cascades. Where applicable, the authors confirm that the experiments Karp CL et al. Cystic fibrosis and lipoxins. Prostaglandins Leukot Essent described here conform with The Physiological Society ethical Fatty Acids. 2005 Sep-Oct;73(3-4):263-70 requirements. This project is supported by NBIP Ireland, MMI, and HEA. Where applicable, the authors confirm that the experiments C134 described here conform with The Physiological Society ethical requirements.

EffectofLipoxinA4inModifyingtheAirwaySurfaceLiquidLayer V. Verriere1, M. Al-Alawi1, O. Mc Cabe1, V. Urbach3, R.W. Costello2 and B.J. Harvey1 C135

1Molecular Medicine, RCSI, Dublin, Ireland, 2Respiratory Medicine, Contribution of Rho kinase to calcium-contraction coupling RCSI, Dublin, Ireland and 3U661, INSERM, Montpellier, France in airway smooth muscle A key aspect of the lung’s innate defence system is the ability P. Mbikou1, A. Fajmut2,3, M. Brumen2,3 and E. Roux1 of the epithelium to regulate the Airway Surface Liquid (ASL) 1INSERM U885, Université de Bordeaux, Bordeaux, France, volume. The ASL is tightly autoregulated by ion and water trans- 2University of Maribor, Maribor, Slovenia and 3Institute Jozef Stefan, port regulation which need the control ion transport. The trans- Maribor, Slovenia porters involve in ion transport are required for effective ciliary beating and muco-ciliary clearance quality. Na+ transport is We investigated theoretically and experimentally the role of closely controlled to maintain an appropriate fluid layer on the Rho kinase (RhoK) in Ca2+-contraction coupling in rat airways. alveolar surface through the Amiloride-sensitive sodium chan- Isometric contraction was measured on tracheal, extra- and nel (ENaC). Lipoxin A4 (LXA4) is an endogenous anti-inflam- intrapulmonary bronchial rings, in response to carbachol and 2+ matory molecule produced from arachidonic acid. LXA4 has 50 mM external KCl (n=6 to 8). [Ca ]i was recorded in freshly been reported to be reduced in inflammatory Cystic Fibro- isolated tracheal myocytes using Indo-1 (n=19 to 25). Values sis(CF)lung1. CF is a severe genetic disease due to the mutation are expressed as mean ± SEM. Statistical comparisons were of the Cystic Fibrosis Transmembrane conductance Regulator done using a Student’s t test. Theoretical modeling consisted (CFTR)gene. The consequence of the mutation is Na+ hyper in a four-state model of the contractile apparatus coupled with absorption and a defect in Cl- secretion, resulting in dehydra- a model of Ca2+-dependent MLCK activation and RhoK-depend- tion of the airway lumen. This limited mucociliary clearance ent MLCP inactivation. Analysis of the time course of contrac- favours chronic infection and inflammation. One of the thera- tion to carbachol (0.3 and 10 μM) showed that force develop- peutic avenues in CF is to explore a way to inhibit the Na+ hyper- ment occured in two phases: (i) a short-time, Hill-shaped absorption. Our hypothesis is that LXA4 treatment increases contraction obtained within 90 s, (ii) followed by a maintained the height of ASL in CF and non-CF cell lines. Materials and or an additional delayed contraction. Hill fitting of the first phase Methods: CF (CuFi-1) and non CF (NuLi-1) cell line are grown showed that the short-time maximal contraction (stFmax) of to confluency on semi-permeable filters in order to obtain a tracheal rings to 10 μM carbachol was 77,8±2.6% of total max- well differentiated polarised epithelium showing high transep- imal force, and the time to obtain half-stFmax was 17.4±1.6 s. 2+ ithelial resistance. Cells are loaded with fluorescent dye Calcein Values of similar range were obtained in bronchial rings. [Ca ]i AM (5μM). A small volume of a dextran solution containing the responses to 10 μM acetylcholine (ACh) consisted in a fast peak Texas Red fluorochrome is applied on the apex of the epithe- followed by a plateau and, in 42% of the cells, superimposed lium in order to label the ASL compartment. The ASL height is Ca2+ oscillations. Exposure to the RhoK inhibitor Y27632 (10 μ 2+ measured using a laser scanning confocal microscopy in live M) did not alter the resting [Ca ]i nor the parameters of the cells in response to LXA4. Results: ASL stabilisation is obtained ACh-induced Ca2+ response. Whatever the concentration of between 12 and 24 hours after the application of the dextran carbachol and the location along the airway tree, Y27632 did solution on the apex of the cells. The ASL of the CF epithelium not modify the basal tension but decreased the amplitude of is reduced when compared to non-CF epithelium. In CuFi and the short-time response, without altering the additional delayed NuLi cell lines, a LXA4 (1nM) basolateral pretreatment signifi- contraction. Calyculin A (MLCP inhibitor) increased the basal cantly increased the ASL stabilized height from a physiological tension, and abolished the effect of RhoK inhibition on carba- baseline height of 5±0.28 μm (n=18) and 7±0.21 μm (n=27) chol-induced contraction. Stimulation by 50 mM external KCl respectively to 15.25±1.18 μm at 15 minutes (n=19) in CuFi solution in the presence of atropine induced a short-time con- cells. This effect is also observed to a level of 9.23±0.56 μm traction followed by a sustained tension, both depending of

76P Oral Communications the presence of external Ca2+. As with carbachol-induced con- 2+ on [Ca ]i by ~50%. Pre-exposure to the cholesterol chelator traction, Y27632 decreased the amplitude of the short-time β methyl- -cyclodextrin (10 mM) blunted E2 effects (35% of peak response, without altering the delayed tension. KN93 (CamK II responses). These novel data suggest that in human ASM, estro- inhibitor) and DIDS (Ca2+-activated Cl- channels) had not influ- gens act via ERs (potentially within caveolae) to non-genomi- ence on the effect of RhoK inhibition. cally produced bronchodilation. Given enhanced ER expression We conclude that Ca2+-dependent but CamK II-independent with inflammation, estrogens may be a novel therapeutic mech- RhoK activation contributes to the early phase of the contrac- anism in airway diseases such as asthma. tile response via MLCP inhibition. On these bases, we inple- mented our previously published model of Ca2+-contraction Supported by NIH grants HL090595 and HL088029, and by the coupling (1). The model explains the time course of the short- Flight Attendants Medical Research Institute. 2+ time contraction and the role of RhoK by Ca -dependent acti- Where applicable, the authors confirm that the experiments vation of MLCK and RhoK, which inactivates MLCP. Oscillatory described here conform with The Physiological Society ethical 2+ and non-oscillatory [Ca ]i responses result in a non-oscillatory requirements. contraction which amplitude is encoded by the plateau value and oscillation frequency. Mbikou P, Fajmut A, Brumen M, and Roux E. Theoretical and experimental investigation of calcium-contraction coupling in airway smooth C137 muscle. Cell Biochem Biophys 46: 233-252, 2006.

H.Crevel and P. Téchoueyres for technical assistance. Grant from Contractileandelectrophysiologicalpropertiesofpulmonary Proteus HC partnership. artery smooth muscle from TASK-1 knockout mice Where applicable, the authors confirm that the experiments B. Manoury, C. Lamalle and A.M. Gurney described here conform with The Physiological Society ethical requirements. The University of Manchester, Manchester, UK In pulmonary artery (PA) smooth muscle cells (SMC), compelling evidence suggests that TWIK-related acid sensitive K+- 1 (TASK-1) channels contribute to the background K+ current C136 that supports the membrane potential (Em). Due to poor selectivity of TASK-1 channel modulators, the role of the channel in Estrogen Effects on Intracellular Calcium Regulation in the functional regulation of PA tone remains elusive. The pur- Human Airway Smooth Muscle pose of this study was to determine the properties of PA from C.M. Pabelick1,2, E.A. Townsend2 and Y.S. Prakash1,2 mice in which the TASK-1 and TASK-3 genes were deleted (TASK1/3 knockout, KO), in comparison with C57BL/6 wild type 1Anesthesiology, Mayo Clinic, Rochester, MN, USA and 2Department (WT) control mice. of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Intra-PAs were isolated from adult male WT and KO mice. Ves- MN, USA sels were mounted on a wire myograph for isometric tension Gender differences in incidence and severity of asthma are clin- recording or processed for gene expression studies using ically observed. Pre-menstrual exacerbations are inconsistent reverse-transcription polymerase chain reaction (RT-PCR). Patch and actually occur in late luteal phase when estrogen levels are clamp experiments in the perforated-patch configuration were lowest while in spite of high sex steroid levels, pregnancy is not conducted on freshly isolated PASMC. RT-PCR confirmed TASK- always associated with worsening of symptoms. Diseases such 1 expression in PA from WT but not KO mice, whereas TASK-3 as asthma are characterized by airway hyperresponsiveness was absent in both strains. The TASK-1/3 KO mice could there- 2+ 2+ that involves altered intracellular Ca ([Ca ]i) responses of air- fore be used to study the role of TASK-1 in mouse PA. If TASK- way smooth muscle (ASM) to agonist stimulation. In this regard, 1 is a key determinant of Em, deletion of the TASK-1 gene might we have recently demonstrated an important role for caveolae be expected to cause depolarisation, thereby stimulating Ca2+ 2+ + in [Ca ]i regulation. In vascular endothelium, estrogen recep- influx and intrinsic tone. The addition of 10mM KCl, the K chan- tors (ERs) are expressed within caveolae, and facilitate vasodi- nel modulators 4-aminopyridine (4-AP, 1mM) and levcro- lation. We hypothesized that estrogens facilitate bronchodila- makalim (10μM), or the L-type calcium channel blocker nifedip- 2+ tion by decreasing [Ca ]i in ASM. In ASM isolated from human ine (1μM) had no significant effect on the tone of PAs from lung surgical waste tissue, we found through Western blot either KO (N=3) or WT mice (N=3). These results imply that Em analysis (n=4) that both estrogen receptors (ERs), ERα and ERβ is not depolarised and that PAs are fully dilated in both groups. are expressed to substantial extents, as are two truncated ER The contractile responses of PA to phenylephrine, serotonin isoforms (ERα-36 and ERα-47). ERα is also expressed within and PGF2α were also similar between the two mouse strains. caveolae. Overnight exposure to 20 ng/ml TNFα substantially + The non inactivating K current (IK(N)), recorded after clamp- increased caveolar ERα (and to a lesser extent ERβ) expression. ing the Em at 0 mV for 5-6 minutes, was similar in PASMC from In ASM cells loaded with the Ca2+ indicator fura-2, acute expo- WT (0.2 ± 0.1 pA/pF, N=4) and KO (0.2 ± 0.1 pA/pF, N=3) mice. sure to 17β-estradiol (17βE ; 1nM) blunted [Ca2+] response to 2 i IK(N) was unchanged in the presence of 10mM tetraethylam- bronchoconstrictor agonists (1 μM ACh, 10 μM histamine) by monium chloride (TEA) to block BKCa channels, but was much ~60% of peak Ca2+ responses. These effects were prevented by smaller than IK(N) recorded from rat PASMC in the same condi- pre-exposure to ER antagonist (ICI 182,780; 20% of peak tions (1.7 ± 0.3 pA/pF, N=4). Unlike the rat counterpart, murine responses). Overnight exposure to TNFα enhanced E effects 2 IK(N) showed no sensitivity to pH changes between 6.3 and 8.4,

77P Oral Communications in either WT or KO mice. No differences were detected in the smooth muscle cells. Overall, these studies indicate a promi- mRNA levels of TASK-2, TASK-5, TWIK-2, Kv1.5 or Kv2.1 between nent role for airway epithelium and epithelium-derived NO in WT (N=3) and KO (N=3) mice. the bronchodilatory effects of estrogens in humans. PA isolated from KO mice failed to develop intrinsic tone and Carey, M.A., et al. Trends Endocrinol Metab 2007; 18: 308-13. displayed unaltered vasoconstrictor responses. The absence of Chambliss, K.L., and Shaul, P.W. Endocr Rev 2002; 23: 665-86. TASK-1 in PA SMC did not affect IK(N) and was not compensated by up-regulation of other K+ channel subunits. The results ques- Supported by NIH grants HL088029 and HL090595, and the tion the importance of TASK-1 in mouse PA and highlight Flight Attendants Medical Research Institute (FAMRI). species differences in the resting K+ conductance. Where applicable, the authors confirm that the experiments Funding provided by the Biotechnology & Biological Sciences described here conform with The Physiological Society ethical Research Council and British Heart Foundation. Prof Bill Wis- requirements. den provided the TASK 1/3 KO mice. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical C139 requirements. Regulation of spontaneous activity in interstitial cells of Cajal of the rabbit urethra by ATP

C138 G.P. Sergeant, E. Bradley, B. Drumm, M.A. Hollywood, N.G. McHale and K.D. Thornbury Estrogen effects in human airway epithelial cells Smooth Muscle Research Centre, Dundalk Institute of Technolgy, Y.S. Prakash1,2, M.A. Thompson1 and C.M. Pabelick1,2 Dundalk, Co. Louth, Ireland Interstitial cells of Cajal (ICC) in the urethra are considered to 1Department of Anesthesiology, Mayo Clinic, Rochester, MN, USA be putative pacemaker cells which are involved in the genera- and 2Department of Physiology and Biomedical Engineering, Mayo tion of spontaneous myogenic tone (Sergeant et al., 2000). Ure- Clinic, Rochester, MN, USA thral ICC are spontaneously active and the frequency of this While gender differences incidence and severity of asthma are activity is modulated by several neurotransmitters, including clinically recognized, the physiological role of sex steroids is noradrenaline and nitric oxide, (Sergeant et al., 2002 & 2006). less clear, with cyclical exacerbations (pre-menstrual asthma) ATP is also considered to be an important neurotransmitter in occurring in late luteal phase when estrogen levels are low, the urethra, therefore experiments were performed to test if while high sex steroid levels (e.g. pregnancy) are not always spontaneous activity in urethral ICC was also regulated by ATP. associated with worsening of symptoms. Similar to endothe- New Zealand white rabbits were humanely killed and ICC were lium in vasculature, airway epithelium is a source of bron- freshly dispersed from the urethra as described previously chodilatory agents such as NO. Using human bronchial rings (Sergeant et al., 2000). ICC were then plated and loaded with and epithelial cells derived from such samples (obtained from Fluo-4 AM for study of cytosolic Ca2+ signals using a spinning surgery), we tested the hypothesis that estrogens facilitate disk confocal microscope. STICs were recorded at -60 mV using bronchodilation by enhancing NO production in airway epithe- the amphotericin B perforated patch technique. 2+ 2+ μ lium, resulting in reduced intracellular Ca ([Ca ]i) in airway ATP (10 M) increased the frequency of STICs in urethral ICC smooth muscle. Values are means with SE. Acute exposure to from 9 ± 1.6 to 26 ± 3.7 min-1 (p<0.05). These effects were β 1 nM 17 -estradiol (E2) relaxed epithelium-intact bronchial mimicked by the P2Y receptor agonist 2-methylthio ADP (2- rings contracted with 1 μM ACh to a significantly greater extent MeSADP, 1 μM), which increased STIC frequency from 7.25 ± (72 ± 9% of max. force) than epithelium-denuded rings (42 ± 0.5 to 26.25 ± 3.2 min-1, n=8 (p<0.05). The broad spectrum 8%; p<0.05, n=4, t-test). E2-induced relaxation was blunted by purinergic receptor antagonist suramin (100 uM) reduced the the NO scavenger PTIO (48 ± 9%). In isolated human airway mean frequency of ATP evoked inward currents from 17.9 ± 2.8 epithelial cells (n=20 per group) loaded with the fluorescent to 1.3 ± 0.66 min-1 (n = 9, p<0.05) and those evoked by 2- ± ± NO indicator DAF-2, 1 nM E2 increased fluorescence levels (28 MeSADP from 31 6.1 to 4.2 2.1 min-1 (n = 6, p<0.05). Sim- ± 3 arbitrary units (au) with 5 ± 2 au baseline), comparable to ilar results were achieved with the selective P2Y1 receptor that induced by 1 μM ACh (32 ± 7 au) or 50 μM ATP (41 ± 4 au). antagonist, MRS2500 (100 nM) which reduced the mean fre- ± In the presence of E2, ACh effects on DAF-2 fluorescence were quency of ATP evoked inward currents from to 17 4.5 min-1 enhanced (69 ± 5 au), while PTIO substantially blunted these to 6 ± 2.5 min-1 (n= 8, p<0.05) and 2-MeSADP induced currents effects (22 ± 6 au). The estrogen receptor (ER) specific agonists from 25.8 ± 4.3 to 7 ± 1.8 min-1 (n= 6, p<0.05). THC (ERα) and DPN (ERα) both induced comparable NO pro- Johnston et al., (2005) showed that STICs in urethral ICC are ± ± duction (31 6 au vs. 38 6 au). Neither ACh nor E2 affected associated with global Ca2+ oscillations therefore we investi- fluorescence of the NO-insensitive dye DAF-4. Western blot gated if this activity was also modulated by ATP and 2-MeSADP. analyses of eNOS phosphorylation at Ser1177 showed an ~5- Application of ATP (10 μM) significantly increased the frequency ± ± fold increase with E2 exposure. Finally, in Transwell plates where of Ca2+ waves from 9 1.4 to 29.1 4.5 min-1 (n = 14, p<0.05) human epithelial cells and airway smooth muscle cells were co- and 2-MeSADP (1 μM) caused an increase from 7.3 ± 1.6 to 17.1 ± ± cultured, exposure of epithelial cells to 1 nM E2 induced 58 2.8 min-1 (n=10, p<0.05). The frequency of the ATP induced 2+ ± ± 7% reductions in ACh-induced elevation of [Ca ]i in airway Ca2+ oscillations was reduced from 27 8.7 min-1 to 6.6 2.1

78P Oral Communications min-1 by suramin (n=7, p<0.05) and similar results were centration-dependent relaxation (10μM: 24 ± 3% relaxation, achieved with MRS2500, which decreased the mean frequency n=15, P<0.01 vs. vehicle; 30μM: 60 ± 5% relaxation, n=12, of ATP induced Ca2+ oscillations from 28.1 ± 4.2 to 10.3 ± 3.9 P<0.01 vs. DMSO vehicle). LY83583-induced relaxation was pre- min-1 (n= 6, p<0.05). vented by the Rho-kinase inhibitor Y27632 (10μM, n=6, These data demonstrate that spontaneous activity in urethral LY83583 relaxation not significant vs. vehicle), but was insen- ICC is modulated by ATP and that this effect is likely to be medi- sitive to blockade by either the antioxidant tempol (LY83583: ated by P2Y receptors. 37 ± 6% relaxation, n=6, P=0.05 vs. vehicle) or SOD (LY83583: Sergeant GP, Hollywood MA, McCloskey KD, Thornbury KD, McHale NG 36 ± 3% relaxation, n=7, P<0.05 vs. vehicle). The underlying pCa (2000). Specialised pacemaking cells in the rabbit urethra. J Physiol. 6.4 constriction was insensitive to LY83583 or vehicle. H2O2 526, 359-66. however, and in contrast to its effect in non-permeabilized MA, Johnston L, Sergeant GP, M.A. Hollywood, K.D. Thornbury and N.G. caused constriction at pCa 6.4 both in the absence (43 ± 7% McHale (2005). Calcium oscillations in interstitial cells of the rabbit ure- enhancement at 15min, n=5, P<0.01 vs. time control) and pres- thra. J Physiol 565, 449-61. ence of U46619 (17 ± 3% enhancement at 15min, n=4, P<0.001 Sergeant GP, Johnston L, McHale NG, Thornbury KD, Hollywood MA vs. time controls). (2006). Activation of the cGMP/PKG pathway inhibits electrical activ- In summary, LY83583 either relaxes or constricts MA, depend- ity in rabbit urethral interstitial cells of Cajal by reducing the spatial ing on the nature of the pre-constriction. LY83583-generated spread of Ca2+ waves.J Physiol 574, 167-81. superoxide more strongly relaxes intact compared to perme- Sergeant GP, Thornbury KD, McHale NG, Hollywood MA (2002). Char- abilized U46619 pre-constricted MA, whereas exogenous H2O2 acterization of norepinephrine-evoked inward currents in interstitial relaxes intact but constricts alpha-toxin permeabilized MA. ROS cells isolated from the rabbit urethra. Am J Physiol Cell Physiol 283, therefore contribute to multiple constriction/relaxation path- C885-94. ways in MA. LY83583-induced Rho-kinase dependent Ca2+- The authors are grateful for support from the NIH (RO1 desensitization in MA does not appear to be mediated via ROS. DK68565), HRB (PD/2005/4 & RP/2006/127). Knock GA, Snetkov VA, Shaifta Y, Connolly M, Drndarski S, Noah A, Pourmahram GE, Becker S, Aaronson PI, ward JPT (2009) Free Radical Where applicable, the authors confirm that the experiments Biology & Medicine 46(5):633-42. described here conform with The Physiological Society ethical requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

C140

Vasomotor responses to reactive oxygen species in rat mesenteric arteries J. Kua, J.P. Ward, P.I. Aaronson and G.A. Knock King’s College London, London, UK Reactive oxygen species (ROS) are important mediators of vascular tone and play a key role in cardiovascular disease. We showed previously that LY83583, a generator of superoxide anion, caused a superoxide dismutase (SOD)-inhibitable constriction of rat pulmonary arteries that was directed primarily viaRho-kinase-mediatedCa2+-sensitization(Knocketal.,2009). In the present study we investigated the vasomotor responses to LY83583 and hydrogen peroxide (H2O2) in intact and alpha-toxin permeabilised U46619 (100 nM)-pre-constricted rat mesenteric arteries (MA), mounted on a wire myograph. In intact MA, LY83583 was applied in the presence of L- NAME (1mM) in order to eliminate superoxide scavenging of NO. Statistical analysis was by Student t-test. Both LY83583 (10μM) and H2O2 (100μM) strongly relaxed U46619 pre-constricted intact MA (LY83583: 86 ± 6% relaxation at 15min, n=10; H2O2: 94 ± 1% relaxation at 5min, n=2), and the former was partially prevented by combined pre-incubation of SOD and catalase (40 ± 14% relaxation at 15min, n=9, P<0.01 vs. LY83583 alone). In contrast, when MA were pre-constricted with 30 mM KCl, LY83583 caused further sustained constriction and no relaxation (119 ± 15% enhancement at 5 min, n=4, P<0.05). In alpha-toxin permeabilized MA (pre-constricted with 100nM U46619 at pCa 6.4), LY83583 caused modest but con-

79P Oral Communications

C141 C142 Automated calcium spark detection algorithm for linescan Human mesenchimal stem cells as a novel approach for images containing Poisson noise radiation-induced vascular malfunction therapy P. Bankhead, N. Scholfield, T. Curtis and G. McGeown A. Soloviev1, I. Prudnikov2, V. Tsyvkin2, S. Tishkin1, Centre for Vision and Vascular Science, Queen’s University Belfast, S. Kyrychenko1, S. Zelensky1 and I. Ivanova1 Belfast, UK 1Institute of Pharmacology and Toxicology, Kiev, Ukraine and The physiological significance of calcium sparks in cardiac, 2Bogomoletz Institute of Physiology, Kiev, Ukraine skeletal and smooth muscle cells has received a great deal of Therapeutic effect of mesenchymal human stem cells trans- attention in recent years. Such studies rely upon the accurate plantation (MSCT) has been evaluated in a whole-body irradi- detection and analysis of brief, localised release events in order ated (6 Gy) rats. Experimental design of the study comprised to quantify changes in spark frequency and properties under large conductance Ca2+-dependent K+ channels (BKCa) activ- different conditions. In order to adequately characterise the ity measurements in aortic smooth muscle cells using patch time course of sparks, images are often obtained using confo- clamp technique in whole-cell modification, non-invasive sys- cal laser scanning microscopy in linescan mode. The manual tolic arterial blood pressure measurement and simultaneous analysis of this data is time consuming and prone to user bias, measurement of contractile force and [Ca2+]i. because the small size of many events and relatively low signal- Bone marrow was aspirated in heparin from the sternum of to-noise ratio make it difficult to discern small amplitude sparks healthy volunteers after informed consent (with 1%lidocaine from noise artifacts. as a local anaesthetic). Mesenchymal stem cells (MSC) were The main source of noise in these images is photon noise, which separated using negative selection procedure with monoclo- follows a Poisson distribution and entails that the noise vari- nal antibodies (Human RosetteSep Mesenchymal Stem Cell ance is larger when the background fluorescence is increased. Enrichment Cocktail, StemCell Inc.). The isolated MSC after Previously published automated spark detection algorithms Ficoll-Hypaque centrifugation were resuspended in MesenCult are limited in at least one of two related ways: (1) sparks are medium (StemCell Inc.) supplemented with appropriate Mes- assumed to occur from a constant baseline, and (2) the noise enchymal Stem Cell Stimulatory Supplements and cultivated is assumed to be Gaussian, with a fixed variance throughout 20 - 32 days in the same medium with additional recombinant the image. These algorithms are therefore inappropriate for human growth factors: SDF-1a, EGF, and PDGF-AA (CHO- reliably identifying sparks in images containing multiple cells grade). After two passages MSC were transplanted intra- with differing baseline fluorescence levels, or occurring on top venously to irradiated rats on the 7th day of post-irradiation in of global calcium elevations. Both situations arise in images of a single dose of 16-20x106 cells per rat. calcium signaling events in the smooth muscle of retinal arte- Whole-body irradiation produced a decrease of BKCa activity riolar segments, as described by Curtis et al. (2004). in aortic myocytes. This was paralleled by a reduction of the We have developed new software that overcomes these issues NO-dependent ACh-induced vascular relaxation and arterial by using a wavelet-based variance stabilisation technique to hypertension development. Thus, the vasorelaxing force of automatically adapt spark detection to changes in baseline flu- BKCa was diminished in irradiated myocytes. Simultaneous orescence. In addition to facilitating the analysis of images for measurements of contractile force and [Ca2+]i showed that which no automated algorithm currently exists, preliminary myofilament Ca2+ sensitivity defined as the ratio of force change tests indicate that the use of a more accurate noise model to [Ca2+]i significantly increased following irradiation. means that our algorithm can also offer improved detection MSCT effectively restored outward currents (from 13±1 to 24±1 accuracy in any linescan containing sparks, particularly at low pA/pF, P<0.05, n=12) mainly due to paxilline-sensitive BKCa signal-to-noise ratios. component, and led to an increase in amplitude of maximal Curtis TM, Tumelty J, Dawicki J, Scholfield CN, McGeown JG. (2004) ACh-induced endothelium-dependent relaxation in irradiated Identification and spatiotemporal characterization of spontaneous ± ± Ca2+ sparks and global Ca2+ oscillations in retinal arteriolar smooth vascular tissues from 43 3% to 87 6% (P<0.05, n=12). MSCT 2+ muscle cells. Invest Opthalmol Vis Sci 45, 4409-4414 normalized myofilament Ca sensitivity (from 0.068±0.007 to 0.030±0.004 mN/nM, P<0.05, n=12) and arterial blood pres- Where applicable, the authors confirm that the experiments sure from 152±4 to 121±2 mm Hg (P< 0.05, n=12). described here conform with The Physiological Society ethical The data obtained suggest that MSC demonstrate a clearly requirements. expressed therapeutic potential to normalize vascular abnormalities induced by ionized irradiation and appear to be worthwhile therapeutic approach in case of vascular malfunction induced by ionizing irradiation. This study was supported by The Physiological Society ‘Centre of Excellence Award Scheme’ 2008. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

80P Poster Communications

Pereira,D.T.B.; Vendramini,R.C.; David,R.B.; Nozaki,P.N.; Menani,J.V.; PC1 De Luca Jr,L.A. Isotonic NaCl intake by cell-dehydrated rats. Physiol- ogy and Behaviour, 76: 501-505, 2002.

Lesions of the commissural nucleus of tractus solitarii Tribollet, E.; Armstrong, W. E.; Dubois-Dauphin, M; Dreifuss, J. J.. Extra- hypothalamic afferent inputs to the supraoptic nucleus area of the rat increase osmotic-induced activation of paraventricular and as determined by retrograde and anterograde tracing techniques. Neu- supraoptic hypothalamic nuclei roscience, 15(1):135-148, 1985. 1 1 1 1 G.T. Blanch , A.H. Freiria-Oliveira , E. Colombari , J.V. Menani , This work was supported by CNPq and Royal Society. D. Murphy2 and D.S. Colombari1 Where applicable, the authors confirm that the experiments 1Physiology and Pathology, FOAr-UNESP, Araraquara, Sao Paulo, described here conform with The Physiological Society ethical Brazil and 2Henry Wellcome Laboratories for Integrative requirements. Neuroscience and Endocrinology, University of Bristol, Bristol, UK

Neurons in the magnocellular division of paraventricular nucleus (PVN) and supraoptic nucleus (SON) of hypothalamus are activated by osmotic stimulus increasing the release of oxytocin PC2 and vasopressin, hormones that induce natriuresis, antidiure- sis and vasoconstriction (McCann et al, 2003). PVN and SON Sympathetic activity enhances inflammatory responses in receive important ascending neural connections from the dorsal root ganglia proximal to a sciatic nerve transection nucleus of tractus solitarii (NTS) (Tribollet et al, 1985). Previ- in rats ously we have demonstrated that rats with lesions in the com- E. McLachlan1,2 and P. Hu1 missural subdivision of the NTS (commNTS) increased water intake, natriuresis and arterial pressure after intragastric 2 M 1Spinal Injuries Research Centre, Prince of Wales Medical Research NaCl load. Considering that the increase in arterial pressure in Institute, Randwick, NSW, Australia and 2University of New South commNTS-lesioned rats is dependent on vasopressin, in the Wales, Sydney, NSW, Australia present study we investigated the effects of commNTS lesions T cells and macrophages invade dorsal root ganglia (DRGs) after on c-fos expression in the PVN and SON produced by intragas- sciatic nerve injury (1). We tested the hypothesis that this tric hypertonic sodium load. immune cell response is influenced by local sympathetic activ- Male Holtzman rats (300-320 g, n = 3-4/group) were anes- ity. L5 DRGs were evaluated one week after left sciatic nerve thetized with ketamine (80 mg/kg of body weight, ip) com- transection in rats in which, 10-14 days previously, L3 and L4 bined with xylazine (7 mg/kg of body weight, ip) and submit- sympathetic ganglia had been removed either bilaterally or only ted to electrolytic (1 mA x 10 s) or sham lesion of the commNTS. ipsilaterally (sympathectomy), or the sympathetic chain had Fifteen days after lesions, rats received 2 ml of 2 M NaCl intra- been cut just above L3 ganglion on both or only the ipsilateral gastrically, which increases plasma osmolality in 4% (Pereira side(s) (decentralization) (n=5 animals in each group). The pro- et al, 2002) or 2 ml of 0.15 M NaCl. Two hours after sodium load, cedures were performed in adult female Wistar rats anaes- rats were deeply anesthetized with pentobarbital (50 mg/kg of thetized with a mixture of ketamine 60 mg/kg and xylazine 10 body weight, ip), perfused with 4% paraformaldehyde and mg/kg, i.p. for nerve lesions and with pentobarbitone 80 mg/kg brains were removed. Imunohistochemistry for c-fos expres- i.p. for exsanguination and perfusion with fixative one week sion was performed in brain slices using DAB staining. Data are later. expressed as means ± SEM and analyzed by one-way ANOVA. Sections of L5 DRGs and adjacent nerve trunks were inves- In sham rats, intragastric 2 M NaCl induced c-fos expression in tigated using fluorescence immunohistochemical techniques magnocellular PVN (70 ± 12 vs. 0.15 M NaCl: 8 ± 2 positive to identify lymphocytes positive for alpha/beta T-cell receptor cells/section – each 150 μm bilateraly, p <0.05), parvocellular (TCR) or CD8 and macrophages positive for major histocom- PVN (27 ± 4 vs. 0.15 M NaCl: 5 ± 1 positive cells/section, p <0.05) patibility complex class II (MHC II), CD68 or CD163. The den- and SON (191 ± 12 vs. 0.15 M NaCl: 2 ± 1, positive cells/section, sity of T-Iymphocytes and CD68+ and MHC II+ macrophages p <0.05). CommNTS lesions increased c-fos expression induced increased after sciatic nerve transection. When ipsilateral sym- by intragastric 2 M NaCl in magnocellular and parvocellular PVN pathectomy or ipsi- or bilateral decentralization preceded sci- (139 ± 22 and 40 ± 27 positive cells/section, respectively, p atic transection, mean alpha/beta TCR+ and CD8+ lymphocytes <0.05 vs. sham) and SON (270 ± 11 positive cells/section, p < and MHC II+ and CD68+ macrophage densities in the lesioned 0.05 vs. sham). Double labeling immunofluorescence demon- DRGs were all ~35% lower than after sciatic transection alone, strated that a significant number of c-fos positive cells in PVN but the density of resident (CD163+) macrophages was simi- and SON in both commNTS- and sham-lesioned rats were oxy- lar in all groups of animals. Bilateral sympathectomy alone did tocinergic or vasopressinergic. not increase immune cell density in the DRGs but this inter- The results show increased number of PVN and SON neurons vention markedly enhanced the subsequent response to sciatic activated by osmotic stimulus in commNTS-lesioned rats, transection. Double labelling for CD8 and CD3 (a pan-T-cell which may have a correlation with the increased natriuresis and marker) revealed that a few CD8+ cells were not CD3+ (i.e. they arterial pressure in commNTS-lesioned rats after hypertonic were macrophages). Analysis of the proportions of CD8-/CD3+ sodium load. (i.e. CD4+) lymphocytes after each intervention revealed that MacCann,S.M.; Gutkowska,J.; Antunes-Rodrigues,J. Neuroendocrine the influx of CD4+ lymphocytes was markedly reduced after control of body fluid homeostasis. Braz. Journal Med. Biol. Research, removal of sympathetic activity. 36: 165-181. 2003.

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It is concluded that local sympathetic activity influences Supported by: FAPESP, CNPq. immune cell invasion into dorsal root ganglia after peripheral Where applicable, the authors confirm that the experiments nerve injury. described here conform with The Physiological Society ethical (1) Hu P & McLachlan EM (2002). Neuroscience 112, 23-38. requirements. This work was supported by the National Health & Medical Research Council of Australia. Where applicable, the authors confirm that the experiments PC4 described here conform with The Physiological Society ethical requirements. Macrophage inhibitory migration factor in the paraventricular nucleus of hypothalamus attenuates hyperosmotic-evoked sympathoexcitation in the rat PC3 E. Colombari1, D.S. Colombari2, H. Li3, C. Sumners3, M.K. Raizada3, D. Murphy2 and J.F.R. Paton1 Effects of exogenous or endogenous hydrogen peroxide 1Physiology & Pharmacology, Bristol Heart Institute, University of centrally on the pressor response to central cholinergic 2 activation Bristol, Bristol, UK, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, UK M.R. Lauar, L.M. Cardoso, D.S. Colombari, E. Colombari, P.M. De and 3Physiology & Functional Genomics, University of Florida, Paula, L.A. De Luca Jr. and J.V. Menani Gainesville, FL, USA Department of Physiology and Pathology, School of Dentistry - Systemic hyperosmotic stimulation (HS) evokes increases in UNESP, Araraquara, SP, Brazil sympathetic nerve activity (SNA) mediated by activation of Recent results from our laboratory demonstrated that intrac- angiotensin II type 1 (AT1) receptors in the hypothalamic par- erebroventricular (icv) injection of hydrogen peroxide (H2O2), aventricular nucleus(PVN), as described by Chen and Toney a reactive oxygen species, reduced water intake and pressor (2001). Recently, macrophage inhibitory migration factor (MIF) responses induced by icv injection of angiotensin II (Lauar et.al., can antagonize the hypertension evoked by angiotensin II act- 2008). Thus, in the present study we investigated the effects ing at the level of the PVN (Li et al, 2006). This inhibitory effect of icv injection of H2O2 or ATZ (3-amino-1,2,4-triazole, a cata- of MIF is due to its intrinsic thiol-protein oxidoredutase (TPOR) lase inhibitor) on the pressor responses induced by icv injec- activity (Sun et al, 2007). In this study, we evaluated the effect tion of the cholinergic agonist carbachol. of virally mediated over expression of either MIF or C60SMIF Male Holtzman rats (280-320 g, n=8/group) were anesthetized (which lacks TPOR activity) in the PVN on the sympathoexci- with ketamine (80 mg/kg of body weight) combine with tation induced by hyperosmolality (HS). Under deep halothane xylazine (7 mg/kg of body weight) and had stainless steel can- anaesthesia, male Wistar rats (65-85 g) were decorticated to nulas implanted in the lateral ventricle (LV). Mean arterial pres- make insentient and perfused intra-arterially (Antunes et al. sure (MAP) and heart rate (HR) were recorded in unanesthetized 2006). HS was induced by raising perfusate osmolality from freely moving rats. A polyethylene tubing (PE-10 connected 290 to 380 mOsmol for 40 s. Adeno associated viruses to a PE-50) was inserted into the abdominal aorta through the employed for over expression of MIF (1.0x 108), C60SMIF (1.0x femoral artery on the day before the experiments under keta- 108) or eGFP (8.3 x 108) were injected bilaterally (500 nl/side) mine + xylazine anesthesia. MAP and HR were continuously into the PVN as was saline as a control. Seven to 10 days later recorded and H2O2 (5 μmol/1 μl) or PBS (vehicle, 1 μl) was the HS-induced sympathoexcitation in both the saline and eGFP injected into the LV 1 min before the injection of carbachol (4 groups (increases of 27 ± 4% and 25 ± 4%, respectively) was not nmol/1 μl) also into the LV, and ATZ (5 nmol/1 μl) or saline was observed in the MIF group (4 ± 5%). Conversely, the HS induced injected into the LV 10 min before the injection of carbachol. SNA response was potentiated (45 ± 6%) in the C60SMIF group. The previous icv injection of H2O2 or ATZ reduced the pressor When MIF injections were located outside the PVN a response responses produced by icv injection of carbachol (12 ± 4 and similar to control was observed. Imunohistochemical analysis 13 ± 4 mmHg, respectively, vs. vehicle or saline: 25 ± 4 and 30 demonstrated that MIF expression in PVN was restricted to neu- ± 4 mmHg, respectively). No significant change on HR was pro- rones some of which were immunopositive for vasopressin. We duced by carbachol alone (-13 ± 13 and -31 ± 15 bpm, respec- propose that MIF acting within the PVN is a major counter reg- tively) or combined with H2O2 or ATZ (-3 ± 9 and -8 ± 17 bpm), ulator of HS induced sympathoexcitation, which is dependent compared with vehicle or saline (-2 ± 16 and 13 ± 13 bpm, on its TPOR activity. Further, enhancing TPOR activity may have respectively). therapeutic potential in restricting salt-induced hypertension. The results show that central injections of H2O2 or ATZ Chen,Q.H.; Toney,G.M. AT(1)-receptor blockade in the hypothalamic reduced the pressor response induced by icv injection of car- PVN reduces central hyperosmolality-induced renal sympathoexcita- bachol, suggesting that exogenous or endogenous H2O2 may tion. Am . J. Physiol., 281(6): R 1844-1853. inhibit central pressor mechanisms activated by central cholin- Li H, Gao Y, Freire CD, Raizada MK, Toney GM, Sumners C. Macrophage ergic activation. migration inhibitory factor in the PVN attenuates the central pressor and dipsogenic actions of angiotensin II. Faseb J, 20(10):1748-50, 2006. M. R. LAUAR, L. M. CARDOSO, D. S. A. COLOMBARI, P. M. DE PAULA, E. COLOMBARI, *J. V. MENANI. Central injection of hydrogen peroxide Sun,C.; Li,H.; Gao,Y.; Matsuura,T.; Upchurch,P.A.; Raizada,M.K.; Sum- reduces angiotensin II-induced pressor responses. Program # 186.9, ners,C. Lack of macrophage migration inhibitory factor regulation is Poster # SS33. 2008 Neuroscience Meeting Planner. Washington, DC: linked to the increased chronotropic action of angiotensin II in SHR neu- Society for Neuroscience, 2008. Online. rons. Hypertension, 49(3): 528-534, 2007.

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Research funded by CNPq (Brazil), CAPES (Brazil), The Royal chronic dehydration, the regulation of SNA transfers to the Society and British Heart Foundation. medulla oblongata, particularly the cNTS. This plasticity seems to be mediated via activation of the AP1 transcription factor; Where applicable, the authors confirm that the experiments if AP1 transcription factor activity is blocked in NTS during dehy- described here conform with The Physiological Society ethical dration, then control of sympathetic activity reverts back to requirements. forebrain regions. Antunes, VR; Yao, ST; Pickering, AE; Murphy, D; Paton, JFR. J. Physiol, 576(2): 569-583, 2006. PC5 Ji,L.L.; Gottlieb,H.B.; Penny,M.L.; Fleming,T.; Toney,G.M.; Cunning- ham,J.T. Exp. Neurology, 203(2): 445-456, 2007. Chronic dehydration switches the control of sympathetic activity from forebrain to hindbrain in the rat Supported by CNPq, Capes, Royal Society and BHF. D.S. Colombari1, E. Colombari2, D. Murphy1 and J.F.R. Paton2 Where applicable, the authors confirm that the experiments 1 described here conform with The Physiological Society ethical Henry Wellcome Laboratories for Integrative Neuroscience and requirements. Endocrinology, University of Bristol, Bristol, UK and 2Department of Physiology & Pharmacology, Bristol Heart Institute, University of Bristol, Bristol, UK An increase in plasma osmolality induced by dehydration causes PC6 an increase in sympathetic nerve activity (SNA). The central Reactive Oxygen Species Mediates Acute Vascular nervous mechanisms underlying the increase in SNA after dehy- Contraction Responses In Rat Mid Cerebral Artery dration are not fully established. Here, we investigated the sequential effects of systemic administration of Losartan (20 S. Zainalabidin, R.M. Wadsworth and P. Coats μM, angiotensin II type 1 (AT1) receptor antagonist), brain tran- SIPBS, Uni of Strathclyde, Glasgow, UK sections and chemical inhibition of commissural nucleus tractus solitarii (cNTS) on ongoing thoracic SNA after chronic dehy- Intro: Pressure-dependent myogenic responses play a signifi- dration (DH; 3 days of water deprivation). cant role in modulating autoregulation of resistance arteries, Subsequent to DH, experiments were performed in the in situ especially in cerebral circulation [1]. Increasing evidence sug- working heart-brainstem preparation of rat (Antunes et al, gests a potential physiological role for NADPH-oxidase and reac- 2006). Under deep halothane anaesthesia (assessed by an tive oxygen species (ROS) in pressure-dependent myogenic absence of a limb withdrawal reflex to noxious pinching), the tone [2]. There may also be potential link between ROS and rat was transected below the diaphragm, decorticated to make actin cytoskeletal dynamics [3]. In this study, we investigated insentient and perfused with oxygenated Ringer’s solution via the potential role of NADPH-oxidase/ROS and actin polymer- the descending aorta. Perfusion pressure, heart rate, phrenic ization in acute pressure-dependent vascular contraction. nerve activity and thoracic SNA were recorded. Data are Method: Adult male Sprague-Dawley rats (12 weeks, expressed as mean ± SEM. 250~300g) were asphyxiated using CO2 and euthanized by cer- In euhydrated (EH) rats (290 mOsmol perfusate), systemic vical dislocation. Pressure-dependent responses were recorded application of Losartan and subsequent pre-collicular transec- in isolated mid cerebral arteries (MCA; Diameter 150 ± 7.81 tion (to remove the hypothalamus) reduced SNA by -20±3% μm) using pressure myography. Pressure-dependent responses and -44±2% respectively (n=5;P<0.05). In contrast, in DH rats were studied by pressure-step (40 to 80 to 120 mmHg). Con- (340 mOsmol perfusate,n=6) Losartan, subsequent pre-col- focal microscopy was used to visualize staining of F-actin using licular and then pontine transections failed to reduce SNA (- phalloidin-FITC (1μM) in pressure-fixed vessel. Statistics analy- 1±4%, -3±8% and -12±4%; P=0.753, P=0.995 and P=0.372, sis was measured by two-way ANOVA for repeated measure ANOVA). However, transection at the medulla-spinal cord junc- and values presented as mean ± SEM. Results & Discussion: Pres- tion reduced SNA by -70±8%. In intact DH, but not EH rats, sure-dependent myogenic tone in MCA control was 11.46 ± reversible inactivation of cNTS using isoguvacine, (a GABAA 3.05, 18.72 ± 2.88 and 23.75 ± 2.61 % at 40, 80 and 120mmHg, receptor agonist; 100 mM, 100 nl), reduced significantly base- respectively (n=14). Pressure-dependent myogenic tone was line SNA (-33±7% in DH; P<0.01 vs 3±8% in EH). Since it was significantly reduced following NAC, DPI and Cyto D incuba- demonstrated that dehydration increases FosB staining in the tion. The antioxidant N-acetylcysteine (NAC, 10mM, n=4) cNTS (Ji et al, 2007), we chronically blocked AP1 transcription reduced myogenic tone to 7.72 ± 1.31, 7.44 ± 2.81, 8.06 ± 1.44 factor activity in the cNTS using a viral vector expressing a FosB % at 40, 80 and 120mmHg respectively. The NADPH-oxidase dominant negative (Ad-CMV-IRESEGFP-dnFosB; 2.9x106 pfu/μl) inhibitor diphenyleneiodonium (DPI, 10μM, n=3) reduced myo- 5-7 days prior to 3 day water deprivation. In these animals, inac- genic tone to 5.93 ± 1.53, 2.00 ± 2.33, -1.15 ± 2.25 % at 40, 80 tivation of the cNTS was ineffective (-6.9±8.7% vs. -49±8%, con- and 120mmHg, respectively. Likewise, the actin polymeriza- trol rats with Ad-CMV-eGFP in NTS; P < 0.05, Student’s t test). tion inhibitor Cytochalasin D (Cyto D, 5μM, n=4) reduced myo- In addition, in DH rats in which AP1 was blocked in the cNTS, genic tone to 9.52 ± 4.32, 3.79 ± 0.67, 0.45 ± 1.78 % at 40, 80 SNA was now decreased after pre-collicular transection (- and 120mmHg, respectively. Time-control experiments 45±8%), a response that was similar to that seen in control EH showed sustained pressure-dependent myogenic tone. NAC rats (-35±6%). and DPI did not have any effect on high-K+ PSS (60mM) con- These data indicate that in the EH rat baseline SNA is depend- striction. The confocal study showed that there was a marked ent on both the hypothalamus and AT1 receptors. Following increase of actin polymerization in vascular smooth muscle

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(VSM) at 120mmHg compared to 40mmHg. The effect of Cyto interceptions.μm-1). Thus, interestingly, innervation density D greatly reduced the fluorescence measured at 120 mmHg. significantly decreased with maturation in males, but not in This further confirmed a dynamic actin polymerization contri- females. bution to the pressure-dependent myogenic tone mechanism Thus, the present results indicate that in juvenile (pre-pubes- in the cerebral arteries. Conclusion: These data suggest that cent) rats, the sympathetic innervation density of an artery with the activation of NADPH oxidase/ROS/ actin polymerization major thermoregulatory function is lower in females than as part of the acute pressure-dependent myogenic constriction males. However, the innervation densities are similar in sexu- mechanism. ally mature males and females because density decreases with Schubert & Mulvany (1999) Clinical Science. 96: 313-326. maturity in males, but not in females. Given sympathetic vaso- Keller et al. (2006) The FASEB Journal. 05-4075fje. constrictor influences are greater in sexually mature pre- menopausal women than in age-matched men (2), we suggest Gokina & Osol (2002) Am J Physiol Heart Circ Physiol 282 : H1410-20. that female reproductive hormones preserve or enhance the Ministry of Higher Education (Malaysia), SIPBS influence of sympathetic nerve fibres on thermoregulatory vessels. Post-menopausally, sympathetic nerve activity is higher Where applicable, the authors confirm that the experiments in females than males (3), so raising the question as to how the described here conform with The Physiological Society ethical menopause affects sympathetic innervation density. requirements. Charkoudian N (2001). Clin Auton Res 11, 295-301. Dart AM et al (2002). Cardiovasc Res 53, 678-687. PC7 Narkiewicz K et al (2005). Hypertension 45, 522-525. Cowen T & Burnstock G (1980). Histochem 66, 19-34. Effect of sex and maturation on sympathetic innervation density of the rat caudal ventral artery (CVA) – a role in Where applicable, the authors confirm that the experiments thermoregulation? described here conform with The Physiological Society ethical requirements. A. Ledsam, A.M. Coney, J.M. Marshall and C.J. Ray School of Clinical and Experimental Medicine (Physiology), Medical School, University of Birmingham, Birmingham, UK PC8 Cutaneous sympathetic vasoconstrictor nerves are tonically active; changes in their activity regulate skin blood flow and Do NADPH oxidase (Nox)-derived reactive oxygen species are important in thermoregulation. Studies in humans have (ROS) modulate muscle vasodilator responses to acute shown that female reproductive hormones have substantial systemic hypoxia or adenosine? influences on thermoregulatory responses (1) and that before the menopause, the vasoconstrictor influences of sympathetic M. Lawless, C.J. Ray, J.M. Marshall and A.M. Coney nerve fibres are greater in females than males (2). Sympathetic School of Clinical & Experimental Medicine (Physiology), University nerve activity to skeletal muscle vasculature increases with age of Birmingham, Birmingham, UK in both women and men, but is lower in women than men in the 20-29 year age range (3). Whether the same is true of cuta- Two major sources of ROS in vascular tissue are xanthine oxi- neous sympathetic vasoconstrictor nerves is not known, nor dase (XO), which generates ROS from the metabolites of adeno- whether any differences between males and females exist pre- sine and Nox1. The vasodilatation that occurs in skeletal mus- puberty. Further, it is not known whether there are differences cle during acute systemic hypoxia is largely mediated by in sympathetic nerve innervation density that might contribute adenosine and nitric oxide (NO)2. During acute systemic to sex-related differences in thermoregulation. Thus, we have hypoxia, ROS levels increase in skeletal muscle, and the hypoxia- investigated the effect of sex and maturation on sympathetic induced muscle vasodilatation is modulated by XO-derived ROS nerve innervation density of the CVA of the tail, the main ther- 3 - which may act directly, or by interacting with NO : O2 moregulatory organ in the rat. decreases NO bioavailability by combining with NO to form per- Four groups (n=6) of Wistar rats were used: juvenile male and oxynitrite. Nox activity is increased in chronic conditions such female (4-weeks) and sexually mature (12-weeks) male and as hypertension4, but Nox expression was also increased by female, approximately equivalent to 10 year-old and 30 year- acute hypoxia in pulmonary artery5. It is not known whether old humans. A length of CVA was removed under anaesthesia ROS generated acutely from Nox can modulate skeletal mus- (Alfaxan 12 mg.kg-1.hr-1 i.v.). The catecholamine-containing cle dilatation. sympathetic nerve fibres were visualised using the glyoxylic Thus, in anaesthetized male Wistar rats (Alfaxan: 3-6ml.kg-1.hr- acid method and quantitative analysis was performed to meas- 1 iv), arterial blood pressure (ABP) and femoral blood flow (FBF) ure surface density of the nerves on the blood vessel (4). Com- were recorded; femoral vascular conductance (FVC) was comparisons were made using Students unpaired t-test, P<0.05 puted (FBF/ABP). Responses evoked by 5-minute periods of being considered significant. - hypoxia (breathing 8% O2) and adenosine infusion (1.2mg.kg The innervation density of the CVA of female juvenile rats was 1.hr-1 ia) were recorded before and after, the Nox inhibitor apoc- significantly lower than that of males (0.049±0.003 vs ynin (13mg.kg-1 iv, Group 1), the NO synthase inhibitor L-NAME 0.056±0.001 interceptions.μm-1). However, there was no dif- (10mg.kg-1 iv, so as to maximize ROS levels) followed by apoc- ference between the innervation densities of the CVA in sexu- ynin (Group 2) and L-NAME followed by a second Nox inhibitor ally mature female and male rats (0.045±0.002 vs 0.047±0.003 DPI (Group 3). The muscle vasodilatation was analysed as the

84P Poster Communications change in the integral of FVC (IntFVC) in arbitrary conductance rats at 20 and 40 weeks of age. Receptor expression was deter- units (CU). Comparisons were made by ANOVA, P<0.05 being mined at mRNA level using RT-PCR (normalized to β-actin). Iso- considered significant. metric contractions were recorded from proximal sections (3- Acute hypoxia induced a fall in ABP (from 103±3 to 5 mm, endothelium-denuded) in response to electrical stimuli 50±4mmHg), and muscle vasodilatation (IntFVC increased from (5 impulses, 1ms duration at 20Hz) delivered every 90 seconds. 2.8±0.4CU by 2.6±0.4CU). In Group 1, apocynin had no effect NPY (100nM) potentiated responses in all groups (20 week con- on the hypoxia-induced increase in IntFVC. As expected, in trol vs diabetic: 39 ± 10%, mean ± S.E., n=8; vs 62±11%, n=6; 40 Group 2, L-NAME increased baseline ABP (to 130±1mmHg) and week control vs diabetic: 56±4%, n=8 vs 69±8%, n=5; P<0.01 in decreased IntFVC (to 1.6±0.2CU) reflecting removal of the effect each case, unpaired Student’s t-test). No differences were of tonic NO release, and the hypoxia-induced increase in Int- detected between time-points. NPY Y1 specific antagonist FVC was attenuated (1.2±0.2CU), but apocynin had no effect BIBP3226 (1μM) reduced responses in all groups (20 week con- on baseline (1.7±0.2CU) or on the hypoxia-induced dilatation trol vs diabetic: 40±7% vs 37±6%, n=8 and P<0.01 in each case; (1.4±0.2CU). Similarly, in Group 3, DPI had no effect on the 40 week control vs diabetic: 32±3 % vs 26±3%, n=5 and P<0.05 hypoxia-induced increase in IntFVC after L-NAME. Moreover, in each case). No differences were detected between groups. adenosine infusion induced similar dilator responses to hypoxia, NPY Y2 agonist PYY3-36 potentiated responses in 20 week old but they were not affected by apocynin or DPI. diabetic artery (P<0.01, 42±5%, n=9) relative to control (1±2%, The pharmacological inhibitors of Nox currently available are n=5). However, this effect was not observed in 40 week old dia- not fully selective and when used individually, may have led to betic artery (12±3%, n=5) relative to control (4±%, n=5). NPY overestimation of the role of Nox. The lack of effect of apoc- Y2 specific antagonist BIIE0246 significantly reduced responses ynin or DPI in the present study leads us to propose that Nox- in 20 week old diabetic artery (P<0.01, 32±6.10%, n=9) relative ± derived ROS do not modulate the vasodilatation induced in to control (3.4 6.25%, n=8). Similar to Y2 agonist, this was not skeletal muscle by acute systemic hypoxia, even when the inhi- apparent in 40 week old diabetic artery (5±1%, n=5) relative bition of NO synthase prevents the scavenging of ROS by NO. to control (3±1%, n=5). ± It may be that a chronic condition, such as chronic hypoxia, is NPY Y1 receptor expression increased (P<0.05; 3.63 0.07, n=5) required increase Nox activity in systemic tissues. in diabetic arteries at 20 weeks relative control (1.28±0.06, n=5). ± Griendling K.K., Sorescu D. & Ushio-Fukai M. (2000). Circ. Res. 86: However, no change in NPY Y1 receptor expression (1.19 0.02, 494-501 n=6) was observed in diabetic artery at 40 weeks relative to control (0.82±0.06, n=5). NPY Y receptor expression was elevated Ray C.J. & Marshall J.M. (2005). J. Physiol. 568:967-978 2 in 20 week old diabetic artery (P<0.01, 3.38±0.1, n=5) relative Pyner S, Coney A. & Marshall J.M. (2003). Exp. Physiol. 88:733-740 to control (1.22±0.04, n=5). In contrast, there was a reduction ± Carlstrom M. et al (2009). Am. J. Physiol. 296: R72-R79 in expression (P<0.05, 0.36 0.02, n=5) in 40 week old diabetic artery relative to its control (1.09±0.01, n=5). Muzaffar S et al (2005). Thorax 60:305-313. These data indicate that enhanced NPY Y1 and Y2 receptor expression and its associated contribution to vasoconstriction Where applicable, the authors confirm that the experiments in a model of Type 1 diabetes has a temporal aspect to its described here conform with The Physiological Society ethical involvement in peripheral vascular dysfunction. requirements. Speirs et al., (2006) Am J Physiol 291: 2327-2333

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC9 requirements.

Contribution of NPY Y1 and NPY Y2 receptors to sympathetic vasoconstriction in diabetic rat tail artery at two time points PC10 P. Dickson, D. Bell, N. Scholfield and C. Johnson Contributionofalteredexpressionofa1a-adrenoceptorsand School of Medicine, Dentistry and Biomedical Sciences, Queen’s P2X1 purinergicreceptorstofunctionalchangesintailarteries Univeristy of Belfast, Belfast, UK of streptozotocin-induced diabetic rats and Akita mice Vascular dysfunction is a common consequence of diabetes P. Dickson, D. Bell, N. Scholfield and C. Johnson mellitus. Alterations in sensitivity/responsiveness to neuro- School of Medicine, Dentistry and Biomedical Sciences, Queen’s transmitters (noradrenaline (NA), ATP and NPY) may underlie Univeristy of Belfast, Belfast, UK functional abnormalities of diabetic blood vessels (Speirs et al., 2006). Previous studies have reported considerable variation Autonomic nervous dysfunction is a common complication of in such alterations, potentially due to sampling at different time- diabetes mellitus. Previously, we have hypothesized that alter- points after induction of diabetes. In this study, contributions ations in sensitivity/responsiveness to noradrenaline (NA), ATP of NPY Y1 and NPY Y2 receptors to sympathetic vasoconstric- and Neuropeptide Y (NPY) may underlie functional abnormal- tion were explored in tail artery of diabetic rats at 20 and 40 ities of diabetic blood vessels (Speirs et al., 2006). In this study, α weeks of age. changes in expression of predominant adrenergic ( 1a-adreno- α Tail arteries were excised from diabetic (60 mg.kg-1 strepto- ceptor: 1a-AR) and purinergic (P2X1) receptors were investi- zotocin, i.p. injection at 8 weeks) and control Sprague-Dawley gated in tail arteries of streptozotocin-treated rats and Akita

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patients with Brugada syndrome and complication of atrial fib- mice (both type 1 diabetes models); expressional studies were rillation (AF) [1]. By using biophysical simulations, we investi- supplemented with functional studies in rat tail arteries. gated (a) the effects of the mutation on atrial electrical action Tail arteries were excised from diabetic (60 mg.kg-1 strepto- potentials (AP) at the cellular level; (b) the effects of the muta- zotocin, i.p. injection at 8 weeks) and control Sprague-Dawley tion on propagation of atrial excitation waves on spatially rats at 20 weeks and Akita mice and controls at 10 weeks. extended virtual human atrial tissues. Receptor expression was determined at mRNA and protein Methods: The Courtemanche et al. [2] model of human atrial level using RT-PCR and Western blotting (normalized to β- cell was modified to incorporate experimental data of Shin et actin mRNA and β-actin respectively). Isometric contractions al. [1] on the mutation-induced loss-of-function in I , which were recorded from proximal sections of rat tail arteries (3- Na resulted in a reduction in the maximal channel conductance. 5 mm length, endothelium-denuded). Agonist concentra- The modified model was implemented to simulate APs under tion response curves were expressed as a % of contraction control and mutation conditions. Characteristics of APs for both induced by 60 mM KCl. conditions were computed that include the resting potential, Maximal contraction induced by NA was greater in arteries of AP amplitude (APA), maximum upstroke velocity (dV/dt ), diabetics (279±17%, mean ± S.E., n=20, P<0.001; 2-way ANOVA, max AP duration at 50 % repolarisation (APD ) and at 90 % repo- Bonferroni post-hoc tests) relative to arteries of age-matched 50 larisation (APD ) and over shoot (OS). Single cell model was control rats (n=20, 179±12%). α -AR expression was not 90 1a then incorporated into an one-dimensional reaction diffusion increased in arteries of diabetic rats relative to controls at mRNA partial differential equation model of human atrial strand, using (1.16±0.16 vs. 1.01±0.05; n=5 each, unpaired students t-test) which the conduction velocity (CV) was determined by pacing or protein level (1.17±0.31 vs. 0.79-±0.15; n=5 each). Maxi- the 1D strand with standard S1-S2 stimulus protocols. mal contraction produced β,γ-methylene ATP was greater in Results: The SCN5A-W1191X mutation resulted in no signifi- arteries of diabetics (P<0.001; 103±9%; n=20) relative to age- cant change in either APD or APD . However, it reduced sub- matched control rats (69.9±11%; n=20). Parallel increases in 50 90 stantially the dV/dt , which reduced from 217.081 mV/ms P2X receptor expression were observed at both mRNA (P<0.01; max 1 in Control to 109.5 mV/ms under mutant conditions. Conse- 4.09 + 0.67; n=5) and protein level (P<0.01; 0.48±0.04) relative quentially the computed OS was also decreased from 24.75 mV to age-matched controls (1.22±0.37; 0.24±0.01; n=5). in Control to 8.5 mV in mutation. In the 1D simulations, the Similarly, P2X receptor mRNA expression was increased in tail 1 measured CV for solitary excitation waves was reduce from 0.27 arteries of diabetic mice compared to age-matched control mm/ms in Control to 0.20 mm/ms in mutation conditions. mice (P<0.05; 0.93±0.15 vs. 0.49±0.18; n=5 each) however in Conclusions: SCN5A-W1191X does not alter the human atrial contrast to the absence of expressional changes in tail arteries AP morphology significantly, but decrease its excitability result- of diabetic rats, α -adrenoceptor mRNA expression was also 1a ing in a dramatic slowing of CV. Reduction of CV shortens the increased (P<0.05; 1.54±0.26; n=5) in tail arteries of diabetic wavelength of atrial excitation waves, allowing 2D and 3D sub- mice relative to age-matched control mice (0.94±0.06; n=5). strates to sustain re-entrant waves, which is pro-arrhythmic. These data indicate that augmented expression of P2X1 recep- α Summary of Results tors, but not of 1a-AR, may contribute to changes in peripheral vessel function in Type 1 diabetes; contractile studies in Akita diabetic mice are awaited to confirm the contribution of α the observed augmented expression of P2X1 receptors and 1a- AR to altered function in diabetes. Speirs et al., (2006) Am J Physiol 291: 2327-2333

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

PC11

Conduction Propagation Dysfunction with No Apparent Changes in Cellular Electrical Action Potentials in Human Atria: A Simulation Study P.R. Law, J. Stott, S. Kharche and H. Zhang School of Physics and Astronomy, The University of Manchester, A. AP profiles under Control (solid line) and SCN5A mutant (dashed line) Manchester, UK conditions. APD90 is not affected and overshoot is reduced. The maximum upstroke velocity is dramatically reduced under mutant conditions. B. CV Aim: Mutations in the SCN5A gene encoding for the α-sub- restitutions under Control (solid line) and SCN5A mutant (dashed line) con- unit of the cardiac sodium channel, INa, might result in dys- ditions. CV under mutant conditions was reduced due to mutation. function of cardiac excitation wave propagation. In this study Shin DJ et al. (2007). Life Sciences 80, 716-724. we computationally evaluated the functional impacts on atrial Courtemanche M et al. (1998). Am J Physiol 275, H301-H321. excitation of a recently identified mutation SCN5A-W1191X in

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This work was supported by two EPSRC (UK) Ph.D. studentships (grant nos. EP/P50158X/1 and EP/P502616/1). Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. Brothwell et al., 2008 JPhysiol 586 739 Faleiro et al., 2004 Neuropsychopharm. 29, 2115 Saal et al., 2003 Neuron 37, 577 PC12 We thank the BBSRC for funding this work Amphetamine administration evokes no change in Where applicable, the authors confirm that the experiments ifenprodil-sensitivity of synaptic NMDA receptors in described here conform with The Physiological Society ethical dopaminergic neurones of rat substantia nigra requirements. F. Suarez1, M. Smith1, A.J. Gibb2 and S. Jones1 1Department of Physiology, Development and Neuroscience, PC13 University of Cambridge, Cambridge, UK and 2Department of Neuroscience, Physiology and Pharmacology, University College Immunomodulatory properties of eicosapentaenoic acid London, London, UK and its derivative docosapentaenoic acid in vivo. Glutamatergic synapses in rodent midbrain dopaminergic neu- B. Grehan, L.C. Kelly and M.A. Lynch rones show an increase in the ratio of AMPA receptor (AMPAR)- mediated excitatory postsynaptic currents (EPSCs) to NMDA Physiology Department, Trinity College Institute of Neuroscience, receptor (NMDAR)-mediated EPSCs following a single dose of Dublin, Ireland amphetamine administered 2-24 hours prior to EPSC record- The aged brain is characterized by an increase in pro-inflam- ings (Saal et al., 2003; Faleiro et al., 2004). This form of synap- matory and oxidative molecules (Griffin et al., 2006, Finkel and tic plasticity may contribute to behavioural adaptations seen Holbrook, 2000), with a concomitant decrease in anti-inflam- in response to amphetamine, and may reflect changes in synap- matory and anti-oxidative mediators (Murali and Panneersel- tic AMPAR expression, NMDAR expression, or both. Previously vam, 2007) and evidence from this laboratory has suggested we have shown that synaptic NMDARs in rat substantia nigra that the polyunsaturated fatty acid, eicosapentaenoic acid (EPA) dopaminergic neurones contain both NR2B and NR2D subunits attenuates some of these age-related changes (Lonergan et al., (Brothwell et al., 2008). We have used whole-cell patch-clamp 2004, Lynch et al., 2007). The aim of the present study was to recordings to determine whether or not the sensitivity of investigate age-related changes in several markers of microglial NMDARs to the NR2B-preferring antagonist, ifenprodil, is activation and to establish whether treatment of aged and changed in dopaminergic neurones following a single dose of young rats with EPA or its metabolite docosapentaenoic acid, amphetamine. Rats aged ~postnatal day (P)6 or ~P13 were (DPA), modulated any change which occurred with age. given a single intra-peritoneal injection of amphetamine (2.5 Groups of young (3-5 months) and aged rats (22-24 months) -1 mg kg ) or saline control, and the locomotor response for 45- were divided into control and experimental groups; the ani- 60 minutes following the injection was computed (number of mals in the experimental group received 200mg/kg/day EPA or infrared beam breaks); locomotor activity was significantly DPA orally for 8 weeks. At the end of this period, animals were greater (P<0.05, t-test) in rats injected with amphetamine com- killed by cervical dislocation and cryostat sections were pre- ± pared with saline at the two ages tested (Table 1; mean SEM, pared from hemisected brains and stained for evidence of n in parentheses). Approximately 24 hours later, midbrain slices change in markers of microglial activation, CD11b, MHC class containing substantia nigra were prepared. In dopaminergic II, and CD68, as well as for expression of activated caspase 3, 2+ neurones voltage-clamped to +40mV (to remove Mg block and the marker of oxidative stress, 8-OHdG. of NMDARs), EPSCs were evoked by electrical stimulation in the There was a marked increase in expression of each of the mark- μ presence of 10 M glycine. In the first set of experiments the ers of microglial activation in sections prepared from aged, total EPSC, consisting of AMPAR- and NMDAR-mediated com- compared with young, rats; this is consistent with previous evi- μ ponents, was recorded and then D-AP5 (50 M) applied to block dence of an age-related increase in microglial activation (Grif- NMDAR-EPSCs. The AMPAR-EPSC / NMDAR-EPSC ratio was signi- fin et al., 2006) and, significantly this effect was attenuated in ficantly greater (P<0.05; ANOVA) in rats aged ~P14 injected sections prepared from aged rats which received EPA or DPA. with amphetamine compared with naïve rats (Table 1). In the Furthermore, the evidence indicated that caspase 3 immunore- second set of experiments NMDAR-EPSCs were pharmacolog- activity was increased with age and that this was also attenu- μ ically isolated and the effect of ifenprodil (10 M) was deter- ated by EPA and DPA. Consistent with earlier evidence of an mined. There was no significant difference in the inhibition of increase in oxidative change in tissue prepared from aged rats, NMDAR-EPSCs by ifenprodil in rats injected with amphetamine we also report that 8-OHdG staining was greater in sections (Table 1). These results suggest that the change in the AMPAR- prepared from aged, compared with young, rats and that this EPSC / NMDAR-EPSC ratio is developmentally sensitive, and that effect was markedly reduced in sections prepared from aged the proportion of synaptic NR2B-containing NMDARs is not rats which received EPA and DPA. This finding is consistent with altered by a single dose of amphetamine. Table 1

87P Poster Communications previous observations which identified an anti-oxidative effect with WT mice and the differences were statistically significant of EPA (Lonergan et al., 2004). at both mRNA and protein levels (p < 0.05; ANOVA). These data demonstrate that the EPA derivative, DPA, like EPA These findings, which indicate that mixed glial prepared from itself, possesses anti-oxidative properties and that this is likely CD200-/- mice were more responsive to LPS than glia prepared to be a consequence of the modulatory effect of the fatty acids from WT mice and are consistent with our previous observa- on microglial activation. tion that interaction of CD200 with its receptor contributes to Finkel, T. & Holbrook, N. J. (2000) Oxidants, oxidative stress and the the maintenance of microglia in a quiescent state. biology of ageing. Nature, 408, 239-47. This work was supported by a grant obtained from the EU. Griffin, R., Nally, R., Nolan, Y., McCartney, Y., Linden, J. & Lynch, M. A. (2006) The age-related attenuation in long-term potentiation is asso- Where applicable, the authors confirm that the experiments ciated with microglial activation. J Neurochem, 99, 1263-72. described here conform with The Physiological Society ethical Lonergan, P. E., Martin, D. S., Horrobin, D. F. & Lynch, M. A. (2004) Neu- requirements. roprotective actions of eicosapentaenoic acid on lipopolysaccharide- induced dysfunction in rat hippocampus. J Neurochem, 91, 20-9. Lynch, A. M., Loane, D. J., Minogue, A. M., Clarke, R. M., Kilroy, D., Nally, PC15 R. E., Roche, O. J., O’Connell, F. & Lynch, M. A. (2007) Eicosapentaenoic acid confers neuroprotection in the amyloid-beta challenged aged hip- Downregulation of CD200 is coupled with the inflammatory pocampus. Neurobiol Aging, 28, 845-55. phenotype in the CNS of mice with experimental Murali, G. & Panneerselvam, C. (2007) Age-associated oxidative macro- autoimmune encephalomyelitis molecular damages in rat brain regions: role of glutathione monoester. J Gerontol A Biol Sci Med Sci, 62, 824-30. B.F. Deighan, A.C. Murphy and M.A. Lynch Department of Physiology, Trinity College Dublin, Dublin, Ireland This work was supported by Amarin Corporation, plc and Trin- ity College Dublin. Microglial activation has been identified as one factor which contributes to the deterioration in function associated with Where applicable, the authors confirm that the experiments experimental autoimmune encephalomyelitis (EAE), the ani- described here conform with The Physiological Society ethical mal model for multiple sclerosis. Microglial activation is char- requirements. acterized by an increase in the expression of cell surface markers such as CD40 and an increase in the production of proinflammatory cytokines such as interleukin 1β (IL-1β). PC14 Recent evidence has indicated that microglial activation is modulated by the interaction between CD200 which is expressed The lipopolysaccharide-induced increase in pro- on neurons and its cognate receptor, CD200 receptor, which is inflammatory cytokine production is exaggerated in mixed expressed on microglia (Lyons et al., 2007). The objectives of glia prepared from mice deficient in CD200 this study were to establish whether any change in microglial activation in the spinal cord of mice with EAE was associated F. Cox and M.A. Lynch with a downregulation of CD200 and to assess whether simi- Trinity College Dublin, Dublin, Ireland lar changes were observed in hippocampus. EAE was induced in C57 mice by injection of myelin oligodro- CD200 is a cell-membrane protein expressed on several cells cyte glycoprotein (MOG), pertussis toxin (PT) and complete including neurons and endothelia. Its cognant receptor, Freund’s adjuvant (CFA), and 48 hours later by an additional CD200R, is primarily expressed on cells of the myeloid line- injection of PT. Clinical symptoms were observed over 21 days age, including microglia. It is believed that engagement of (Reinke et al., 2007) and symptoms consistent with the onset CD200 with its receptor can lead to immunosupression, thus of EAE were observed after 10 days and these progressed to restraining myeloid cells from tissue-damaging activation. hindlimb weakness thereafter. At the end of the 21 day period, Recent evidence from this laboratory has indicated that there mice were sacrificed and spinal cord and hippocampus were is an age-related decline in CD200 expression in the hip- removed. Tissue was prepared for analysis of CD40 and CD200 pocampus and it is suggested that this decrease could con- mRNA by QPCR and for analysis of pro-inflammatory cytokines tribute to the enhanced pro-inflammatory profile observed in by ELISA. the brain of aged rats. We observed an increase in the expression CD40 mRNA in the The action of the inflammatory stimulus lipopolysaccharide hippocampus and the spinal cord of mice with EAE compared (LPS) was investigated in mixed glia prepared from neonatal with control mice (p<0.05, ANOVA, n=6). We further report C57BL/6 wild-type (WT) and CD200-/- mice. Mixed glia were an increase in IL-1β protein in the hippocampus and spinal cord incubated in the presence or absence of LPS (1μg/ml) for 24 of mice with EAE mice (p<0.05, ANOVA, n=6). The increase in hours and following this, the supernatant was collected for CD40 mRNA was associated with a decrease in CD200mRNA analysis of the pro-inflammatory cytokines IL-1β, IL-6 and TNF- and protein in the spinal cord of EAE mice (p<0.05, ANOVA, n=6) α by ELISA and analysis of mRNA expression of these cytokines providing further evidence of an inverse correlation between by Q-PCR. LPS significantly increased IL-1β, IL-6 and TNF-α at microglial activation and expression of CD200. mRNA and protein levels in glia prepared from WT and CD200- The data highlight the importance of the CD200 receptor-ligand /- animals (p < 0.05; ANOVA; n=6). However LPS increased pro- interaction in the inflammatory phenotype associated with the duction of pro-inflammatory cytokines to a greater extent in symptoms of EAE. mixed glial cultures prepared from CD200-/- mice compared

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Reinke EK, Lee J, Zozulya A, Kaman J, Muller WA, Sandor M, Fubry ric analysis for cAMP was carried out in hippocampal slice Z.Short-term sPECAM-Fc treatment ameliorates EAE while chronic use homogenates to investigate if A1Rs were activated after TNF- hastens onset of symptoms.(2007) α treatment (3ng/ml) and re-oxygenation. Unexpectedly, an Journal of Neuroimmuology. May 186 (1-2) 86-93. increase in cAMP was observed in TNF-α treated slices Lyons A, Downer ED, Crotty S, Nolan YM, Mills KH, Lynch MA. CD200 (20.6±2.3nM versus 11.4±1.5 nM controls; n=4; p< 0.05), an ligand receptor interaction modulates microglial activation in vivo and effect reversed by the p38 mitogen-activated protein kinase in vitro: a role for IL-4.(2007). Journal of Neuroscience. August 1 27(31) inhibitor (SB203580; 1 μM) (8.3±1.4 nM; n=4; p< 0.05). These 8309-13 data show that TNF-α can modulate synaptic transmission after I would like to acknowledge the support of the Health Research a hypoxic exposure, an effect that does not seem to involve A1R Board (HRB) in Ireland and Trinity College Dublin. activation during re-oxygenation. [1] Burkovetskaya ME, Levin SG, Godukhin OV (2007). Neuroprotec- Where applicable, the authors confirm that the experiments tive effects of interleukin-10 and tumor necrosis factor-alpha against described here conform with The Physiological Society ethical hypoxia-induced hyperexcitability in hippocampal slice neurons. Neu- requirements. roscience letters 416(3), 236-40. EU Marie Curie Action

PC16 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Modulation of synaptic transmission by tumor necrosis requirements. factor-alpha after hypoxic exposure in rat hippocampal slices L. Batti and J.J. O’Connor UCD, Conway Institute, Dublin, Ireland PC17 The brain is highly dependent on oxygen supply in order to maintain synaptic function and conductivity. Although a Calcium signalling during sperm-female tract interaction hypoxic event may lead to hippocampal neuronal death, an T.J. Connolly1,2, D.J. Smith1,2, S. Publicover3,2 and J. Kirkman- early neuroprotective response to this insult is the activation Brown1,2 of adenosine receptors (A R). The role of pro-inflammatory 1 1 1Clinical and Experimental Medicine, University of Birmingham, components in an ischaemic/hypoxic episode is still contro- 2 versial, although deleterious effects of pro-inflammatory Birmingham, UK, Center for Human Reproductive Science, Birmingham Women’s Hospital, Edgbaston, Birmingham, UK and cytokines in the area of injury are well documented. Recent evi- 3 dence suggest a role for tumor necrosis factor-alpha (TNF-α) School of Biosciences, University of Birmingham, Edgbaston, in protecting neuronal cells against hyperexcitability induced Birmingham, UK by hypoxic insults [1]. In the present study we have investigated The events during migration of human sperm through the the modulatory actions of TNF-α on synaptic transmission dur- female reproductive tract remain almost completely unchar- ing and after hypoxia. We have also investigated the role of A1R acterised. Not only are sperm known to be sensitive to prod- in this effect. Hippocampal slices (350 μm) were obtained from ucts of the female tract but animal data suggests the female P21 male Wistar rats that were humanely killed under anes- tract alters gene expression after exposure to sperm (1), imply- thetization (4% isoflurane, by inhalation). fEPSPs were elicited ing two-way communication. We have used single-cell fluo- from the Shaffer collateral pathway in the CA1 region every 30 rescence imaging to observe calcium signalling in both human s. Long-term potentiation (LTP) was induced with 3 trains of sperm and human reproductive tract cells upon initial contact 100 stimuli applied at 100Hz every 30 s. Statistical analysis was and during sperm adhesion and release. performed using Mann-Whitney test. All results are represented Explants and primary cell lines were prepared from donated as mean±S.E.M. All fEPSP slope measurements are presented human reproductive tract tissue removed during surgery (2). as a percentage of baseline recordings. Following a 2 hr hypoxic An immortalised oviductal cell line (OE E6/E7) has been used ± exposure with N2 perfusion (PO2 37 2 mmHg above the slice; as an internal standard (3). Human sperm were harvested via 30.8±10.4% compared to controls), the fEPSP returned to con- a modified swim-up technique. Briefly, sperm were selected by trol levels. Inhibition of A1Rs (8-cyclopentyl-1,3-dipropylxan- their ability to migrate through a viscous medium (~ 140 cen- thine; DPCPX; 200 nM) significantly reversed the hypoxia- tipoise) into sEBSS media, then washed and resuspended in induced synaptic depression (85.7±4.3%, versus controls; n=5, sEBSS media containing 0.3% FBS (charcoal stripped) at a con- p<0.05) but impaired the maintenance of LTP after re-oxy- centration of 1 x106 cells per ml. Sperm were incubated for at genation (96.4±10.6% versus 143.8±8.2% in controls, 1 hr post least 3 hours at 37oC 6% CO2 before use. tetanus; n=5; p<0.005). DPCPX also attenuated the hypoxic- To investigate whether there was rapid cell signalling occurring induced depression of the pharmacological isolated NMDA in human female tract cells upon exposure to sperm, tract cells fEPSP (40.5±5.2% versus 61.2±2.7%, n=5 p<0.05, 30 min after were labelled with 7.6 μM Calcium Green-1, AM for 1h at 37oC hypoxic exposure). 30 min TNF-α treatment (3ng/ml) during 6% CO2 to monitor intracellular calcium levels. Sperm were hypoxic exposure attenuated the recovery of the fEPSP after labelled with 5 μM Syto64, a red fluorescent nuclear dye, to re-oxygenation (61.0±7.2% versus 86.7±6.6%; n=5; p<0.05). allow tracking of sperm movement and contact with cells. Raw LTP induced after re-oxygenation was not affected by pre-treat- intensity values were imported into Microsoft Excel and nor- ment with TNF-α during hypoxia (138.0±8.3%, compared to malised. Values are percentage change in fluorescence ±S.E.M. controls, n=5). Liquid Chromatography and Mass Spectromet-

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Initial data with tract cells show that explants demonstrated ing. Electrical recordings were performed using perforated- transient responses of 9±5%(n=3) and an average peak dura- patch technique. Data are presented as mean±S.E.M. RT-PCR tion of ~ 20s. Primary tract culture responses were 15±10% analysis conducted on 500 RVSMCs collected under the micro- (isthmus, n=3), 7±4% (ampulla, n=3), both with similar peak scope with glass micropipette confirmed the expression of durations of ~ 30s. OE E6/E7 cells had larger responses of genes encoding P2X1-R, P2X4-R, P2Y2-R and P2Y6-R. The purity 22±10%(n=6) and generated transients with an average peak of phenotype of SMCs collected for RT-PCR analysis was con- duration of ~ 35s. Human sperm bound to and interacted with firmed by expression of the genes encoding SMC marker (SM- cells from all tract zones. Sperm swimming patterns appear to MHC) but not the markers for fibroblasts and endothelial cells be modified when in the presence of reproductive tract cells. (CD34), neurons (PGP9.5) and pericytes (NG2). Selective stim- We then investigated whether calcium signals occurred within ulation of P2X-Rs with 10 μM α,β-methylene adenosine 5’- 2+ sperm whilst in contact with tract cells. Sperm were loaded triphosphate (AMP-CPP) evoked [Ca ]i transient initiated by μ 2+ with 7.6 M Calcium Green-1, AM for 1h at 37oC 6% CO2. sub-plasmalemmal [Ca ]i upstroke (SPCU) [5]. Combination Detailed observations of signals in attached cells and responses of confocal Ca2+ imaging with electrical recordings revealed to progesterone exposure are currently being examined. Pre- that: (1) peak of both the AMP-CPP –induced action potential 2+ liminary evidence suggests that certain calcium signals may and IP2X preceded the peak of [Ca ]i transient; (2) kinetics of relate to observed motility changes. Research into these IP2X was consistent with predominant contribution of P2X1-Rs; 2+ processes may lead to a better understanding of the basic repro- (3) the amplitude of the [Ca ]i transient detected under volt- ductive biology, which will allow the development of rational age-clamp (Vh=-60 mV) was reduced by about 50% in com- treatments and diagnostics. parison to that observed under current-clamp. These observa- AllprocedureswereinaccordancewithLocalEthicsCommittee tions suggest that both Ca2+ entry through voltage-gated Ca2+ guidelines and patient and donors provided informed consent. channels (VGCCs) and Ca2+ release from intracellular stores also 2+ Georgiou AS et al. (2007) J Proteome Res. 6(12), 4656-66. contribute to AMP-CPP –induced [Ca ]i mobilisation. This was Pacey AA et al. (1995) Hum Reprod. 10(2):360-6. confirmed with selective pharmacological agents. AMP-CPP –induced [Ca2+] transient was attenuated by 56.9±3.3% (n=26) Lee YL et al. (2001) Mol Reprod Dev. 59(4), 400-9. i following depletion of intracellular Ca2+ stores by 10-min incu- This work was funded by the Infertility Research Trust and sup- bation with 10 μM cyclopiazonic acid and by 34.0%±3.3% (n=14) μ 2+ ported by the Centre for Human Reproductive Science. following block of VGCCs with 5 M nicardipine. The [Ca ]i transient remaining in the presence of both drugs had a peak Where applicable, the authors confirm that the experiments amplitude 31.4%±4.3% of that in control (n=16) and resulted described here conform with The Physiological Society ethical from direct Ca2+ entry though P2X-Rs. requirements. Navar LG et al. (1996) Physiol Rev 76, 452-536. Majid DSA et al. (1999) J Am Soc Nephrol 10, 492-498. White SM et al. (2001) Am J Physiol 280, F1054-F1061. PC18 Gordienko DV et al. (1994). Am J Physiol 266, F325 - F341. 2+ Electrical events and mechanisms of [Ca ]i mobilisation Gordienko DV et al. (2008). Cell Calcium 43, 122-141. induced by P2X receptor stimulation in rat renal resistance artery myocytes Supported by BHF (PG/08/062/25382 and FS/06/077). O. Povstyan1,2, M. Harhun1 and D.V. Gordienko1,2 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical 1Ion Channels and Cell Signalling Centre, St. George’s University of requirements. London, London, UK and 2Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, O.O.Bogomoletz Institute of Physiology, Kyiv, Ukraine Apart from being released as a co-transmitter from sympathetic PC19 nerve terminals, interstitial ATP acting upon P2Y and P2X purinoceptors has been shown to be an important paracrine Downregulation of ACh secretion in reinnervated mouse regulator of renal preglomerular microvascular function [1]. neuromuscular junctions involving PKC activity and voltage- + Indeed, inactivation of P2 receptors in renal resistance blood dependent K channels vessels inhibits autoregulatory behaviour [2]. Stimulation of P. Bogatcheva and O. Balezina P2X purinoceptors (P2X-Rs) in renal vascular smooth muscle 2+ Human and Animal Physiology, Moscow State University, cells (RVSMCs) increases [Ca ]i, thus triggering the myocyte 2+ Moscow, Russia contraction [3]. In this study we related the dynamics of [Ca ]i changes induced by selective P2X-R stimulation to correspon- On early stages of adult skeletal muscle reinnervation multiple ding changes in the cell membrane potential and the kinetics axonalinputspermusclefiberexist.Theseexcessiveinputsneed of P2X-R mediated cationic current (IP2X), and analised the tobeeliminateduntilonlyoneendplateperfiberremains.Synap- 2+ mechanisms of purinergic [Ca ]i mobilisation in RVSMCs at tic elimination includes decrease of synaptic activity and sub- sub-cellular level. Experiments were conducted on single sequentretractionofthesilentterminals[1].Previouslywehave RVSMCs freshly isolated from arcuate and interlobular arter- shown that Ca2+ entering the terminal through L-type Ca2+- 2+ ies dissected from rat kidney [4]. Changes of [Ca ]i in RVSMCs channels triggers the release of intracellular Ca2+ from ryan- loaded with Fluo-4 were visualised using fast x-y confocal imag- odinestores,whichinturnleadstodepressionofAChrelease

90P Poster Communications in reinnervated neuromuscular junctions [2]. Ca2+-dependent this disease. Previous studies have shown that oxidative stress protein kinase C which is known (along with other substrates) impairs hormone-evoked Ca2+ signalling and induces an irre- 2+ 2+ tomodulatetheactivityofvoltage-dependentK+-channels(Kv) versible increase in [Ca ]i (Ca overload) (Bruce et al, 2007). was considered as a possible target for this Ca2+ signal. This oxidant-induced Ca2+ overload response coincided with Study of electrical activity of regenerating motor synapses was inhibition of the PMCA and mitochondrial depolarisation. More- performed 11 days after the mechanical crushing (nembutal over, this oxidant-induced PMCA inhibition could occur with- (50 mg/kg) i.p. for general and 0.5% lidocaine hydrochloride out impairment of mitochondrial Ca2+ handling or ATP deple- s.c. for local anaesthesia) of n. peroneus communis, supplying tion and was attenuated by inhibitors of the mitochondrial m. extensor digitorum longus in adult mice. Spontaneous and permeability transition pore (Baggaley et al, 2008). evoked synaptic transmission and its sensitivity to Kv-channels Several studies have demonstrated that insulin can activate and Ca2+-dependent protein kinase C modulation was exam- pro-survival pathways and protect from pancreatic cell injury. ined. Mann-Whitney test was performed for statistical analy- Therefore the aim of the current study was to test the effects sis. All data are presented as mean±S.E.M. Blockade of protein of insulin on oxidant-mediated impairment of Ca2+ homeosta- kinase C, with chelerytrine (4 μM) and bisindolylmaleimide I (1 sis and inhibition of the PMCA. Pancreatic acinar cells were iso- μM) showed a significant increase in quantal content (QC) of lated from Sprague-Dawley rats by collagenase digestion. The ± ± 2+ EPSPs, up to 58 7% and 66 9% respectively (n=71, p<0.05). effect of hydrogen peroxide (H2O2) on resting [Ca ]i was tested This increase was very similar to effects of L-type Ca2+-chan- on fura-2-loaded cells treated with or without insulin (1- nels blocker nifedipine (10 μM): it caused rapid 60±5% eleva- 100nM). In addition, we utilised an in situ Ca2+ clearance assay tion of QC (n=60). But nifedipine applied after chelerytrine was in which the PMCA activity was pharmacologically isolated. A not able to affect amplitude or QC of EPSP`s. QC was 12±1 in paired experimental design was used to directly compare clear- ± ± control, 18 2 under influence of chelerytrine (p<0.05) but 17 2 ance phases in which H2O2 was applied during the second clear- under influence of both chelerytrine and nifedipine (n=65). ance phase and the rate normalised to the first clearance phase. Upregulation of QC caused by blocking the ryanodine recep- All data are presented as mean values ± standard error. μ tors with ryanodine (5 M) was prevented by adding chelery- Insulin pre-treatment (1nM) had no effect on the H2O2 induced ± ± 2+ μ ± trine to the bath solution: 8 1 in control, 15 1 after incubation increase in resting [Ca ]i (50 M: 0.47 0.14 ratio units, n = 3 with ryanodine (p<0.05), 10±1 with both ryanodine and chel- control and 0.40 ± 0.15 ratio units, n = 4 insulin treated). How- erytrine (n=67). These results suggest that activation of pro- ever,1nMinsulincausedapparentshiftintheproportionofcells μ tein kinase C may be triggered by Ca2+ influx from the intra- that recovered following H2O2 treatment (100 M: 36% of total cellular ryanodine stores, which in turn is activated by Ca2+ cells, n = 5 control and 56% of total cells, n = 6 insulin treated) entering the terminal through L-type Ca2+-channels. Applica- and an increase in the magnitude of recovery from the H2O2- tion of Kv-channels blocker 4-aminopyridine (6 μM) was found mediated Ca2+ overload response (50μM: 27.13 ± 0.93%, n = 3 to greatly increase QC of EPSP`s in newly formed synapses: control and 57.21 ± 9.13%, n = 4 insulin treated). Furthermore, ± ± from 19 2 in control to 30 2 (p<0.05, n=63). Pre-incubation H2O2 inhibitedthePMCAinaconcentration-dependentmanner with chelerytrine prevented further enhancement of synaptic (500μM: rate = 18.44 ± 4.75%, n = 6 compared to control: rate = transmission by 4-aminopyridine. EPSP`s QC reached 17±2 in 91.70 ± 12.54%, n = 8). This inhibition was attenuated in cells control, 30±2 after incubation with chelerytrine (p<0.05) and treated with 1nM insulin (500μM: rate = 44.53 ± 9.13%, n = 4). 30±2 with both chelerytrine and 4-aminopyridine (n=70). Insummary,thesedatasuggestthatinsulincouldprotectagainst The data obtained demonstrate that increased activity of Kv oxidant-induced Ca2+ overload and oxidant-induced inhibition of channels controlled by PKC may be the mechanism of Ca2+- the PMCA. This may have important implications for the preven- dependent inactivation in newly formed synapses. tionofnecroticcelldeathassociatedwithpancreatitis.Futurework WyattR.M.,Balice-GordonR.J.//J.Neurocytol.(2003)32(5-8):777-94. will involve elucidating the molecular mechanisms of this insulin- Balezina O.P. et al.// Bull. Exp. Biol. Med. (2007) 143(2):171-4. mediatedprotectionoftheoxidant-inducedinhibitionofthePMCA. The work is supported by RFBR grant 07-04-00920-a. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

PC20

Insulin protects against oxidant-induced impairment of Ca2+ homeostasis and plasma membrane Ca2+-ATPase (PMCA) in pancreatic acinar cells P. Mankad, A. James, T. Leggett and J. Bruce University of Manchester, Manchester, UK Pancreatitis is an inflammatory disease of the exocrine pancreas, characterised by auto-digestion of the pancreas and necrotic cell death. Oxidative stress and impairment of intra- 2+ cellular calcium ([Ca ]i) homeostasis has been implicated in

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC21 requirements. Inhibition of skeletal muscle differentiation by tumour necrosis factor-a is reversed by the omega-3 polyunsaturated fatty acid eicosapentaenoic acid: a mechanism associated PC22 with inhibition of nuclear factor-kB and upregulation of peroxisome proliferator activated receptor g The effect of increased fructose and/or salt intake on maternal liver and plasma lipids in rats P. Magee 1, S. Pearson2 and J.T. Allen1 C. Gray1, M.E. Symonds2, S.M. Gardiner3 and D.S. Gardner1 1Biomedical Sciences Research Institute, University of Salford, 1 Salford, UK and 2Centre for Rehabilitation & Human Performance School of Veterinary Medicine & Science, Nottingham University, 2 Research, Institute for Health & Social Care Research, University Nottingham, UK, School of Clinical Sciences, Nottingham 3 of Salford, Salford, UK University, Nottingham, UK and School of Biomedical Sciences, Nottingham University, Nottingham, UK Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid with anti-inflammatory and anti-cachetic (1, 2) prop- The prevalence of non-alcoholic fatty liver disease (NAFLD) in erties which we reported to be protective against the damag- the developed world has markedly increased. NAFLD is a multi- ing effects of TNF-α during skeletal muscle differentiation (3). factorial disease but a specific causal link has been made to Inflammatory cytokines such as TNF-α may contribute to mus- increased fructose consumption(1). A major concern for women cle wasting through inhibition of myogenic differentiation via living in a contemporary western society is excess nutrient avail- a nuclear factor-κB (NF-κB)-dependent pathway (4). Thus, we ability, especially salt and simple sugars (such as fructose). The hypothesised that EPA may exert its actions downstream of present study has therefore examined the effect of increased TNF-α through NF-κB-mediated effects on gene transcription. maternal salt and fructose intake on maternal metabolism and C2C12 (or C2C12 stably transfected with an NF-κB reporter con- growth. 32 virgin Sprague Dawley rats (~180g) were randomly struct)myoblastsweredifferentiatedbycultureingrowthmedium divided into 4 dietary groups; 1) control diet (CD, n=8) fed puri- containing2%horseserum.EPA(50μM)wasaddedatthestartof fied chow and tap water, 2) salt diet (SD, n=8) fed purified chow differentiation and myotube formation allowed to progress for containing 4% NaCl and tap water, 3) fructose diet (FD, n=8) up to 48 hours in the presence or absence of TNF-α(20ng/ml). At fed purified chow and 10% fructose in tap water and 4) fruc- various time-points, whole cell lysates were prepared for meas- tose & salt diet (FSD, n=8), fed 4% NaCl purified chow with 10% urement of NF-κBactivationbyluminescence.Inparallel,total fructose in tap water. Animals were fed ad libitum for 28 days RNA was extracted and cDNA synthesised for quantitative real- prior to conception, mated and maintained on experimental timePCRtranscriptionalanalysisofperoxisomeproliferatoracti- diets until day 20 of gestation, whereupon they were eutha- vated receptor (PPARγ), a downstream target for TNF-α. nized. Blood samples were taken 14 days prior to conception In response to TNF-α treatment, NF-κB activation was signifi- and at day 20 of gestation for analysis of protein, fat and car- cantly (p<0.05) enhanced. However, EPA treatment significantly bohydrate metabolism using an auto analyser (RX-IMOLA, Ran- (p<0.05) attenuated this TNF-α-mediated activation of NF-κB dox). Prior to feeding the experimental diets, there was no signi- ± at all time-points up to 24 hours after treatment. Furthermore, ficant difference between the body weights of dams (220 10g). whereas TNF-α treatment alone downregulated PPARγ expres- During pregnancy all animals gained a similar amount of weight ± sion, normalised against β-actin, by 2-fold (p<0.05), EPA co- (115 6g). Prior to pregnancy, fructose intake increased triglyc- ± treatment reversed this inhibition, increasing PPARγ expression eride (P<0.001) in FD (1.064 0.08mmol/l) and FSD ± ± by 2-fold (p<0.05) at 12 hours. (0.911 0.11mmol/l) when compared to CD (0.72 0.08mmol/l ± In summary, activation of NF-κB by TNF-α inhibits skeletal mus- ). NEFA was also increased (P<0.001) in FD (0.67 0.04 mmol/l) ± cle differentiation but is blocked by administration of EPA and when compared to CD (0.55 0.04mmol/l). Maternal liver wet this is associated with upregulation of PPARγ expression. These weight was specifically increased 20-25% (P<0.001) by the con- ± ± data support a protective role for EPA, particularly where patho- sumption of fructose, FD (15.22 0.32g), FSD (14.64 0.32g) ± logically high levels of TNF-α may be present and support fur- vs. CD (12.17 0.37g). In addition, a significant redistribution ther work to elucidate its mechanisms of action. of adipose tissue in the dams consuming excess fructose was observed; relative to CD, the gonadal depot in FD was reduced 1. Smith, H et al., 1999 Cancer Res 59(21): 5507-13. (FD, 4.91±0.51g vs. CD, 7.15±0.59) but perirenal depot 2. Smith, H. and M. J. Tisdale, 2003. Apoptosis 8(2): 161-9. increased (FD, 2.96±0.11g vs. CD, 1.98±0.13). All data was 3. Magee P et al, 2008 Lipids Health Dis. 7: 24. analysed as a 2x2 factorial design with fruc- 4. Langen R et al, 2001 The FASEB Journal. 15:1169-1180 tose+salt+fructose*salt included (general analysis of variance). Data is shown as estimated marginal means ± SEM for plasma Thanks to Ramon Langen for the C2C12 stably transfected with samples and organ weights (CD: n=8, SD: n=8, FD: n=8, FSD: κ an NF- B reporter construct n=8). The data indicate that increased maternal intake of fructose in water and of salt in diet has marked effects on the dam’s metabolism. The excess energy intake as a result of increased simple sugar consumption leads to significant alterations in

92P Poster Communications lipid metabolism, hepatomegaly and a shift in adipose tissue if the decrease in PPARβ expression shown with these forced distribution. Intra-hepatic triglyceride concentrations in the exercises is beneficial or harmful as such a down-regulation is enlarged livers and the possible consequences of these changes not observed after voluntary exercise. Whether these responses in maternal tissues on later growth and metabolism of adult are necessary to induce subsequent adaptations observed with offspring are currently being investigated. chronic exercise remains to be elucidated. In heart, the preser- Metabolic disturbances in non-alcoholic vation of PPARβ and ERRα expression may be a protection. Our fatty liver disease. Christopher D. Byrne, Rasaq Olufadi, Kimberley d. results showed that exercise loads (intensity and duration) Bruce,Felino R. Cagampang and Mohamed H. Ahmed Clinical Science should be considered while recommending exercises for pre- (2009) 116, 539–564 (Printed in Great Britain) vention or treatment of metabolic diseases. Where applicable, the authors confirm that the experiments (1) Handschin C, Spiegelman BM. The role of exercise and described here conform with The Physiological Society ethical PGC1alpha in inflammation and chronic disease. Nature. 2008; requirements. 454:463-9. (2) Luquet S, Lopez-Soriano J, Holst D, Fredenrich A, Melki J, Ras- soulzadegan M, Grimaldi PA. Peroxisome proliferator-activated recep- PC23 tor delta controls muscle development and oxidative capability. FASEB J. 2003; 17:2299-301. Exercise-induced stress differentially alters expression of nuclear receptor genes in mouse skeletal muscle and heart (3) Billat VL, Mouisel E, Roblot N, Melki J. Inter- and intrastrain variation in mouse critical running speed. J Appl Physiol. 2005; 98:1258-63. A. Rousseau1,J.Murdaca1,V.L.Billat2,P.A.Grimaldi1 and L. Mille-Hamard2 Where applicable, the authors confirm that the experiments 1INSERM U907, Université de Nice Sophia-Antipolis, Nice, France described here conform with The Physiological Society ethical and 2INSERM U902, Université d’Evry Val d’Essonne, Evry, France requirements. The transcriptional peroxisome proliferator-activated recep- α tor-gamma coactivator-1 alpha (PGC1 ) is described as the PC24 master regulator of skeletal and cardiac muscle metabolism, mediating the beneficial effects of exercise (1). Its expression increases with oxidative stressors. PGC1α has been shown to Exercise for a precocious detection of impairment in mdx regulate muscle peroxisome proliferator-activated receptor mice model beta (PPARβ) and estrogen related-receptor alpha (ERRα) L. Mille-Hamard, E. Henry and V.L. Billat expressions. We have previously described an increase in muscle PPARβ expression with exercise training (2). It is still unclear Universite Evry-Val d’Essonne, UBIAE INSERM U902, Evry, France if PGC1α has a redundant or a counteractive role with PPARβ and ERRα in response to metabolic challenges. We investigated The mdx mouse with essential dystrophin deficiency is an these genes expression in both skeletal and cardiac muscles established animal model of Duchenne’s muscular dystrophy after exhaustive aerobic exercises. Heat shock protein 70/72 (DMD) in human. However, hindlimb muscle of mdx mice does (HSP70/72) expression is used as a biomarker of the cellular not exhibit severe and progressive muscle weakness before response to stress induced by exercise loads. Two groups of FVB 15 month, which is late compared to DMD patients, excepted mice performed an exhaustive run on a treadmill at critical (CS; for the diaphragm that is severely affected earlier by the dis- n=10; speed: 23.7 ± 3.4 m.min-1; duration: 55 ± 28 min) or peak ease (Dupont-Versteegden, 1996). More over respiratory func- speed (PS; n=10; speed: 32.3 ± 4.5 m.min-1; duration: 37 ± 30 tion seems to be decreased at muscle level, mdx muscle mito- min), whereas one group of mice remained at rest (Control; chondria had only 60% of maximal respiration activities of n=10). Mouse performances were individually determined by control mice skeletal muscle mitochondria (Kuznetsov et al., pre-test evaluations (3). Mice were sacrificed 2 hours after the 1998). However how the diaphragm and these less efficient end of exercise. One-way ANOVA test was used for statistical mitochondrion affect the whole body performance (running analysis. Exercise induced a significant 12 fold increase in mus- velocity and maximal oxygen consumption) in young and cle HSP70/72 expression after exercising at CS, whereas the older mice was not established. As mice are animals with increase after exercising at PS was not as strong. We reported mostly anaerobic displacements and mdx mice are not here that in contrast to PGC1α, which was up-regulated, PPARβ severely impaired before 15 months, we hypothesized that and ERRα expressions were down-regulated in skeletal muscle running performance should not be greatly affected conver- in response to exercise. Our findings provided evidence to sup- sly to maximal oxygen consumption in mice between 5 and port the notion that PGC1α on one hand, and PPARβ and ERRα 9 months. on the other hand, have distinct roles in the regulation of mus- Six mdx mice of 5 months and 6 mdx mice of 9 months of age cle adaptation to exercise, which may depend on the magni- were tested on a treadmill inserted in a metabolic chamber tude of the exercise-induced stress. In cardiac muscle, the (Columbus Instrument). Exercise protocols were performed to increase in HSP70/72 expression was not significant and PPARβ determine maximal (Vpeak) and critical velocity (CS) as well and ERRα expressions remained unchanged. However, PGC1α as VO2max (Billat et al. 2005, Ferreira et al., 2007).and the cor- was increased and the magnitude was significantly higher after responding velocity (vVO2max) Results are compared with 7 CS than after PS suggesting that cardiac response to exercise control mice aged 5 and 9 months. is subtle and different from skeletal muscle. It is questionable

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Results showed that at 5 months VO2max of mdx mice was not different from control (49.38 ± 5.48 vs 48.14 ± 3.24 ml.kg- PC26 0.75.min-1, p = 0.639). However mdx mice performed higher Vpeak (26.20 ± 2.26 vs 19.86 ± 2.27, p = 0.001)and vVO2max Modulation of the staircase response in mouse myocardium was higher (25.5 ± 2.26 vs 15.57 ± 3.21, p < 0.001). Surpris- by isoprenaline ingly VO2max did not deacreased between 5 and 9 months old mdx mice. Even more the decreased of other parameters Y. Lin, C.H. Fry and R.I. Jabr of performance between 5 and 9 months (vVO2max, CS, Postgraduate Medical School, University of Surrey, Guildford, Vpeak) were more severe in mdx than in control mice (Fig- Surrey, UK ure 1). These results indicate that all parameters of performance in A negative staircase response is associated with a high intra- mdx mice were affected differently and physiological speeds cellular Na concentration in human and animal myocardium decreased before the hindlimb muscle impairment. The metab- (Shattock & Bers, 1989). The mechanisms underlying this phe- olism of running mdx mice differed marquedly from control nomenon remain unclear but has been postulated to involve and this can explain beneficial effects of exercice previously the relative phosphorylation of intracellular sites. β-agonists demonstrated (Kaczor et al., 2007). Exercise allowed to under- act via protein kinase-A (PKA), and such sites in turn can be a line defficiency precociously (9 months) compared with other target for the Ca-dependent phosphatase, calcineurin. The in vivo parameters used in the litterature (around 15 months). potential role of PKA and calcineurin in modifying the staircase Further analyses are in progress to determine the respective response was explored by examining the effects of the cal- part of the diaphragm and skeletal muscle energetics impair- cineurin inhibitor cyclosporin-A, alone or in combination with ment responsible for this severe decrease of performance in isoprenaline. mdx mice. Thus exercise can be a usefull tool for pathological Isolated mouse left ventricles strips (<1 mm diam) were super- models, included in the mdx model. fused with Tyrode’s solution (24 mM NaHCO3/5% CO2, pH 7.40, 37∞C), attached to an isometric force transducer and field- stimulated between 0.1-6.0 Hz (values normalised to 1 Hz values=100%). Interventions, isoprenaline (10 μM + 10 μM ascorbic acid) and the calcineurin inhibitor cyclosporin A (10 μM) were added to the superfusate. Data are expressed as mean ± S.E.; *p〈0.05; **p〈0.01; ***p〈0.001 by paired Students’s t- tests. Multiple group comparisons used ANOVA. An increase of stimulation frequency decreased significantly isometric peak tension across the range: e.g. at 0.4, 1.2, 2.0 and 6.0 Hz (n=7) normalized tensions were 136.0±12.2%*; ± ± ± Figure 1 : Change in performance parameters (percentage) of control (n = 93.6 1.6%**; 74.9 6.7%***, 64.4 15.6%*, respectively. 7) and mdx (n = 6) mice at 9 months compared with 5 months. VO2max: Superfusion with the β-adrenergic agonist, isoprenaline (Iso) maximal oxygen consumption. Vpeak: maximal velocity reached. vVO2max: generated a positive inotropic effect at all frequencies. How- velocity associated with VO2max. CS: critical speed. ever, the negative staircase associated with increasing fre- Exercise and clenbuterol as strategies to decrease the progression of quency was abolished: e.g. values at 0.4, 1.2, 2.0 and 6.0 Hz muscular dystrophy in mdx mice. Dupont-Versteegden EE. J Appl Phys- (n=7) were not significantly different from each other: iol. 1996 Mar;80(3):734-41. 156.9±21.4%; 134.5±25.0%; 118.3±19.4%, 150.2±32.4%, Impaired mitochondrial oxidative phosphorylation in skeletal muscle respectively. A quantitative index of the force-frequency rela- of the dystrophin-deficient mdx mouse. Kuznetsov AV, Winkler K, tionship calculated the tension ratio at 1.2 and 0.4 Hz (T1.2/T0.4). Wiedemann FR, von Bossanyi P, Dietzmann K, Kunz WS. Mol Cell Isoprenaline increased T /T from 0.64±0.04 to 0.86±0.22*, Biochem. 1998 Jun;183(1-2):87-96. 1.2 0.4 n=6. Pretreatment with cyclosporin A (CysA) alone for 15 min Low intensity training decreases markers of oxidative stress in skeletal ± ± did not affect T1.2/T0.4 (0.61 0.07; 0.70 0.06; control vs CysA). muscle of mdx mice. Kaczor JJ, Hall JE, Payne E, Tarnopolsky MA. Free Moreover, the combination of CysA and Iso (0.81±0.05; n=5) Radic Biol Med. 2007 Jul 1;43(1):145-54 was not different from the action of Iso alone. Inter-andintrastrainvariationinmousecriticalrunningspeed.BillatVL, Our data demonstrate a negative force-frequency relationship Mouisel E, Roblot N, Melki J. J Appl Physiol. 2005 Apr;98(4):1258-63. in mouse myocardium over a wide range of frequencies, which Maximal lactate steady state in running mice: effect of exercise train- was flattened with isoprenaline. This action of isoprenaline was ing. Ferreira JC, Rolim NP, Bartholomeu JB, Gobatto CA, Kokubun E, unaffected by CysA. We hypothesise that isoprenaline and cal- Brum PC. Clin Exp Pharmacol Physiol. 2007 Aug;34(8):760-5. cineurin act at different intracellular phosphorylation sites. Where applicable, the authors confirm that the experiments We thank the British Heart Foundation for support described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

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Vartiainen MK et al. (2003). J Biol Chem 278, 34347-34355.

PC27 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Twinfilin-1 mRNA in isolated adult rat cardiomyocytes requirements. O. McAvinchey, D. Goldoni and A. Collins Queen’s University, Belfast, UK It has recently been discovered that the expression profile of PC28 microRNA changes in the diseased heart (1, 2). Some of the dysregulated microRNAs have been shown to affect the expres- A physiological mode of heart perfusion in vivo and in vitro sion of important cardiomyocyte genes, resulting in patho- G. Hao1, Y. Song1, M. Boyett1, X. Yang2, Y. Du2, A. Luo3, M. Tang3 physiology characteristic of the disease (3). It is likely that there and Z. Shui1,2 are similar effects of other microRNAs that are dysregulated in heart disease. It has recently become possible to study the 1Cardiovascular Research Group, University of Manchester, effects of specific microRNAs on cardiomyocyte physiology Manchester M13 9NT, UK, 2Departments of Cardiovascular Surgery by transfecting adult rat cardiomyocytes with microRNA mim- and Cardiology, Union Hospital, Wuhan, China and 3Department ics (1). Fluorophore-tagged mimics have been used as a posi- of Physiology, Tongji Medical College, Huazhong University of tive control to indicate successful transfection, although a pos- Science and Technology, Wuhan, China itive control for functional activity of transfected microRNA Langendorff mode of heart perfusion is widely used in bio- would enhance these studies. Analysis of microRNA gene tar- medical research. It is a constant retrograde perfusion with geting has shown that miR-1 destabilises the mRNA of several unnatural pressure in four chambers, which causes abnormal target genes, with the actin-binding protein twinfilin-1 (Twf- heart conditions. For example, rabbit heart rate in vivo is 1; PTK9) being the most strongly affected (4). Twf-1 mRNA 205~220/min at rest, whereas the heart rate of Langendorff down-regulation would therefore be a good indicator of miR- perfused rabbit heart is 120~150/min (Döring & Dehnert, 1 function. Twf-1 mRNA is known to be present in mouse heart 1988). Furthermore, the preload of left ventricle in Langendorff (5), but its presence in adult rat cardiomyocytes has not been perfused heart is highly increased as the aortic valve is unable reported. We have detected Twf-1 mRNA in isolated adult rat to work as normal, which may cause acute congestive heart cardiomyocytes using quantitative real-time PCR (qPCR) on an failure. To investigate arrhythmia in the isolated hearts from Applied Biosystems 3700 machine. Adult male Sprague-Daw- rabbit and rat, we have developed a physiological mode of heart ley rats were anaesthetised by intraperitoneal injection of pen- perfusion in vitro that simulated the working heart in vivo as tobarbital (100 mg/kg). Hearts were removed and perfused shown in Figure 1. In this system, the regular flow through the with Kreb’s buffer containing collagenase and hyaluronidase heart was maintained, and each chamber was automatically to isolate cardiomyocytes, which were seeded onto laminin- controlled and filled with changing volume and pressure as treated cover slips (8 mm diameter) and maintained in culture usual. As a result, normal heart rate in vivo could be kept in vitro medium. The number of cells on each cover slip was estimated for more than 24 hours. The pressure in each chamber was by counting cells in 6 fields of view. The TaqMan® Gene Expres- adjustable and controllable, which was useful to study those sion Cells-to-CT™ Kit (Applied Biosystems) was used for cell arrhythmia associated with stretch and/or pressure. To inves- lysis and reverse transcription. A Twf-1 TaqMan® assay tigate the role of autonomic nerve system in atrial fibrillation, (Rn01407564_g1, Applied Biosystems) was used for qPCR. Con- the system was also used to carry out the experiments in vivo trol reactions without reverse transcription (RT-) were used to with intact autonomic innervation of the heart perfused phys- estimate the maximum number of cardiomyocytes per sam- iologically with Tyrode solution as shown in Figure 2. Based on ple that could be fully lysed using the Cells-to-CT™ protocol, characteristics of the system, this mode is valuable to func- as indicated by the elimination of genomic DNA by DNAse I tional studies on arrhythmia that is a systemic disorder at organ treatment. RT- assays from ~1400 or more viable cardiomy- level and highly related to the pressure in heart chamber. ocytes (n=4) produced amplification products, indicating incomplete cell lysis. RT- assays were negative (below amplification threshold after 40 cycles) in lysates from ~1000 or fewer viable cardiomyocytes (n=4), while assays from these lysates gave amplification products after reverse transcription (RT+). Within-sample ΔCt values for Twf-1 vs glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Rn01775763_g1, Applied Biosystems) were 3.7, 3.4 and 2.4 for 3 separate samples. These results establish the principle that quantitation of Twf-1 mRNA relative to an endogenous control gene such as GAPDH could be used as a functional assay for microRNA transfection in adult rat cardiomyocytes, using miR-1 as a positive control. Thum T et al. (2007). Circulation 116, 258-267. van Rooij E et al. (2006). Proc Natl Acad Sci U S A 103, 18255-18260. Luo X et al. (2008). J Biol Chem 283, 20045-20052. Lim LP et al. (2005). Nature 433, 769-773. Figure 1. A physiological mode of heart perfusion in vitro

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Pressure transducer (PT), pulmonary artery (PA), aorta (A), superior vena Chemical hypoxia was induced by switching the perfusion buffer cava (SVC), left atrial appendage (LAA). Pulmonary veins and inferior vena to one containing 2.5mM NaCN and no glucose. Cardiomy- cava were tied. ocytes were viewed on a monitor and the effects of metabolic inhibition were noted. The times taken for the cells to cease contraction and to enter a state of rigor were recorded. Con- centrations of ATP were measured in myocardial biopsies from the same aged rats, using HPLC. Data expressed as mean ± standard error. Results: The lengths of time for cardiomyocytes to both cease contraction and enter rigor were significantly shorter for cardiomyocytes from 21 day old rats compared to all other age groups (p<0.0001, ANOVA, n=30). Times taken to cease contraction were (in minutes) 12±2, 6±2, 17±3for 14, 21 and adults respectively. This trend was mirrored in the times taken to enter rigor for all ages. Since going into rigor would depend on availability of myocardial ATP, we measured the concentration of ATP in myocardial biopsies from all age groups. Mean ATP levels in freshly excised hearts were 14.4±1.8, 12.5±1.6, 17.1±0.8 nmol/mg protein Figure 2. Physiological heart perfusion in vivo with intact autonomic in 14, 21 days and adult respectively. ATP was significantly innervation lower at 21 days compared to adults (p=0.024, unpaired t In accordance with the Animals (Scientific Procedures) Act 1986 and the test, n=5). Guide for the Care and Use of Laboratory Animals (NIH Publication No. Conclusion: This work demonstrates that the age-related 85~23, 1985), anaesthesia was induced and maintained with Hypnoval (1 mg/kg, I.V.) and pentobarbitone (2 mg/5 min, I.V.) before and during dis- response to chemical hypoxia is likely to be due to differences section of the vagus nerves, and an overdose of pentobarbitone (60 mg, in the basal levels of myocardial ATP. I.V.) was given before opening the thoracic cavity (André Ng et al. 2001). The descending aorta and inferior vena cava were connected, whereas SVC This work is supported by the BHF. was tied. Where applicable, the authors confirm that the experiments Döring HJ & Dehnert H (1988). The isolated perfused heart, Bio- messtechnick-Verlag, ISBN3-924638-04-7. described here conform with The Physiological Society ethical requirements. André Ng G et al. (2001). Exp Physiol 86.3, 319–329.

This work is supported by the British Heart Foundation and the National Natural Science Foundation of China. PC30

Where applicable, the authors confirm that the experiments Differences between functional properties of atrium and described here conform with The Physiological Society ethical pulmonary vein region requirements. Z. Shui1,2, G. Hao1, Y. Song1, M. Boyett1, J. Liu2, C. Xia2, X. Yang2, Y. Du2, A. Luo3 and M. Tang3 1Cardiovascular Research Group, University of Manchester, Manchester M13 9NT, UK, 2Departments of Cardiovascular Surgery PC29 and Cardiology, Union Hospital, Wuhan, China and 3Department of Physiology, Tongji Medical College, Huazhong University of Age-related differences in response to chemical hypoxia of Science and Technology, Wuhan, China isolated perfused cardiomyocytes The pulmonary vein sleeves (PVS) play an important role in atrial H. Lin, S. Martin, N. Shukla, J. Jeremy and S. Suleiman fibrillation (AF) (Nattel, 2002). However, the functional properties of the PVS region have not been well characterized (Ald- Bristol Heart Institute, University of Bristol, Bristol, UK hoon et al. 2009). In this study, a set of monophasic action potential (MAP) and modified bipolar electrodes and three force Aim: The heart undergoes stages of postnatal development transducers were used to simultaneously record MAP and extra- during which it responds differently to cardiac insults. Whether cellular potentials and mechanical contraction at left and right such age-related differences are due to developmental atriums and PVS region of rabbit heart perfused physiologically changes in single heart cells is not presently known. In this in vitro, when the electrical stimulation was applied every ten study we investigated the effect of chemical hypoxia on freshly sinus rhythms with decreasing delay from 200 ms to 0 ms isolated cardiomyocytes from different postnatal ages of the between the tenth depolarization time of right atrium action rat heart. potential and the stimulation as shown in Figure 1. The results Methods: Cardiomyocytes were enzymatically isolated from showed that: [i] the mechanical relative refractory period was 14, 21 day and adult rat hearts. Isolated cardiomyocytes were significantly longer (38±7 ms, 53±9 ms, n=8) at left and right perfused with HEPES buffer (1mM Ca2+) and field stimulated at ο atriums than at the PVS region; [ii] the mechanical absolute 0.2Hz in a chamber under an inverted microscope at 34 C. refractory period was remarkably longer (12±3 ms, 16±5 ms,

96P Poster Communications n=8) at left and right atriums than at the PVS region; [iii] the a longer period (4-6 days) of dietary nitrate consumption on delay between the electrical stimulation and contraction was resting blood pressure in healthy normotensive humans. significantly shorter (45±14 ms, n=8) at right atrium than at Nine healthy males (mean ± SD, age 26 ± 7 years, body mass the PVS region; and [iv] burst pacing (7 Hz) induced atrial fib- 81.6 ± 6.2 kg) volunteered to participate in this ethically- rillation in the PVS region, but not in the left and right atrial free approved study. In random order, the subjects consumed 500 walls. Therefore, it is concluded that the heterogeneity of func- mL of either beetroot juice (BTJ, containing 11.2 ± 0.6 mM of tional refractory period at PVS region may facilitate reentry for- nitrate) or blackcurrant squash (as a placebo, PLC) per day for mation as an important factor in the initiation and maintenance six consecutive days. The conditions were separated by a wash- of atrial fibrillation. out period of 7 days. Resting blood pressure was measured using an automated blood pressure machine and blood samples were drawn to determine plasma [nitrite] on days 4, 5 and 6 of each condition. Data were analysed using repeated-measures ANOVA. On days 4-6, plasma [nitrite] was significantly increased by beetroot juice consumption but was unchanged following placebo consumption (BTJ: 160 ± 80 vs. PLC: 80 ± 60 μM; P<0.05). Beet- root juice consumption led to a significant reduction in systolic blood pressure on day 4 (BTJ: 124 ± 2 vs. PLC: 132 ± 5 mmHg; P<0.01) with the difference remaining significant on days 5 and 6. Diastolic blood pressure was not significantly affected by the intervention on day 4 (BTJ: 71 ± 9 vs. PLC: 71 ± 7 mmHg) or on subsequent days. These data extend the earlier report of Webb et al. (2008) by showing that increased dietary nitrate consumption (in the form of 500 mL/day of beetroot juice) results in a sustained Figure1.Functionalrefractoryperiodofatriumandpulmonaryveinregion reduction in systolic blood pressure in healthy normotensive humans. We suggest that dietary nitrate reduces blood pres- A, B and C, recordings of contraction force of right atrium (RA), pulmonary vein region (PV) and left atrium (LA). D and G, traces of MAP action poten- sure by increasing nitric oxide availability and enhancing vasodi- tial of RA and LV. E and F, bipolar electrograms of PV and LA. H, gray lines latation in the microcirculation (Lundberg & Govoni, 2004). indicate the time of right atrium action potential (RAAP) depolarization; Increased dietary nitrate consumption might be considered a black lines with higher amplitude show electrical stimulation at RA. natural strategy to maintain or enhance cardiovascular health. Nattel S (2002). Nature 415, 219-226. Lundberg JO, Govoni M (2004). Inorganic nitrate is a possible source for Aldhoon B et al. (2009). Physiol Res. (Epub ahead of print). systemic generation of nitric oxide. Free Radic Biol Med 37, 395-400. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid This work is supported by the British Heart Foundation and the R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia National Natural Science Foundation of China. A (2008). Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Where applicable, the authors confirm that the experiments Hypertension 51, 784-790. described here conform with The Physiological Society ethical requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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Influenceofdietarynitrateonrestingbloodpressureinhumans PC32 A.M. Jones1, S.J. Bailey1, J.R. Blackwell1, N. Benjamin2 and Measurement of cortical voluntary activation of the trapezius P.G. Winyard 2 muscle using transcranial magnetic stimulation in man 1School of Sport and Health Sciences, University of Exeter, Exeter, L. Duffell, M. Kulkarni, M. Catley and P.H. Strutton UK and 2Peninsula College of Medicine and Dentistry, Exeter, UK Biosurgery and Surgical Technology, Imperial College London, It has recently been reported that resting blood pressure was London, UK significantly reduced 2-3 hours following the ingestion of a dietary nitrate load (500 mL of beetroot juice), (Webb et al. The ability to perform tasks of daily living is dependant upon 2008). The mechanism for this effect is obscure but likely related the capacity of the central nervous system to voluntarily acti- to the bioconversion of nitrate to nitrite and thence to the vate muscles over prolonged durations. This neural drive is potent vasodilator molecule, nitric oxide (Lundberg & Govoni, termed voluntary activation (VA) and can be measured using 2004). Dietary-induced reductions in blood pressure poten- twitch interpolation1. It has been reported that VA is reduced tially have important implications for public health. The pur- following both maximal and prolonged submaximal contrac- pose of the present investigation was to assess the influence of tions, reflecting central fatigue.

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The trapezius muscle is activated submaximally for prolonged periods during tasks of daily living such as bag carrying, but it PC33 remains to be established if this task causes central fatigue. It has been reported that VA of trapezius can be measured using Assessment of the physiological loads and subjective electrical stimulation (ES) of the motor nerve2; however this discomforts of respirator at constant work load exercise technique provides no information regarding the changes under prolonged condition occurring at a cortical level following a fatiguing task. Tran- W. Ho, W. Hsu, Y. Zhang, K. Tian and S. Guo scranial magnetic stimulation (TMS) of the motor cortex might provide this information, and the aim of this study was to iden- Department of Occupational Safety and Health, China Medical tify if TMS can be used to asses cortical VA of trapezius. University, Taichung city, Taiwan With ethical approval and written informed consent eleven Literatures indicated the respirators are required and in use in healthy subjects were recruited. Electromyographic recordings only20-30%oftheworkphases[4].Itispostulatedthatwork were made from the right trapezius, ES was delivered to the wearing respirator at constant load under prolonged condition right accessory nerve and TMS applied to the left motor cor- may cause additional physiological strain due to restriction of tex. Subjects sat on a chair with their feet suspended, and a heat loss from the respiratory system [1,3] and the anaerobic force transducer was positioned over their right shoulder. Max- respiration. This study aimed to evaluate the effects of wearing imum voluntary contractions (MVC) of the right trapezius were respirators at constant work load under prolonged conditions. carried out to identify target forces of 10, 30, 50, 70, 90 and Eight male and eight female physically fit university students 100% MVC. Subjects performed contractions to these target [3] voluntarily participated in the study, which was approved forces in a random order during which ES or TMS was applied. by the IRB of CMU. A full-face respirator and a control quarter- For ES, superimposed twitch (SIT) size decreased linearly as con- 2 face mask were employed. The evaluated independent vari- traction strength increased (mean r =0.75, p<0.05 (n=10)). For ables included gender, work time (10 and 50 min) and respira- TMS this relationship was polynomial across all contraction tor type. Besides, for the prolonged exercise condition, the data strengths (mean r2=0.67, p<0.05 (n=9)) or linear >70% MVC 2 during initial 10 minute (D1) and last 10 minute (D5) were also (mean r =0.62, p<0.05 (n=8)). VA during MVCs of the trapez- analyzed. The evaluated dependent variables were summa- ius was 81.0 (12.9)% when measured with ES (n=8) and 78.1 rized. Subjective rate of perceptual exertion (RPE) were meas- (13.3)% when measured with TMS (n=8). SITs evoked by TMS ured with a 10 point Borg scale questionnaire. were significantly larger than by ES at 10-90% MVC (p<0.05). All subjects carried out the basic aerobic data measurement Maximally evoked direct motor responses to ES (M-waves) were and 4 experiments at 3 different days with at least one day off significantly larger than motor evoked potentials (MEPs) at con- between the experiment days. On the first day the maximal ≤ traction strengths 90% MVC (p<0.05). There was a polynomial oxygen consumptions were measured using a revised protocol relationship between contraction strength and both M-wave 2 2 [2]. The total 4 experiment conditions were completely ran- (r =0.89) and MEP (r =0.99) amplitudes with ES and TMS, domized. The physiological variables were analyzed by repeated respectively. measure ANOVAs . The subjective RPE was analyzed using The non-linearity of the SITs to TMS at contraction strengths of Wilcoxon test. <70% MVC may reflect lowered levels of corticospinal excitabil- The ANOVAs and some descriptive statistics are summarized. ity, as supported by smaller MEP amplitudes, at these strengths. Results indicated that working for constant load under pro- A similar result has been found using TMS in other muscle 3,4 longed condition significantly increased minute ventilation groups . TMS can be used to assess cortical VA of the trapez- (p<0.001), oxygen consumption (p<0.001), working pulse ius and this technique could be useful to assess the changes 5 (p<0.05),metabolic rate (p<0.001) and total RPE (p<0.001). that occur at a cortical level as a result of central fatigue . While comparing the D1 with D5, the similar trends were found Merton PA. (1954). J Physiol 123, 553–64. more significantly especially for female subjects. The oxygen Taylor JL et al. (2008). Electromyogr Kinesiol (in press). consumption were found significantly increased from Todd G et al. (2004). J Appl Physiol 97, 236–42. 1.09±0.04 (D1) to 1.86±0.04 liter/min (D5). It is suggested that Lagan J et al. (2008). Clin Neurophysiol 119, 2839-45. the work-rest ratio should be reconsidered under constant load prolonged work and micro break may be helpful which need Todd G et al. (2003). J Physiol 551, 661–71. further study. GSK & Imperial College London Chongvisal, P. (1980). Effects of Wearing Respirators in Hot Environ- ments. Ph.D. Dissertation. U. of Cincinnati. Where applicable, the authors confirm that the experiments Heus R, den Hartog EA, Kistemaker LJA, van Dijk WJ, Swenker G (2004). described here conform with The Physiological Society ethical Influence of inspiratory resistance on performance during graded exer- requirements. cise test on a cycle ergometer. Appl Ergonomics 35, 583-590. Lovhevaara, V.A. (1984). Physiological effects associated with the use of respiratory protective devices. Scand J Work Environ Health 10, 275-281. Vihma, T. (1981). Health hazards and stress factors in small industry- Prevalence study in the province of Uusimaa with special reference to the type of industry and the occupational title as classification for the description of occupational health problem. Scand J Work Environ Health 7, suppl 3, 149.

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This research was supported by NSC of Taiwan, 96-2628-E-039- 001-MY3. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

± FIO2, fraction of inspired oxygen. Values are mean S.D.; two-way repeated measures ANOVA. n=9 for each group. PC34 New KJ et al. (2008). J. Physiol 586P. New KJ et al.(2009) IUPS Abstract. Regulation of Post-Exercise Haemodynamics Following Hyperoxia in Man: Role of Adrenergic Vasoconstriction Where applicable, the authors confirm that the experiments K.J. New1, B. Davies5, J. Hooper3, C. Templeton4, G. Ellis4 and described here conform with The Physiological Society ethical D.M. Bailey2,5 requirements. 1Exercise Physiology, Swansea Metropolitan University, Swansea, UK, 2Neurovascular Laboratory, University of Glamorgan, Pontypridd, UK, 3Clinical Biochemistry, Royal Brompton Hospital, PC35 London, UK, 4Cardiology, Royal Glamorgan Hospital, Llantrisant, UK and 5Health and Exercise Science, University of Glamorgan, Muscle deoxygenation changes with repeated Wingate Pontypridd, UK power tests Hyperoxic exercise attenuates post-exercise vasodilatation inde- J. Neary1, S.J. Neary1 and M.A. Holmes2 pendent of reduced NO (New et al, 2008) but coincident with 1Kinesiology & Health Studies, University of Regina, Regina, SK, a diminished bioavailability of atrial natriuretic peptide (New Canada and 2School of Health and Sport Sciences, University of the et al, 2009). The present study investigated the influence of Sunshine Coast, Maroochydore, QLD, Australia adrenaline (AD) and nor-adrenaline (NA) on post-exercise haemodynamics. 9 males, mean arterial pressure (MAP) = 106 This study examined the changes in muscle deoxygenation ± 5 mmHg (50 ± 10 yr), not on medication, were studied fol- (HHb) following repeated 30s Wingate power tests to deter- lowing 30-minutes of cycle exercise at 70% maximal oxygen mine the muscle power-deoxygenation relationship. It was consumption in hyperoxia (50% O2) and normoxia (21% O2). hypothesized that tissue HHb would correlate with changes in Subjects were followed post-exercise for 2-hours. Left ventric- anaerobic power, reflecting the energy contribution following ular haemodynamics were assessed via echocardiography and repeated Wingate trials. Muscle HHb was used to reflect O2 uti- systemic vascular resistance (SVR)/vascular conductance deter- lization, and total blood volume (tHb) was calculated from the mined by the quotient of MAP/Q and Q/MAP, respectively. oxy-Hb and HHb signal (HbO2 + HHb). Eleven competitive male Peripheral venous blood was sampled from an antecubital vein cyclists (mean ± SD age, height, mass =21.8±7.6 yr; 177.5±5.7 pre-, immediately post-, 1-hour post- (P1) and 2-hours post- cm; 73.7±10.4 kg) volunteered to perform three 30s anaero- (P2) exercise and metabolite concentrations corrected for bic Wingate power tests (Wn1, Wn2, Wn3) with 60s rest plasma volume shifts. AD and NA were determined via reverse- between trials on a Monark cycle ergometer. The resistance phase high-performance liquid chromatograpy using electro- was set at 90 g/kg. Tissue oxygenation was monitored contin- chemical detection. Data were analysed with a two-way uously from the right vastus lateralis muscle using dual wave- repeated measures ANOVA and post-hoc Bonferroni-corrected length near infrared spectroscopy (NIRS). Mean (n=11) maxi- paired samples T-tests. mal power (Watts) was significantly reduced with each Hyperoxic exercise blunted post-exercise haemodynamics by consecutive Wingate, from Wn1 (598±135W), to Wn2 attenuating the reductions (from normoxic baseline) in SVR (475±74W), to Wn3 (446±97W), with the fatigue index = 40- (21 ± 20.7 vs. 38 ± 19.3 %, 11 ± 8.2 vs. 19 ± 5.5 % and 11 ± 8.9 44%. Muscle HHb closely tracked these power changes but did vs. 15 ± 9.6%, P<0.05 vs. normoxic exercise at post, P1 and P2 not reach peak muscle HHb until 20s after the start of each respectively) and MAP (3 ± 4 [elevation] vs. 0.5 ± 5 mmHg, 3 ± Wn test. As well, muscle HHb was reduced and remained below 3 vs. 6 ± 4 mmHg, 3 ± 3 vs. 4 ± 3 mmHg, P<0.05 vs. normoxic baseline during the recovery period between Wingate tests. exercise at post, P1 and P2 respectively) (Paired samples T- Average 30s power was significantly (ANOVA; p<0.05) reduced tests). AD and NA concentrations were invariant between con- from Wn1 (474±79W) to Wn2 (377±62W) to Wn3 (338±61W), ditions P>0.05, Paired samples T-tests; Table 1). as was HHb for Wn1 (0.273±0.26OD), Wn2 (0.176±0.26OD), These results indicate that an augmented adrenergic response and Wn3 (0.108±0.25OD). A significant correlation was found is not a principle governor of the attenuated vasodilatation. between anaerobic power vs. HHb for Wn1 (r= -0.79) and Wn2 Whether changes in alpha- or beta-receptor sensitivity play a (r= -0.78), but not for Wn3 (r= -0.29). Significant correlations role requires further investigation, although, it appears that were also found for tHb change vs. anaerobic power the deleterious influence of hyperoxia on post-exercise haemo- (r=0.72–0.87). These results suggest that 1) NIRS can track dynamics has non-adrenergic origins. changes in muscle HHb and tHb during anaerobic power test- Table 1. Systemic Venous Concentration of AD and NA ing and that decreases in muscle power correlated with muscle HHb and tHb; and 2) the muscle began to deoxygenate at the onset of maximal exercise during the Wingate tests sug-

99P Poster Communications gesting that aerobic metabolism contributed significantly to We thank the Management of UBTH and the Sickle Cell Cen- energy production. ter, Benin City, Nigeria, for allowing us access to the patients, and to all our subjects for their cooperation. Special thank you to the subjects that volunteered for this study. Financial Support: NSERC(JPN); Research Funding, University Where applicable, the authors confirm that the experiments of the Sunshine Coast (MAH). described here conform with The Physiological Society ethical requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC37

Muscarinic stimulation enhances spontaneous electrical PC36 activity in the rabbit urethra through activation of M3 receptors Thirst perception in dehydrated sickle cell disease patients A. De Faoite, K.D. Thornbury, N.G. McHale, G.P. Sergeant and in steady state M.A. Hollywood 1 1 1,2 1 L.F. Obika , J.O. Ozoene , M.E. Enosolease , O.I. Ajayi and Smooth Muscle Research Centre, Dundalk Institute of Technology, 1 F.O. Agoreyo Dundalk, Ireland 1Department of Physiology, School of Basic Medical Sciences, Although urethral tone is spontaneous, it can be modulated by University of Benin,, Benin City,, Edo, Nigeria and 2Department of inhibitory nitrergic and excitatory noradrenergic and cholin- Haematology,, College of Medical Sciences, University of Benin,, ergic nerves (Thornbury et al., 1992). The purpose of this study Benin City,, Edo, Nigeria was to identify which acetylcholine receptor subtype was Liberal fluid intake is one of the key management strategies in responsible for increasing tone in the proximal rabbit urethra sickle cell anaemia (SCA) patients in steady state, but less work and examine how their stimulation affected spontaneous elec- has been done on the patient’s desire to drink water. Using trical activity. the Visual Analogue Scale (Thompson et al, 1986), we studied Rabbits were humanely killed with pentobarbitone (I.V.) and thirst perception (TP) in twenty euhydrated SCA patients and the proximal 3 cm of the urethra was removed. The urothelium twenty eight control (HbA) subjects, as well as during dehy- was removed before the preparation was pinned out on a sili- dration in thirteen SCA patients and nine HbA subjects. Con- con rubber base and superfused with Krebs solution at 35-37oC. sent was obtained from all subjects and the protocol approved For isometric tension recording experiments, circular strips of by the Ethics Committee of the University of Benin Teaching urethra were adjusted to a resting tension of 5 mN and super- μ Hospital (UBTH), and Sickle Cell Center, Benin City, Nigeria. Dur- fused with Krebs solution at 35-37oC containing 100 M NO- μ ing euhydration, TP was significantly higher in male SCA ARG and 1_ M guanethidine. patients compared to the HbA subjects (8.67 + 0.23 cm, n = When contractions were elicited by transmural nerve stimula- 11 vs. 3.12 + 0.74 cm, n = 16, p<0.05). In females, TP in SCA tion (0.5 Hz – 4 Hz, 50 V, 0.3 ms pulse width, 60 s duration) via patient was lower than in HbA subjects, but this was not sta- electrodes placed at either end of the organ bath, frequency tistically significant (3.20 + 0.55 cm, n = 9, vs. 4.33 + 1.0 cm, n dependent increases in tone were observed and these were μ =12). Thus, male SCA patients, but not female patients are in a blocked by tetrodotoxin (1 M, n=4). The peak amplitude of state of relative dehydration. After 13 hours of dehydration, TP the neurogenic contractions evoked by stimulating at 4 Hz was ± ± was reduced in both male (7.88 + 1.10 cm, n = 5 vs. 6.46 + 1.8 significantly reduced from 19.1 4.6 mN to 7.2 2.3 mN (p<0.05, ± μ cm, n = 5, ns) and significantly in female (9.3 + 0.33 cm, n = 4 mean SEM, paired t-test) by atropine (1 M), suggesting that vs. 3.64 + 0.80 cm, n = 8, p<0.05). Thus, while dehydration muscarinic receptors were involved in this response. Methoc- μ increased TP in HbA subjects, in marked contrast, it reduced TP tramine (1 M, M2 blocker) had little effect on these responses in SCA patients. Fluid intakes after dehydration in SCA patients (n=6). However, the M1/M3 muscarinic receptor antagonist (male: 760 + 150 ml and female: 513 + 40 ml), were not signi- 4-DAMP (100 nM) significantly reduced the neurogenic con- ± ± ficantly different from the control HbA subjects in both male tractions evoked by 2 Hz and 4 Hz from 1.2 0.3 and 2.3 0.4 ± ± (720 + 195 ml) and female (623 + 24 ml). mN to 0.4 0.2 and 1.0 0.4 mN (p<0.05, n=6) respectively, sug- It can be concluded that female SCA patients do not have nor- gesting that acetylcholine mediates its effects on tone via acti- mal response to dehydration with regards to TP after a period vation of M1 or M3 receptors. of dehydration. Since dehydration stimulates the release of When the proximal urethra was impaled with sharp micro- vasoactive hormones like vasopressin, this may explain why electrodes, regular spontaneous slow waves which had spikes female patients are less prone to crisis (Stringer et al., 2005) superimposed upon a plateau were recorded as demonstrated than their male counterparts. previously (Bradley et al., 2004). Carbachol increased the frequency of the slow waves from 3.1±0.3 min-1 (n=9) to 3.3±0.8 Perucca, J., Bouby, N., Valeix, P. & Bankir, L. (2007). Sex differences in ± ± ± Renal and Cardiovascular Function: Physiology and Pathophysiology. (n=3), 5.9 0.5 (n=6), 6.8 0.8 (n=4), 12.3 2.2 (n=4) min-1 in Am. J. Physiol. Regul. Integr. Comp. Physiol., 292, R700-R705, 2007. response to 10 nM, 100 nM, 300 nM and 1μM carbachol respectively. To examine if this effect was via stimulation of M1/M3 Thompson, CJ. Bland, J., Burd, J & Bayliss, PH. (1986). The osmotic thresholds for thirst and vasopressin release are similar in healthy man. receptors, we examined the effects of 300 nM carbachol on Clin. Sci., 71: 651-656. electrical activity before and during incubation of the tissue

100P Poster Communications with 4-DAMP (100 nM). In five experiments, carbachol (300 influx from the extracellular medium (ionotropic function of nM) increased slow wave frequency from 4.1±0.6 min—1 to Ca2+ channel) and Ca2+ release from the SR through a 7.1±1.6 min-1 (p<0.05). In the presence of 4-DAMP, frequency metabotropic pathway. was 3.8 ± 0.5 min-1 compared to 4.0±0.6 min-1 during 4-DAMP Reference: Del Valle-Rodríguez A., López-Barneo J and Ureña J. Ca2+ and carbachol. channel-sarcoplasmic reticulum coupling: a mechanism of arterial These data suggest that muscarinic agonists increase tone by myocytecontractionwithoutCa2+influx.EMBOJ.22:4337-4345(2003) modulating the frequency of slow waves in the proximal ure- This work was partially supported by FISS grant PI060137 thra via activation of M1/M3 receptors. Thornbury K. D., Hollywood M. A. and McHale N. G. (1992). J. Physiol. Where applicable, the authors confirm that the experiments 451: 133-144 described here conform with The Physiological Society ethical Bradley, E.J., Anderson, U. A., Woolsey, S. M., Thornbury, K. D., McHale, requirements. N. G. & Hollywood, M. A. (2004). Am.J. Physiol (Cell) 286, C1078-C1088.

The authors acknowledge grant support from the NIH (NIDDK Grant No: R01 DK68565). PC40

Where applicable, the authors confirm that the experiments Identification of a novel pH-gating mutation in the Kir1.1 described here conform with The Physiological Society ethical (ROMK)channelusingayeastgeneticcomplementationassay requirements. J.J. Paynter1, L. Shang1,2, M. Bollepalli3, T. Baukrowitz3 and S.J. Tucker1,2 1Department of Physiology, Anatomy &Genetics, Oxford University, PC39 Oxford, UK, 2Biological Physics Group, Clarendon Laboratory, Department of Physics, Oxford University, Oxford, UK and 3Institute Ca2+ channel-induced contraction in basilar artery is of Physiology II, Friedrich Schiller University, Jena, Germany mediated by metabotropic Ca2+ release from sarcoplasmic reticulum Gating of inwardly rectifying potassium (Kir) channels by intracellular pH is important for many aspects of channel physiol- M. Fernandez-Tenorio1,2, C. Porras2, A. Castellano1,2, J. López- ogy and pathophysiology. In mammalian Kir1.1 (ROMK) an Barneo1,2 and J. Ureña1,2 intra-subunit H-bond between a lysine in TM1 (K80) and a back- bone carbonyl group in TM2 (A177) at the helix bundle cross- 1Fisiologia Médica y Biofísica, Universidad de Sevilla, Sevilla, Spain ing is thought to control the kinetics of the gating motion of and 2Instituto de Biomedicina de Sevilla, Sevilla, Spain the TM helices in response to pH and PIP2. However, the exact We have described in rat basilar artery that L type Ca2+ chan- pH gating mechanism is not fully understood and further stud- nel activation can activate Ca2+ release from sarcoplasmic retic- ies are needed to determine which other residues are impor- ulum (SR) in the absence of extracellular Ca2+ through a tant for Kir1.1 pH sensitivity. metabotropic pathway (mechanism denoted as Calcium-Chan- In this study, we exploited a yeast genetic complementation nel Induced Calcium Release, CCICR) (Del Valle-Rodriguez et assay to screen for gain of function (GoF) mutations in Kir1.1 al., 2003). The calcium-release mechanism depends on the con- which might alter pH gating, given that the intracellular pH in formational change of L-type Ca2+ channels and the down- yeast is highly acidic and predicted to inhibit Kir1.1 function. stream activation of the G protein/phospholipase C (PLC) cas- However, the screening of a random-mutated Kir1.1 library in cade, leading to synthesis of InsP3 and Ca2+ release from the SGY1528 K+ uptake deficient yeast yielded only two mutants SR. Because previous results were obtained in isolated myocytes (K80M and K80I) which were already known to decrease pH bathed in free Ca2+ medium, the aim of this work was to study sensitivity. We therefore used a chimeric channel (30C) which the functional role of CCICR in physiological conditions (i.e. in contained the transmembrane domain of Kir1.1 and the cyto- arteries bathed in medium containing Ca2+). Experiments were plasmic domain of Kir4.1. This chimera showed no comple- done in rat cerebral arteries obtained from anaesthetized mentation in yeast even with the mutation K80M. However, (sodium pentobarbital, 50 mg/Kg, ip.) animals of weight 250- upon screening of a randomly mutated library a large number 300 g (30 rats and 100 arterial rings). Isometric force and of novel gain of function (GoF) mutations were found. cytosolic Ca2+ concentration ([Ca2+]i) were measured in rat Positive GoF mutant clones were verified and sequenced. These basilar arterial rings and in intact arteries. Ca2+ channel acti- mutations were found to occur in many regions of the Kir1.1 vation produced an initial rapid rise in cytosolic Ca2+ and a sec- transmembrane domain, including known gating sensitive ond plateau phase that was maintained until the end of the regions and those that may interact with PIP2, with only one stimulus. Concomitant with this change in [Ca2+]i, a reduction mutation identified in the Kir4.1 section of the 30C chimera. of the arterial diameter was detected. Both signals were tran- Because these mutations might permit channel function in sient when Ca2+ was eliminated from the extracellular medium. yeast by reducing pH sensitivity these GoF mutations were Cyclopiazonic acid, ryanodine and U73122, inhibitors of SR made individually in the Kir1.1 channel and their pH-sensitiv- Ca2+ pump, ryanodine receptors and PLC respectively, reduced ity measured electrophysiologically. the maintained phase of contraction whereas the transient Many mutants had only a modest effect on Kir1.1 pH-sensitiv- component was not significantly affected. Our results suggest ity. However, one novel mutant in TM2 had a dramatic effect that in physiological conditions Ca2+ channel-induced con- reducing pH sensitivity to a similar extent to the K80M muta- traction of the basilar artery can be mediated by both Ca2+ tion. In addition, the whole cell currents of each GoF mutation

101P Poster Communications were recorded in neutral conditions and correlated with the with PCL-Q, confirm an additive effect PCL-Q constituents, i.e. screening results. These results showed the validity of this yeast the effects of PCL and Q simply summarize. genetic complementation assay to screen for pH-gating muta- The data obtained in this study clearly indicate that the ability tions in K+ channels. Further electrophysiological studies on of PCL-Q to restore BKCa channel activity damaged following these mutations would help to understand the mechanism by irradiation can be realized due to additive action of liposomal which these mutations affect pH-gating. form of lecithin itself and Q encapsulated into lipid vesicles. Encapsulation of flavonoids appears to be worthwhile thera- Where applicable, the authors confirm that the experiments peutic approach in case of ionizing irradiation accident as well described here conform with The Physiological Society ethical in patients which underwent external radiotherapy. requirements. This study was supported by a Physiological Society ‘Centre of Excellence Award Scheme’. Where applicable, the authors confirm that the experiments PC41 described here conform with The Physiological Society ethical requirements. Comparative study of quercetin-filled liposomes, free quercetin and ‘empty’ liposomes regenerative effects on BKCa channels activity in rat aortic smooth muscles cells under ionized irradiation PC42 S. Kyrychenko, S. Tishkin and A. Soloviev Molecular mechanisms behind the acute oxygen sensing of potassium channels in primary human pulmonary artery Department for Experimental Therapeutics, Institute of smooth muscle cells Pharmacology and Toxicology, Kiev, Ukraine N. Chandran1, B. Tang2, Z. Bálint1, J. Lindenmann4, E. Stacher3 It is well known that exposure to excess levels of radiation leads and A. Olschewski*1 to vascular contractile anomalies due to depression of the 1 endothelium-dependent vasodilatation and increase of the Experimental Anesthesiology, University Clinic of Anesthesiology vasoconstriction. These effects of radiation realized through in and Intensive Care, Medical University of Graz, Graz, Austria, 2 a large conductance Ca2+-activated K+ channels (BKCa) activ- Department of Pulmonology, University Clinic of Internal Medicine, 3 ity in vascular tissue. Both ‘empty’ phosphatidilcholine lipo- Medical University of Graz, Graz, Austria, Institute of Pathology, 4 somes (PCL) and free quercetin (Q) are known to repair radia- Medical University of Graz, Graz, Austria and Department of tion-induced vascular tone abnormalities, but Q is rather toxic Thoracic and Hyperbaric Surgery, University Clinic of Surgery, and low soluble agent. Medical University of Graz, Graz, Austria So, the goal of this study was to compare effects of low toxic Acutehypoxicinhibitionofpotassium(K+)channelsisacriticalstep and high soluble phosphatidylcholine liposomes filled with in regulatory processes designed to link lowering of oxygen levels quercetin (PCL-Q) and PCL, Q as well its simple mixture (PCL+Q) tocellularresponses.Inthepulmonarycirculationthevasculartone on functional activity of BKCa channels in rat thoracic aorta in response to hypoxia is determined in part by oxygen-sensitive smooth muscle cells (SMCs) after ionizing irradiation. ionchannelsbutthemolecularmechanismbehindisstillunknown. SMCs of thoracic aorta were obtained from anesthetized intact Methods:Primarysmoothmusclecellswereisolatedfromhuman and irradiated (6 Gy) male Wistar rats (200-250 g) (100% sur- pulmonaryarteries(hPASMC)frompatients(n=20)undergoing vival rate (dose for LD50, 10 Gy)). The effects of whole body γ- lung surgery for lung cancer without a history of pulmonary vas- irradiation (6 Gy) and quercetin-filled PCL on BKCa channels culardiseaseorarterialhypoxemiaasdescribedearlier(Olschewski functional activity were studied using patch-clamp technique et al 2006). The study protocol for tissue donation was approved in whole-cell configuration. by the “Institutional Review Board” of the Medical University of BKCa current density (paxilline-sensitive current) in control GrazinaccordancewithnationallawandwithguidelinesonGood SMCs were 25±2 pA/pF at +70 mV (n=20). Ionizing irradiation ClinicalPractice/InternationalConferenceonHarmonization.Writ- decreased BKCa current density in SMCs to 13±1 pA/pF (n=15, ten informed consent was obtained from each individual patient. P<0.05) and 5±1 pA/pF (n=17, P<0.05) on the 9th and 30th Hypoxia-induced changes in ion conductance and in intracel- days, respectively. lular calcium homeostasis of hPASMC were investigated by PCL-Q (3,4 μg/ml by quercetin and 124,0 μg/ml by lecithin), switching the perfusing medium from normoxia to moderate being added to the external solution, effectively increased BKCa hypoxia (pO2 of 25-35 mmHg). The whole-cell patch-clamp current density in post-irradiated SMCs – to 24±3 pA/pF and technique was used as previously described (1). Changes in 17±2 pA/pF (n=7, P<0.05) on the 9th and 30th days, respec- intracellular calcium were measured by using fura-2am loaded tively. The total effect PCL-Q appears to be the result of addi- hPASMC. Immunofluorescence of phosphorylated tyrosine tive action of Q and PCL. The ratios of therapeutic effectiveness kinase in normoxia and after 30 minutes of hypoxia was per- of PCL-Q, Q and PCL, being expressed quantitatively, may be formed using phosphospecfic antibody. Intergroup differences presented as 1.0:0.6:0.4. The substances studied was without were assessed by a factorial analysis of variance with post hoc effect on the BKCa channels activity in control rat aorta SMCs. analysis with Fisher’s least significant difference test. Experimentally obtained for PCL-Q and theoretically expected Results: Voltage-activated (Kv), calcium-sensitive (KCa) and for Q+PCL dose-response curves demonstrate good coinci- non-inactivating TASK-1 K+ currents were reversibly inhibited dence. I/V curves obtained on the 9th and 30th days of post by moderate hypoxia. The reduction of Kv current measured at irradiation using simple mixture of PCL and Q in comparison +50 mV was 43±3% (n=8, p<0.01) and 56±5% for KCa (n=7,

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operated rats (mean±s.e.m, 0.23±0.05 to 0.09±0.03 relative to p<0.01). TASK-1 current was blocked to 51±3% (n = 22, p<0.001) housekeeping gene U6, n=6, P = 0.03 using students t-test). We at 0 mV. PP2, a selective inhibitor of Src thyrosine kinases abol- also studied the expression of the Repressor Element 1-Silenc- ished the effect of hypoxia (n=25), whereas PP3 (n=16), Ro-31- ing Transcription factor (REST), a transcription factor that can 8220 and Gö 6983 (PKC inhibitors, n=18), KT 5720 (PKA bind to KCNQ DNA in vitro and reduce M channel expression in inhibitor, n=15), Y-27632 (Rho-kinase inihibitor, n=16) or com- cultured DRG neurons (Ooi et al, 2009). REST mRNA expression pound C (AMP kinase inhibitor, n=20) failed to attenuate the was increased in the L4 and L5 DRG of PSNL injured rats in com- hypoxia-induced reduction of the currents. Hypoxic challenges parison to sham REST expression (0.008±0.002 to 0.019±0.004 induced a transient increase in intracellular calcium concen- relative to U6, n = 6, P = 0.007). In addition KCNQ2 mRNA was tration (n=50). The effect of hypoxia was abolished when decreased in the ipsilateral nerve neuroma of PSNL injured rats hPASCM were preincubated with PP2 (n=40). In hPASMC a redis- in comparison to sham sciatic nerve KCNQ2 mRNA expression tribution of phosphorylated tyrosine kinase fluorescence to the (1x10-4±0.4x10-4 to 1x10-5±0.3x10-5 relative to U6, n = 6, P = plasma membrane was observed under hypoxic conditions, 0.04). Preliminary immunohistochemical analysis of PSNL rat whereas it was localized in the cytosol in normoxic cells (n=3). L4 DRG showed a decrease in Kv7.2 protein expression 15 days Conclusion: Our data indicate that the thyrosine kinase (src) following injury in comparison to sham operated rats. In sum pathway is required for the acute response to hypoxia of K+ our data suggest that transcriptional down-regulation of M channels and intracellular calcium homeostasis in primary channel expression in nociceptive sensory neurones can con- human PASMC. tribute to increased excitability of peripheral nerves induced Olschewski A et al: Impact of TASK-1 in human pulmonary artery smooth muscle cells. Circ Res 28;98:1072-80 (2006) by nerve injury. Linley et al, (2008). Inhibition of M current in sensory neurons by exoge- The study was supported by European Union and by the Med- nous proteases: a signalling pathway mediating inflammatory noci- ical University of Graz. ception. J. Neurosci. 28,111240-9. Where applicable, the authors confirm that the experiments Ooi et al, (2009). Regulation of KCNQ2/3 channels by the transcriptional repressor REST in nociception. Poster Comm. Biophysics Boston described here conform with The Physiological Society ethical requirements. Passmore et al, (2003). KCNQ/M currents in sensory neurons: significance for pain therapy. J. Neurosci. 7,860-9. Where applicable, the authors confirm that the experiments PC43 described here conform with The Physiological Society ethical requirements. Nerve injury induces down-regulation of M channel expression in peripheral sensory neurons K.E. Rose, C. Dalle, B. Robertson and N. Gamper PC44 IMSB, University of Leeds, Leeds, UK ATP independent translocation of STIM1 and formation of Neuropathic pain is a severe health problem, which is further STIM1-Orai1 complexes complicated by the lack of effective therapy. Search for new treatments has recently focused on ion channel candidates, C.M. Walsh, G. Lur, M. Chvanov, L.P. Haynes, S.G. Voronina, since expression and activity of many ion channels relevant to O.V. Gerasimenko, O.H. Petersen, O.H. Petersen, R.D. Burgoyne pain signalling have been found altered following nerve injury. and A.V. Tepikin There is compelling evidence for the role of non-inactivating, The Physiological Laboratory, University of Liverpool, Liverpool, UK subthreshold Kv7 (KCNQ, ‘M type’) potassium channels in pain Stromal interacting molecule one (STIM1) is currently known transmission, as the membrane excitability of small nocicep- to be a calcium sensor protein located in the endoplasmic retic- tive DRG neurons increases following pharmacological or recep- ulum (ER). Depletion of the ER calcium store triggers translo- tor-induced inhibition of M current (Passmore et al, 2003; Lin- cation of STIM1 into subplasmalemmal puncta where it acti- ley et al, 2008). Immunohistochemistry in adult rat DRG vates calcium channels and initiates store operated calcium revealed that Kv7.2 protein is expressed within subpopulations entry (SOCE). We show that inhibition of ATP production of CGRP-, IB4- and TRPV1-positive neurons. Behavioural tests induced a slow calcium leak from the ER that was followed by demonstrated that acute in vivo inhibition of M channels formation of subplasmalemmal STIM1 puncta. Depletion of expressed in nociceptive neuronal terminals of rat hindpaw cytosolic ATP also initiated the loss of phosphatidylinositol 4,5- induced by the intraplantar injection of specific Kv7 blocker, bisphosphate (PI(4,5)P2) from the plasma membrane. Although XE991 (10 nmoles/site), resulted in spontaneous nocifensive STIM1 puncta formed by inhibition of ATP synthesis co-localised behaviour not observed in rats injected with vehicle. We next with clusters of ORAI1 channels these complexes were ineffi- studied a possible role of M channels in the development of cient to facilitate calcium influx. Restoration of ER calcium lev- chronic pain following peripheral nerve injury. To this end we els in the absence of ATP permitted STIM1 re-translocation from investigated the expression of KCNQ2 in the DRG and sciatic subplasmalemmal puncta to the ER. Therefore we suggest that nerve neuroma of Partial Sciatic Nerve Ligation (PSNL) oper- dynamic re-arrangement of STIM1 and the formation of STIM1- ated rats. Under general anesthesia with 2% isoflurane, the ORAI1 complexes is an ATP independent process that can occur nerve was exposed, partially ligated and sectioned. Real-time under conditions of PI(4,5)P2 depletion. RT-PCR experiments revealed that the level of KCNQ2 mRNA within PSNL injured rat L4 and L5 DRG was significantly decreased 30 days following nerve injury in comparison to sham

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC47 requirements. Effect of equal-volume creatine supplementation with different dosing frequency on muscle hypertrophy and PC46 strength in young adults D.G. Candow1, K. Mueller1, J. Lazorko1, J. Lewis1, J. Neary1 and Neuromuscular fatigue in exhausting exercise at VO2max: D. Burke2 influence of two modalities 1 2 G. Sarre, H. Petot and V. Billat Kinesiology, University of Regina, Regina, SK, Canada and Human Kinetics, St. Francis Xavier University, Antiogonish, NS, Canada LEPHE, Evry, France Creatine (Cr) supplementation during resistance training (RT) It is commonly admitted that time-to-exhaustion corresponds increases muscle mass and strength. However, it is unknown to about six minutes for an exercise performed at an intensity whether the volume and frequency of Cr ingestion will influ- allowing eliciting 100% of the VO2max. Although many works ence the adaptations from RT. This study determined the effects have tried to identify the limiting factors in time-to-exhaustion of equal-volume Cr supplementation with different dosing fre- at VO2max by investigating energetic or metabolic parame- quency on muscle hypertrophy and strength. Young healthy ters, the determinants underlying this performance key factor adults (N=22; 11 male, 11 female; 21±3 years) were random- remain unclear. The purpose of this study will be to address this ized to supplement with Cr during 2 days/week of RT (Cr2, question through a neuromuscular approach. Our hypothesis 0.15g/kg-1, N=11, 6 male, 5 female) or 3 days/week of RT (Cr3, will be that a neuromuscular invariant could explain exercise 0.10g/kg-1, N=11, 6 male, 5 female). Creatine was consumed breakout in subjects performing a maximal exercise up to voli- (in equal amounts) immediately before and immediately after tional exhaustion. each RT session. The RT program consisted of 2 sets (Cr2) or 3 11 physically trained subjects got involved in this study. Each sets (Cr3) of 10 repetitions (10RM) to muscle fatigue for 9 of them had to perform in a random order 2 pedalling exercises whole-body exercises. Prior to and following Cr supplementa- designed to elicit 100% of their individual VO2max, separated tion and RT, measurements were taken for muscle hypertro- by 1 week recovery. Each exercise was lead up to exhaustion. phy (elbow and knee flexors and extensors; ultrasound), and The first one was performed at a constant power correspon- muscle strength (1-repetition maximum leg press and chest ding to the maximal aerobic power (MAP). During the second press). Repeated measures ANOVA (mean ± SD) showed crea- one, external power output initially corresponded to the MAP, tine supplementation increased muscle hypertrophy and but was secondarilly adjusted so that VO2 remains maximal strength over time (p<0.05). There was a greater change in despite decreased load. leg press strength in the Cr2 group (Cr2: 223±93Ç362±220kg, Neuromuscular testings were performed before and immedi- 62% vs. Cr3: 168±54Ç238±112kg, 42%, p=0.05). There were ately after each exercise in order to assess neuromuscular no differences between groups for changes in muscle size of fatigue generated by each condition. the elbow flexors (Cr2: 2.9±0.9cmÇ3.2±1.0cm, 10% ; Cr3: Power output during exercise performed at a variable load was 2.7±0.6Ç3.0±0.6cm, 11%), elbow extensors (Cr2: modelized in regard to MAP, power at individual anaerobic 3.4±1.2Ç4.3±0.7cm, 26%; Cr3: 3.0±1.2Ç3.7±0.9cm, 23%), threshold and time-to-exhaustion at MAP. knee flexors (Cr2: 5.0±1.0Ç5.4±0.7cm, 8%; Cr3: Time-to-exhaustion significantly increased in variable load con- 4.7±1.1Ç5.4±0.8cm, 15%), and knee extensors (Cr2: dition. Neuromuscular fatigue was not significantly different 4.3±1.0Ç4.7±1.4cm, 9%; Cr3: 3.7±0.7Ç3.9±0.7cm, 5%), or between the two conditions. chest press strength (Cr2: 109±66Ç128±73kg, 17%; Cr3: Constant neuromuscular fatigue between the two conditions 77±49Ç85±42kg, 10%). These results suggest that the volume suggests that this parameter could play a key role in volitional of Cr ingestion may be more important than the frequency of exhaustion during maximal exercise. Cr ingestion for improving lower-body muscle strength in young It seems possible to dramatically increase time-to-exhaustion healthy adults. at VO2max. This result could be of interest in field performance training. RIVALUS Inc., Canada. VO2max could be elicited at a submaximal external power output, what could be an important result in field rehabilitation. Where applicable, the authors confirm that the experiments Further investigation is required to better understand under- described here conform with The Physiological Society ethical lying mechanisms in fatigue during maximal metabolic load. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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Where applicable, the authors confirm that the experiments PC48 described here conform with The Physiological Society ethical requirements.

Effects of b2-agonists on force in anoxic rat EDL muscle. The role of the Na+/K+-pumps

A. Fredsted, H. Gissel and T. Clausen PC49

University of Aarhus, Örhus C, Denmark Relationship between changes in force and linear dimensions of Rectus Femoris muscle in man using ultrasound imaging Fatiguing stimulation of isolated rat skeletal muscle leads to 1 2 1 2 1 depolarization of the cellular membrane and a considerable S. Delaney , P.Worsley , M. Warner , M. Taylor and M. Stokes loss of force (1) suggesting excitability of the membrane as an 1School of Health Sciences, University of Southampton, important mediator of the fatigue. Na+/K+- pump activity Southampton, UK and 2School of Engineering Sciences, University β increases dramatically in response to 2-agonist addition, of Southampton, Southampton, UK improving restoration of the Na+, K+ homeostasis and the con- BACKGROUND tractile performance of a working muscle. Reduced availabil- Ultrasound imaging (USI) is used increasingly for assessing mus- ity of oxygen to the muscles has been shown to increase the cle activity during contraction in research and clinical practice rate of fatigue in isolated muscle (2) and isolated diaphragm [1]. The relationship between changes in cross-sectional shape muscle strips (3). The present study examines the effect of β - 2 and linear dimensions that occur during contraction varies in agonists on force in anoxic rat EDL muscle and the role of the different muscles and cannot be assumed. Both linear and curvi- Na+/K+- pumps in mediating this effect. linear relationships have been found for increases in muscle EDL muscles were prepared from 4 wk old Wistar rats and thickness with force, e.g. in calf [2] and abdominal [3,4] mus- mounted on holders for isometric contractions. Muscles were cles. The relationship therefore needs to be characterized in a stimulated intermittently at 40 Hz for 15 min (10 sec on, 30 sec particular muscle before USI measurements can be used as an off, 10 V, 0.2 ms pulses) during anoxic conditions (95% N , 5% 2 indirect indicator of force. This exploratory study aimed to CO ) and force recovery was followed during reoxygenation 2 examine the relationship between incremental force produc- (95% O , 5% CO ) for up to 240 min. β -agonists (salbutamol 2 2 2 tion of quadriceps and linear dimensions of the rectus femoris (10-5 M) or salmeterol (10-6 M)) were added either 15 min prior (RF) muscle in a group of young men. to the anoxic stimulation protocol or during recovery. Intra- METHODS cellular Na+ content was measured at the end of the reoxy- In 14 healthy males, aged 22-27 years (mean 24.8), isometric genation period. The statistical significance of any difference force of quadriceps was measured using a Biodex dynamome- between the control group and the treated group was ascer- ter with the knee flexed to 90 degrees, during maximal volun- tained using two-way Anova and the post hoc Bonferroni test. tary contractions (MVC) and randomly at 10, 20, 30, 50 and Stimulation during anoxia leads to rapid force decline and only 75% of MVC, held for 3 seconds each. An ultrasound scanner partly recovery during the following reoxygenation period. (Aquilla, ESAOTE; 6 MHz linear transducer) was used to image Addition of salbutamol or salmeterol prior to the anoxic stim- RF at mid-thigh at rest and during contractions. Two muscle ulation protocol improves force by 32 ± 10% (P<0.001, n=7) and dimensions (depth or thickness and width) were measured 40 ± 7% (P<0.001, n=4), respectively, within the first 2-3 min offline. Pearson’s correlation was used to examine the rela- of the fatiguing protocol, hereafter force declines rapidly in all tionship between change in muscle dimension (normalized as groups. In the following reoxygenation period force in the con- percentage) and force. Differences in mean dimensions trol muscles reaches 27 ± 3% of initial force, whereas force in between force levels were examined by repeated measures salbutamol treated muscles increases up to 42 ± 5% (P<0.05, analysis of variance and paired samples T-test with Bonferroni n=7) of initial force. Addition of salbutamol to control muscles correction (p<0.008). at 150 min in the reoxygenation period improves force by 102 RESULTS ± 16% (P<0.001, n=12). Muscles treated with a β -agonist also 2 As force increased, there was a curvilinear decrease in RF width show decreased intracellular Na+ content indicating that the (r= -0.92; p<0.01), with decreases in width greatest at low Na+/K+-pumps are involved. Inhibition of the Na+/K+- pumps forces, <30% MVC (Fig 1). Resting width (mean=4.41cm, by ouabain or 2-deoxyglucose abolishes the positive effect of SD+0.07) was significantly different to that at all contraction salbutamol on force. No detectable increase in the release of levels (p<0.05). Significant differences were also found between an intracellular enzyme (LDH) is observed during this anoxic contraction levels (p<0.008), except between 20% - 30% MVC, protocol indicating that the observed force loss is not due to and 75% - MVC (p>0.008). At MVC, width was 3.27cm loss of cellular integrity. (SD+0.47), approximately 25% smaller than at rest. Thickness The main findings are that force during and following an anoxic of RF increased with contraction but only changed minimally period can be improved by addition of β -agonists (eg. salbu- 2 (p>0.05), from a mean of 2.34cm (SD+0.32) at rest to 2.57cm tamol or salmeterol) most likely due to a stimulating effect on (SD+0.3) at MVC. the Na+/K+- pumps. CONCLUSIONS Mikkelsen et al., Am J Physiol Regul Integr Comp Physiol 290, R265- Dynamic assessment of RF in young males can be made by R272, 2006 measuring decreases in width on USI scans to reflect increases Jones et al., Clin Sci 65, 193-201, 1983. in force, which are most evident a low forces. Muscle thickness cannot be used as a measure of contractile activity of RF. van der Heijden et al., Am J Physiol 276, L474-L480, 1999

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± ± These findings are relevant to healthy young males and larger (98 5% vs. 57 9%, control n=6 vs. H2O2 n=7 at 2 min, P<0.05, ± ± numbers are needed to calculate predictive regression equa- ANOVA) and hypoxia (60 7% vs. 15 6%, control n=7 vs. H2O2 tions. The relationship also warrants investigation in healthy n=6 at 2 min, P<0.05, ANOVA). Co-incubation of H2O2 with DFX males and females of different ages and habitual activity, as significantly improved the fatigue index of the muscle strips ± ± well as populations with muscle dysfunction. compared to H2O2 alone in hyperoxia (83 3% vs. 57 9%, H2O2 Whittaker J, Teyhen D, Elliot JM et al. (2007) JOSPT 37(8),435-449 plus DFX n=6 vs. H2O2 n=7 at 2 min P<0.05, ANOVA) but this MaganarisCNBaltzopoulosV,SargentAJ.(1998)JPhysiol512(2),603-614 improvement did not reach statistical significance in hypoxia. In summary, H O increased fatigability of a pharyngeal dila- Hodges P Pengel LH, Herbert RD. (2003) Muscle Nerve 27,682-692 2 2 tor muscle but failed to show any effect on muscle force at both McMeekenJM,BeithID,NewhamDJetal.(2004)ClinBiomech19,337-342 ∞ ∞ 27 C and 35 C. Muscle strips co-incubated with H2O2 and DFX The authors thank DePuy, a Jonson & Jonson Company, for finan- showed a marked improvement in muscle endurance partially reversing the effects of H O alone. DFX prevents the forma- cial support and the participants who volunteered to take part 2 2 tion hydroxyl radicals from H O and therefore the effects of in this study. 2 2 H2O2 on muscle fatigue are presumably partially attributed to Where applicable, the authors confirm that the experiments H2O2 itself and partially to an effect of hydroxyl radicals. described here conform with The Physiological Society ethical Bradford, A. et al, (2005) Respir. Physiol. Neurobiol.; 147(2-3): 223-34 requirements. Dunleavy, M. et al, (2008) Adv. Exp. Med. Biol.; 605:458-62

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC50 requirements. Effects of hydrogen peroxide on rat sternohyoid muscle endurance PC51 C. Shortt and K.D. O’Halloran University College Dublin, Co.Wicklow, Ireland Dihydrotestosterone (DHT) modulates force production in isolated mouse skeletal muscles by regulating the Obstructive sleep apnoea (OSA) is characterized by recurrent phosphorylation of myosin light chains by ERK 1&2 collapse of the upper airway during sleep, subjecting the patient to recurrent bouts of intermittent hypoxia. Chronic intermit- M.M. Hamdi, T. Wileman and G. Mutungi tent hypoxia (CIH) has been shown to induce upper airway mus- Biomedical and Clinical Research Institute, University of East Anglia, cle dysfunction1, perhaps via the production of reactive oxy- Norwich, UK gen species2. First, we examined the effect of hydrogen peroxide (H2O2) on contractile properties of the rat sternohy- In a recent study we showed that treating isolated intact mouse oid muscle at varying temperatures. Second, we investigated fast- and slow-twitch skeletal muscle fibres with physiological the effect of H2O2 in the presence of an iron chelator, deferox- levels of dihydrotestosterone (DHT), for one hour, leads to an amine (DFX) on skeletal muscle contraction. DFX prevents the increase in maximum isometric tension (Po) in the fast twitch generation of hydroxyl radicals from H2O2. fibres and a decrease in Po in the slow twitch fibres (Mutungi, Adult male Wistar rats were anaesthetized with 5% isoflurane 2008). However, in that study the molecular mechanisms and killed by spinal transection. Sternohyoid muscle strips were underlying these effects were not investigated. Therefore, the mounted isometrically in water-jacketed tissue baths at 27∞C primary aim of this study was to investigate the molecular ∞ or 35 C and either bubbled with a hyperoxic (95% O2/5% CO2) mechanisms underlying the effects of DHT on Po in mouse or anoxic (95% N2/5% CO2) gas mixture. Studies were con- skeletal muscle fibre bundles. ducted under control conditions (no drug), in the presence of All the experiments were performed at 20∞C using small mus- 1mM H2O2 or 1mM H2O2 plus 1mM DFX. Strips were set to opti- cle fibre bundles isolated from either the extensor digitorum mal length and force-frequency relationship was assessed by longus (edl, a mainly fast twitch muscle) or soleus (a predom- stimulating the muscle every two minutes at 10, 20, 30, 40, inantly slow twitch muscle) of adult female CD-1 mice 49 ± 4 60, 80 and 100 Hz for 300ms. Fatigue was induced by repeated (n=8; S.E.M) days. The mice were humanely killed and all the tetanic contractions (40Hz, 300ms duration) every 2 seconds experiments conformed to the Animals (Scientific Procedures) for2minutes. Act 1986. The fibre bundles were treated for 1hr with either ∞ ρ At 27 C, H2O2 had no significant effect on muscle force under 630 g/ml DHT dissolved in absolute ethanol (experimental) or hyperoxic (22±3 vs. 17±2 N/cm2, mean±SEM, control n=5 vs. the vehicle only (6.3μL/100ml; controls). In another experi- H2O2 n=5 at 100 Hz, P>0.05, ANOVA) or hypoxic conditions. ment, the bundles were pre-treated for 30 minutes with H2O2-treated muscle strips exhibited increased fatigue under inhibitors of either the epidermal growth factor receptor [EGFR]) ± ± η μ hyperoxic conditions (63 3% vs. 39 3%, control n=6 vs. H2O2 (100 M AG1478) or the androgen receptor (AR) (3 Mflutamide n=6 at 2 min, P<0.05, ANOVA). Similar results were seen in and 1μM cyproterone acetate) before treatment with DHT plus ± ± hypoxia (63 7% vs. 25 2%, control n=7 vs. H2O2 n=5 at 2 min, the inhibitor for 1hr. At the end of each experiment, the bun- P<0.05, ANOVA). At a more physiological temperature of 35∞C, dles were processed for immunoblotting and the changes in H2O2 again did not alter sternohyoid contractile force under the phosphorylation of extracellular signal regulated kinases hyperoxic or hypoxic conditions. Fatigue was increased in H2O2- 1&2 (pERK1&2) and myosin light chains (pMLCs) were probed treated muscle strips compared to control strips in hyperoxia using antibodies against the phosphorylated proteins.

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The findings show that treating the fibre bundles with DHT human P-gp where THC caused increased P-gp ATPase activ- led to a 2-3 fold increase in the phosphorylation of ERK 1&2 in ity1. Extrapolating these in vitro data, we postulate that THC both fibre types and MLC in the fast twitch fibres only. More- stimulation of placental P-gp activity would have a marked over, this increase was blocked by AG1478 but not by flutamide effect on the exposure of the fetus to other drugs which are and cyproterone acetate. From these results we suggest that substrates of this MDRP, taken by women using cannabis in DHT modulates force production in mammalian skeletal mus- pregnancy. cles by regulating the phosphorylation of MLCs by ERK1&2. Zhu H J, Wang J S, Markowitz J S, Donovan J L, Gibson B B, Gefroh H A, Mutungi G. (2008). The effects of androstanolone (a synthetic DHT) DeVane C. L. Characterization of P-glycoprotein inhibition by major on maximum isometric force in intact mouse skeletal muscle fibres. cannabinoidsfrommarijuana.J.PharmExpTher(2006)317(2)850-857 Proc Physiol Soc 11, C56. Funding from the Manchester NIHR Biomedical Research Cen- This research was funded by the University of East Anglia and tre is acknowledged. a PhD studentship to MMH from the Malaysian Government. DEA is funded by an NIHR fellowship Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

PC52 PC53 Interaction of D9-tetrahydrocannabinol and Buprenorphine with Multidrug Resistance Proteins in Human Placenta Vascular reactivity of early pregnancy human placental chorionic plate arteries D. Thajam, C.P. Sibley and D.E. Atkinson M. Wareing Maternal and Fetal Health Research Group, University of Manchester, Manchester, UK Maternal and Fetal Health Research Centre, The University of Manchester, Manchester, UK Drug misuse by pregnant women is a leading preventable cause of fetal and neonatal morbidity and mortality. It has been sug- Background: We have previously demonstrated that placen- gested that in both the UK and the USA 10-16% of pregnant tal chorionic plate arteries (CPAs) at term exhibit a biphasic women use illicit drugs. The level of exposure of any one fetus (transient contraction followed by maintained relaxation) is likely to be determined by the capacity of the placenta to response on exposure to histamine [1]. Here we wished to act as a barrier. The multi drug resistance proteins (MDRPs) P- determine if early pregnancy CPAs displayed similar vascular glycoprotein (P-gp) and breast cancer resistance protein (BRCP) reactivity. are located on the maternal facing (microvillous) plasma mem- Method: Placentas (N=5) were obtained following elective brane of the placenta. In this location, these proteins may act medical or surgical termination of pregnancy. Gestational age to prevent xenobiotics in maternal plasma reaching the fetus was estimated from date of last menstrual period and confirmed - at toxic concentrations. The aim of the present study was to by ultrasound dating. Biopsies were placed into ice-cold HCO3 determine whether two commonly used substances i.e. Δ9- -buffered physiologic salt solution (PSS). Arteries were dissected tetrahydrocannabinol (THC) and buprenorphine (BUP) inter- from the chorionic plate, mounted onto a wire myograph, nor- ∞ act with P-gp and/or BCRP in human placental tissue. malised at 0.9 of L5.1kPa and equilibrated (37 C; 20 mins; Term placentas were collected (with ethics committee approval 5%O2/5%CO2 ~7% O2). Contraction was assessed with 120mM and informed consent) within 30mins of delivery. Small vil- potassium solution (KPSS) and the thromboxane-mimetic lous fragments were dissected and used to measure accumu- U46619 (10-10 to 2x10-6M). Histamine hydrochloride (HIS; 10- 3 3 6 lation of H-vinblastine (a P-gp substrate) and H-mitoxantrone M) was added to pre-contracted vessels (EC80 dose of the (a BCRP substrate) in the presence or absence of 20μM THC or thromboxane-mimetic U46619 for 30 mins) for 60 mins. Exper- BUP. Accumulation at 10 mins (initial rate) and 120 mins (equi- iments with HIS were also performed in the presence of librium) was measured in all cases. indomethacin (I; 10-5M) and indomethacin plus Nω-nitro-L- BUP had no significant effect on either vinblastine (n=5 pla- arginine, (IN; both 10-5M). Arterial relaxation to donated nitric centas) or mitoxantrone (n=3) accumulation at 10 or 120 mins. oxide (NO) was assessed using sodium nitroprusside (SNP; 10- THC caused a significant (p<0.05 Wilcoxon signed rank test, 5M). All data are expressed as median (range). n=5) decrease in accumulation of vinblastine at 120 mins (50%) Results: Normalised luminal internal diameters were 486 (210- and there was a trend towards reduced accumulation (37%) at 1008) μm (n=20 arteries). Maximal arterial contraction to KPSS 10min (P=0.06 n=5). THC had no significant effect on mitox- and U46619 was 2.0 (0.4-5.0) kPa and 3.2 (1.2-6.8) kPa (n=20 antrone accumulation although there was a trend towards arteries from N=5 placentas) respectively. HIS induced a bipha- increased accumulation at both time points (10 mins 21% and sic response in pre-contracted CPAs; a transient contraction 120mins 38% p=0.06 n=4). to 105 (102-118) % of EC80 U46619-induced contraction fol- The reduced accumulation i.e. increased efflux of vinblastine lowed by a maintained relaxation to 95 (23-95) % of EC80 in the presence of THC suggests stimulation of P-gp activity U46619-induced contraction. Contraction and relaxation to by this component of cannabis. These data are consistent with HIS alone were not significantly affected by pre-incubation with previous observations in insect cell membranes containing I or IN (P<0.05; Friedman’s test followed by Dunn’s post hoc

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test). SNP induced a significant relaxation in pre-contracted blood vessels stained positively for KV9.3, but staining was CPAs; a maximal relaxation to 37 (2-71) % of EC80 U46619- absent in capillary loops of terminal villi. Vascular smooth mus- induced contraction. cle cell staining was only apparent in larger diameter vessels. Conclusion: Our preliminary data suggest that 10-6M HIS elic- Expression was not significantly altered in tissue from women its a biphasic response in early pregnancy CPAs; the pattern of with fetal growth restriction. response is similar to that seen in term CPAs but the size of the Expression of KV9.3 suggests human placental tissues may responses are reduced compared to term [1]. The ability to respond to oxygenation fluctuations via alterations in cell mem- assess placental vascular reactivity in early pregnancy is essen- brane potential. This may have important implications for syn- tial for the development of therapeutic agents to treat patholo- cytiotrophoblast cell turnover, nutrient transport and for blood gies associated with increased placental vascular resistance [2]. flow in fetoplacental blood vessels. Mills, Taggart, Greenwood, Baker & Wareing (2007). Placenta 28: Bai et al (2006). Journal of the Society for Gynecoligic Investigation 1158-64. 13: 30-39. Mills, Wareing, Bugg, Greenwood & Baker (2005). European Journal of Wareing et al (2006). American Journal of Physiology (Reg Int Comp Clinical Investigation 35: 758-64. Physiol) 291: R437-446. Supported by Tommy’s [Let Talk Baby] Charity. Hulme et al (1999). Circulation Research 85: 489-497. Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

PC54 PC55 Expression of an electrically silent voltage-gated potassium channel in the human placenta Metabolic Footprint Analysis of Placental Perfusates G.K. Fyfe, J. Corcoran, S. Panicker, R.A. Garside, P.N. Baker, Investigating Hypoxia and Altered Haemodynamics in R.L. Jones and M. Wareing Pre-eclampsia Maternal and Fetal Health Research Group, School of Clinical and S. Kuruvilla1, W.B. Dunn2, M. Brown2, H.S. Elizabeth1, Laboratory Sciences, The University of Manchester, Manchester, UK P. Brownbill 1, P.N. Baker1 and I.P. Crocker1

Potassium (K) channels are important for normal placental func- 1Maternal and Fetal Health Research Group, The University of tion. Activity and expression of a number of K channel isoforms Manchester, Manchester, UK and 2The Manchester Centre for has been reported in syncytial and vascular tissues [1,2]. mRNA Integrative Systems Biology, The University of Manchester, expression of KV9.3, a voltage-gated (KV) channel linked to tis- Manchester, UK sue responses to oxygenation [3], has been demonstrated in whole placental homogenate by RT-PCR. However, the chan- Pre-eclampsia is a vascular complication of human pregnancy, nel’s electrophysiological properties and the lack of a human with symptoms of hypertension and proteinuria manifesting isoform specific antibody have hampered further investiga- from mid-gestation. This multi-system disorder affects 3-5% tions. We aimed to produce an antibody to human KV9.3, with of pregnancies and is a major cause of maternal and fetal mor- a view to examining protein expression and localisation in tality and morbidity. Although its exact cause is unknown, evi- human placental tissues. dence points to placental derived pathogenic factors, liberated Term placentas were collected from uncomplicated singleton in response to placental hypoxia or aberrant utero-placental pregnancies or from women whose pregnancies were affected haemodynamics. In previous studies we have mimicked these by fetal growth restriction. An affinity-purified polyclonal anti- placental conditions in an in vitro dual placental perfusion body was raised in rabbit to residues 131-147 of the human iso- model and have highlighted the liberation of placental factors, form of KV9.3. Channel expression in samples of placental which induce both metabolic and apoptotic changes in cultured homogenate was confirmed by Western blotting. Protein vascular endothelial cells [1]. In this study we used metabolomic expression and localisation was investigated using standard techniques to confirm placental adaptations and identify small immunohistochemical techniques. Scored data were assessed molecules in response to these aberrant conditions. using Mann-Whitney U-test with a threshold of P<0.05 indica- Human placenta lobules from uncomplicated term deliveries tive of statistical significance. were dual perfused with Earle’s Bicarbonate Buffer through can- KV9.3 expression was confirmed by Western blotting. A single nulae, creating a physiological representation of fetal and discrete band was observed at ~61kDa; signal was ablated by maternal blood flow in the placenta. Two separate experiments antibody pre-absorption (10X excess of blocking peptide) and were performed. The first to investigate altered oxygen ten- incubation with pre-immune serum. Immunohistochemical sions, in which placentae were perfused under hypoxic (<3%O2, staining was assessed by three independent researchers using n=5) and normoxic conditions (6%O2, n=4). The second, to an arbitrary scoring system, blinded to the identity of the tis- determine effects of altered intra-placental haemodynamics, sue. Strong staining was observed in syncytiotrophoblast, par- in which maternal flow rates were increased from 14 to ticularly localised to the microvillous membrane. Endothelial 45ml/mins, with a concomitant increase in intra-placental pres- cells within intermediate and stem villus and chorionic plate sures (33±11 to 64±14 mmHg, n=7). The metabolic footprint

108P Poster Communications of the maternal perfusate was analysed by gas chromatogra- muscles were randomly assigned to groups. After a 30 min incu- phy-time of flight-mass spectrometry. Metabolites were iden- bation period, force-frequency relationship was determined by tified by comparing the retention time and mass spectral data electrical field stimulation with stimulus frequencies ranging to libraries of metabolites. 10-100Hz. Forces in all trials were expressed relative to peak Ten metabolites were differentially expressed between high force measured at the beginning of each experiment (% of ini- and low flow rate perfusions at a statistically significant level tial). of P<0.01 (Kruskal-Wallis test). Of those chemically identified, All gas treatments caused significant decreases in sternohy- hexadecanoic acid, octadecanoic acid and ribitol and/or xyli- oid muscle force compared to control (e.g. 89±9 vs. 28±4*, tol, were significantly elevated under high flow conditions. Pyru- 61±7* and 60±8*, mean±SEM at 100Hz, % of initial, control vic acid was one metabolite significantly decreased in response (n=7) vs. H (n=8), IH (n=9) and HR (n=8), *p≤0.01 ANOVA). Like- to hypoxic insult. Throughout these experiments technical pre- wise, muscle performance during repeated stimulation (40Hz, cision remained high, indicating that the observed differences 300msec, every 2 sec for 150 sec), performed after force-fre- were likely to have originated from true biological variance. quency trials, was significantly impaired by all gas treatments. We conclude that metabolomic analysis provides a novel Antioxidant treatment with Tempol partly ameliorated the approach to detecting and identifying placentally-derived low decline in muscle force in all groups (e.g. 86±9 vs. 44±7*, 70±5 molecular weight compounds. Our experiments were consis- and 69±13, mean±SEM at 100Hz, % of initial, control (n=7) vs. tent with biology and implied that placental stress can alter H (n=8), IH (n=9) and HR (n=8), *p<0.05 ANOVA). Additionally, energy metabolism through the differential expression and lib- Tempol rescued sternohyoid forces during the fatigue trial in eration of fatty acids and sugar alcohols. It further remains to IH and HR treated muscles such that they were not different discriminate these factors, with the aim of identifying bio- from control values. markers or bioactive elements pertinent to the pathogenesis Our results indicate that acute hypoxia and of pre-eclampsia. hypoxia/reoxygenation impairs pharyngeal dilator muscle func- Hutchinson ES et al. (2007). Reproductive Sciences 14, 118A. tion. Antioxidant treatment ameliorates these effects suggesting that reactive oxygen species-dependent mechanisms IPC is supported by a personal award from the National Insti- are implicated. As upper airway muscle dysfunction is impli- tute for Health Research. cated in OSA, we speculate that antioxidant supplementation Where applicable, the authors confirm that the experiments might prove useful as an adjunct therapy in the treatment of described here conform with The Physiological Society ethical the disorder. requirements. Supported by the Health Research Board, Ireland (RP/2006/140). Where applicable, the authors confirm that the experiments PC56 described here conform with The Physiological Society ethical requirements. Intermittent Hypoxia Impairs Rat Upper Airway Muscle Function: Protective Effects of a Superoxide Scavenger S. Coyle-Rowan and K.D. O’Halloran PC57

University College Dublin, Dublin, Ireland Effects of Hypoxia on Geniohyoid Muscle Force and Endurance in Young Lean and Old Obese Male Rats Upper airway muscle dysfunction is implicated in obstructive sleep apnoea, a debilitating respiratory disorder associated with E. Lucking and K.D. O’Halloran cardiovascular and neurocognitive morbidities. Hypoxia-reoxy- University College Dublin, Dublin, Ireland genation is a central feature of the disorder due to recurrent apnoea. We wished to characterize the effects of hypoxia (H), Age, obesity and male sex are independent risk factors for the intermittent hypoxia (IH) and hypoxia-reoxygenation (HR) on development of obstructive sleep apnoea (OSA). Upper airway pharyngeal dilator muscle function in adult male rats. As reac- dilator muscle fatigue is implicated in the pathophysiology of tive oxygen species are implicated in skeletal muscle dysfunc- OSA. As antioxidant capacity decreases with age, we specu- tion, we hypothesized that antioxidant treatment would ame- lated that old obese rats would show decreased endurance and liorate the deleterious effects of (intermittent) hypoxia. hypoxic tolerance. Rats were killed humanely, under 5% isoflurane, by cervical Young (2-3 month old), lean (268±10g), male Wistar rats spinal cord transection. Isometric contractile properties of iso- (n=14) and old (19-20 months), obese (822±41g), male Wis- lated sternohyoid muscle strips were examined at 35OC under tar rats (n=14) were used in this study. Rats were killed control (95%O2/5%CO2) or test conditions i.e. [H = humanely, under 5% isoflurane, by high cervical spinal cord tran- 95%N2/5%CO2; IH = 3 cycles of 5 min control/5 min H; or HR = section. Isometric contractile properties of isolated geniohy- 15 min H/15 min control) in vitro in standard physiological salt oid muscles were examined at 350C under control solution with or without 10mM Tempol (a superoxide scav- (95%O2/5%CO2) or hypoxic (95%N2/5%CO2) conditions. Force- enger). All muscle strips were set to optimum length (i.e. length frequency relationship was determined at stimulus frequen- producing maximum isometric twitch force). After an equili- cies ranging 10-100Hz. Curve-fitting analysis was employed bration period, peak tetanic force at 100Hz was determined allowing us to determine the values for min, max, slope and under control conditions before drug or gas treatment. Next, EF50 (i.e. stimulus frequency producing 50% of peak force).

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Fatigue was assessed by repeated stimulation of the muscle specific VEGF ligands in vitro, human pulmonary microvascu- (40Hz, 300ms, every 2 sec for 3 min). Two-way analysis of vari- lar endothelial cells were grown under sterile cell culture con- ance (with Bonferroni correction) was employed to test for sta- ditions until confluent. A single wound was created in the cell tistically significant effects of age and hypoxia. monolayer by scraping with a sterile pipette tip, the cells treated Geniohyoid muscle max force was 6.9±1.5 and 4.2±0.6 N/cm2, with vehicle or recombinant proteins (PlGF, VEGF A, VEGF B) mean±SEM, control (n=7) and hypoxia (n=7). Corresponding and the wound assessed 24 hours later. The percentage reduc- values in young rats were 4.0±0.9 and 1.2±0.3 N/cm2 (p>0.05 tion in wound width was then calculated. ANOVA). The EF50 values were left-shifted in old compared to VEGF A mRNA expression in the hypoxic lung was not altered young animals (p<0.01 in hypoxia alone). Geniohyoid 3min at any time point examined in vivo (over a 3 week interval). fatigue index in old rats was 48±4% and 7±1%, [% of initial force, VEGF B and PlGF mRNA expression was significantly increased control (n=7) and hypoxia (n=7)]. Corresponding values in at 7 and 14 days, with PlGF remaining augmented following 21 young rats were 78±6% and 24±4%. There was a statistically days exposure to chronic hypoxia. VEGF A (8ng/ml) augmented significant effect of age (p<0.001) and hypoxia (p<0.01), but the mean (±SEM) rate of wound healing over vehicle alone. A no significant interaction. Force potentiation in the early phase low concentration of PlGF (40ng/ml) potentiated the rate of of the fatigue trial was observed in all groups but was signifi- VEGF A induced wound healing. However, a higher concentra- cantly greater in old compared to young animals in hyperoxia tion of PlGF protein (160ng/ml) did not alter the rate of VEGF [186±9% vs. 146±8%, % of initial force, old (n=7) vs. young (n=7), A induced wound healing. VEGF B (20ng/ml) inhibited the p<0.01] but not hypoxia. Taken together, muscle performance actions of VEGF A. (i.e. average force/initial force x 100) during repeated stimula- These results suggest that the interaction between VEGF tion was not significantly different in old and young animals, ligands is complex and is critically concentration dependent. though values were noticeably lower in hypoxia (62±6% vs. Thus, further in vivo experiments are required to fully elucidate 79±2%, % of initial, old vs. young). the role of members of the VEGF family in hypoxic pulmonary This study illustrates that age (and/or) obesity causes plastic- angiogenesis. ity in an upper airway dilator muscle. Though intrinsic fatigability of the geniohyoid decreased significantly with age both in hyperoxia and hypoxia, it should be noted that the muscles from old obese rats generated more force under hypoxic conditions than young animals, and this was largely maintained during repeated stimulation. We conclude that increased pharyngeal collapsibility associated with age and obesity relates more to neurogenic than myogenic impairment. Supported by the Health Research Board, Ireland (RP/2006/140). ± Where applicable, the authors confirm that the experiments Figure 1: Mean ( SEM) percentage wound closure over a 24 hour period in response to VEGF A alone (8ng/ml), VEGF A and PlGF (40ng/ml or 160ng/ml). described here conform with The Physiological Society ethical * signifies a significant increase in wound healing compared to vehicle, sig- requirements. nifies significant increase compared to VEGF A alone (P<0.05, ANOVA, post hoc Student Newman Keuls). N=6 per group. Funding: Health Research Board and Higher Education Author- PC58 ity (PRTLI) VEGF family members can inhibit angiogenesis in the Where applicable, the authors confirm that the experiments hypoxic lung described here conform with The Physiological Society ethical requirements. M. Sands, K. Howell, C. Costello and P. McLoughlin School of Medicine and Medical Science, Conway Institute of Biomolecular and Biomedical Science, Dublin 4, Ireland PC59 We have recently shown, for the first time, that angiogenesis occurs in the pulmonary circulation in response to chronic Oxidant/antioxidant status in hypoxic rats after submission hypoxia, a common complication of chronic lung diseases. Much to deep hypothermia is known about the role of members of the vascular endothelial growth factor (VEGF) family in mediating neovascularisa- T. Carbonell, N. Alva and J. Palomeque tion in the systemic circulation. However, to date their role in Physiology, University of Barcelona, Barcelona, Spain hypoxia-induced pulmonary angiogenesis remains less clear. Male specific pathogen free Sprague Dawley rats (n= 7-8 per The use of deep hypothermia as a protective factor in hypoxia group) were exposed to normoxia or chronic hypoxia (10% O2) has been controversial. (Matthew et al., 2002; Riess et al., 2004). for 1, 3, 7, 14 and 21 days. Following the exposure period, the The purpose of this study was to describe the blood acid-base rats were deeply anaesthetised (70mg.kg-1 sodium pentobar- parameters and the profile related to oxidative stress, malon- bitone (i.p.)) and killed by exsanguination via the femoral ves- dialdehyde, nitric oxide and glutathione in plasma, in deep sels. The right lung was removed and flash frozen for later analy- hypothermic Sprague Dawley rats (21.5∞C)breathing room air sis of mRNA by real-time PCR. To examine the interactions of or hypoxic air (10% O2 in N2 ), compared with normothemic

110P Poster Communications animals also breathing room air or hypoxic air. Rats were anaes- Old (18-20 month), obese (902±33g), male rats were killed by thetized I.P. (intraperitoneally) with sodium pentobarbitone cervical spinal cord transection under 5% isoflurane. Ster- (60mg/Kg b.w.) and maintained with respiratory aid. The ani- nohyoid (SH) muscles were dissected and removed for study. mals were humanely killed with an I.P. overdose of anaesthetic. Isometric contractile and endurance properties were examined Data were analyzed by the two-way ANOVA using the Student- in physiological salt solution at 35OC under either hyperoxic ≤ Newman-Keuls test to identify significant differences (p 0.05). (95%O2/5%CO2) or hypoxic (95%N2/5%CO2) conditions in vitro. Hypoxia exposure results in an oxidative stress with an increase Experiments were carried out in the absence (control) or pres- in malondialdehyde, nitric oxide and a decrease in glutathione; ence of Tempol (10mM). Muscles were set to optimum length however, if hypothermia is previously applied the situation is (i.e. length producing maximum isometric force). Force was reversed with a stabilization of the malondialdehyde and an measured in response to electrical field stimulation at stimu- increase in the antioxidant glutathione (Table 1). lus frequencies ranging from 10-100Hz and was expressed as On the other hand, the determination of pH, Pco2 and the ratio a function of muscle cross-sectional area (i.e. specific force). [OH-/H+] discarded a respiratory imbalance but showed a mild We also examined muscle performance in response to repeated metabolic acidosis in the hypothermic groups (Table 2). stimulation (40Hz, 300ms, every 2 seconds for 2 minutes). We proposed metabolic acidosis as a mechanism which Hypoxia was associated with significant decreases in SH mus- explains this protection since it helps to keep the intracellular cle force [peak force was 27±2 vs. 18±1, mean±SEM N/cm2, reducing power, preserving glutathione and avoiding intra- hyperoxia (n=8) vs. hypoxia (n=8), p<0.05 ANOVA]. Tempol res- cellular alkalinisation. cued force in hypoxia-treated muscle strips [peak force = 24±2 Table 1. Plasma oxidative stress-indicators N/cm2, (n=8), p<0.05 vs. hypoxia]. Interestingly, the positive inotropic effect of Tempol was also observed in hyperoxia [32±2 N/cm2, (n=8), p<0.05 vs. hyperoxia] suggesting that the inotropic effect was not dependent on the bath PO2, and was more likely related to a beneficial effect of scavenging of free radicals produced by endogenous metabolism and muscle con- Values are the mean ± SEM traction. We observed improved muscle performance during Table 2. Acid base parameters the fatigue trial under both hyperoxic and hypoxic conditions, owing largely to increased force potentiation during the early phase of the trial in Tempol-treated muscles [e.g. 8±1 vs. 15±2 N/cm2, hypoxia (n=8) vs. hypoxia+Tempol (n=8) at 20s of the 2min trial, p<0.05]. Insummary,themainfindingofthisstudywasthatTempolhas a significant positive inotropic effect on SH muscle in old obese malerats.Pharmacotherapyisconsideredaviableclinicaloption Matthew, et al. (2002). Can J Physiol Pharmacol 80, 925-933. in the treatment of OSAS and agents that improve pharyngeal Riess, et al. (2004). Cardiovasc Res 61, 580-590. dilator muscle function might prove useful, since these mus- clesplayapivotalroleinthemaintenanceofpharyngealairway This work was supported by research grant PI 081389 calibre. We conclude that antioxidant treatment may be bene- Where applicable, the authors confirm that the experiments ficial as an adjunct therapy in the treatment of OSAS. described here conform with The Physiological Society ethical Supported by the Health Research Board (Ireland) requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC60

The superoxide scavenger Tempol improves pharyngeal dilator muscle function in old obese male rats PC61 J.R. Skelly and K.D. O’Halloran Respiratory muscle plasticity following chronic intermittent UCD School of Medicine and Medical Science, University College hypoxia in male but not female Wistar rats Dublin, Dublin 4, Ireland 1 2 1 Age, obesity and male sex are major risk factors for the devel- J.R. Skelly , A. Bradford and K.D. O’Halloran opment of obstructive sleep apnoea/hypopnoea syndrome 1UCD School of Medicine and Medical Science, University College (OSAS). Pharyngeal dilator muscle dysfunction is implicated in Dublin, Dublin 4, Ireland and 2Department of Physiology and the pathophysiology of OSAS. We wished to examine the effects Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, of acute hypoxia on contractile properties of an upper airway Ireland dilator muscle in old obese male rats. Furthermore, we wished to test the hypothesis that Tempol, a superoxide scavenger, Upper airway muscle dysfunction is implicated in obstructive would protect against hypoxia-induced impairment of pha- sleep apnoea – a debilitating respiratory disorder associated ryngeal dilator muscle function. with cardiovascular and neurocognitive impairments. Chronic

111P Poster Communications intermittent hypoxia (CIH), a central feature of sleep apnoea of pain processing given that noxious stimuli drive powerful results in oxidative stress/injury and tissue dysfunction. We motor responses, which have important consequences for wished to characterise the effects of CIH on ventilation and pha- survival. ryngeal dilator muscle function in male and female rats. We The aim of the current study was to examine the effects of neu- tested the hypothesis that sex differences would exist in the ronal activation in the ventrolateral periaqueductal grey effects of CIH on the respiratory system. (vlPAG), which is a source of descending control and is impli- Adult Wistar rats were exposed to CIH (90s air/90s N ; 2 cated in coordinating passive coping behaviours, on responses 5%O at nadir) or sham treatment for 8 hours/day for 9 days. A 2 of spino-olivary projection neurones in the dorsal horn to both subset of animals in both groups received chronic antioxidant innocuous and noxious peripheral stimulation. treatment in the form of the superoxide scavenger Tempol Experiments were carried out in alphaxalone-anaesthetised (1mM in the drinking water). Following treatments, ventilation (Alfaxan, 15-30mg.kg-1.hr-1, i.v) male Wistar rats (280-300g; was assessed by whole body plethysmography. Subsequently, n=12). Extracellularly recorded antidromically activated (Lip- the animals were killed humanely and isometric contractile ski, 1981) spino-olivary neurones were characterised by their properties of the sternohyoid (SH) muscle were examined at responses to peripheral stimulation and the effects of descend- 35OC under control (95%O /5%CO ) or hypoxic (95%N /5%CO ) 2 2 2 2 ing control following neuronal activation in the vlPAG were conditions in vitro. Force-frequency relationship was measured investigated. Application of low threshold (brush, tap, joint at stimulus frequencies ranging 10-100Hz. movement) and high threshold (noxious pinch) mechanical In male rats, ventilatory drive (V /T ) was significantly increased T i stimuli to receptive fields on the hindlimb revealed four classes [2.4±0.1 vs. 2.8±0.2 sham (n=7) vs. CIH (n=7), p<0.001, Stu- of neurones (Class 1, n=1; Class 2, n=4; Class 3, n=3; Class 4, dent’s t test]. SH peak force was decreased in CIH-treated male n=4; Menetrey et al, 1977). Responses of i) Class 2 and 3 neu- rats [23±1 vs. 16±1 N/cm2, sham (n=8) vs. CIH (n=8), p<0.001 rones to noxious pinch (3.6N), ii) Class 1 and 2 neurones to ANOVA]. Chronic antioxidant treatment with Tempol amelio- innocuous pinch (0.6N) and iii) Class 4 neurones to joint move- rated IH-induced muscle impairment. Conversely, in female ment were monitored before and after microinjection of an rats, CIH treatment had no effect on ventilatory drive or SH peak excitatory amino acid (50mM DL-homocysteic acid; 60-80nl) force [21±1 vs. 20±1 N/cm2, sham (n=8) vs. CIH (n=8), p>0.05 into the vlPAG at sites that evoked depressor responses. ANOVA]. Additionally, we noted that female SH muscle exhib- Activation of the vlPAG produced a significant decrease in nox- ited greater tolerance to low in vitro PO [force at 100Hz was 2 ious pinch-evoked responses of Class 2 and 3 neurones (to 21.0±1.2 vs. 18.5±2.0 N/cm2, hyperoxia (n=8) vs. hypoxia (n=8), 35.6%, P<0.05, and 1.2%, p<0.01, of control respectively; p>0.05 ANOVA] compared to male SH muscle [22.7±0.8 vs. Kruskal-Wallis). In contrast, activation in the vlPAG increased 9.5±0.6 N/cm2, hyperoxia (n=8) vs. hypoxia (n=8), p<0.001 the responses of Class 2 spino-olivary neurones to innocuous ANOVA]. pinch (to 123.5%; n=2). Interestingly, responses of Class 4 spino- The main finding of this study is that CIH causes functional olivary neurones to joint movement were significantly plasticity in the respiratory system. We observed CIH-induced increased (to 216%, p<0.001; Kruskal-Wallis) following acti- muscle dysfunction in male rats – an effect that was amelio- vation of the vlPAG. rated by antioxidant treatment. Female rats were unaffected The data reveal differential descending control exerted by the by CIH and female SH muscles showed greater hypoxic toler- vlPAG on responses of spino-olivary neurones to sensory stim- ance in vitro. This suggests a greater antioxidant capacity in uli of different behavioural significance, which could contribute females compared to males preventing hypoxic maladaptation to passive coping behaviours coordinated by the vlPAG. both in vivo and in vitro. Our results may have relevance to Lipski J (1981) Antidromic activation of neurones as an analytic tool in obstructive sleep apnoea, which is more prevalent in men than the study of the central nervous system. J Neurosci Methods 4:1-32. pre-menopausal women. Menetrey D, Giesler GJ Jr, Besson JM (1977) An analysis of response Supported by the Health Research Board (Ireland). properties of spinal cord dorsal horn neurones to nonnoxious and noxious stimuli in the spinal rat. Exp Brain Res. 27:15-33. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical This work was supported by the BBSRC. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC62

Midbrain control of spino-olivary neurones: a role in passive coping behaviour? J.L. Leith, S. Koutsikou, R. Apps and B.M. Lumb Department of Physiology & Pharmacology, University of Bristol, Bristol, UK Studies of descending control of spinal nociception have focused primarily on sensory and autonomic functions and have neglected investigation of the control of pain pathways that convey inputs to motor control centres, such as the olivo- cerebellar system. This is a critical gap in our understanding

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Fitzpatrick R & McCloskey DI (1994). Proprioceptive, visual and vestibu- PC63 lar thresholds for the perception ofsway during standing in humans. J Physiol, 478, 173-186. Vestibular and somatosensory influences on the position Fitzpatrick R, Rogers DK & McCloskey DI (1994). Stable human stand- of balance during human standing ing with lower-limb muscle afferents providing the only sensory input. A. Butler and R. Fitzpatrick J Physiol, 480, 395-403. Prince of Wales Medical Research Institute, Randwick, NSW, NH&MRC Australia funded research. Australia Where applicable, the authors confirm that the experiments Studies in which subjects balanced a ‘virtual body’ to exclude described here conform with The Physiological Society ethical vestibular input suggest that proprioceptive input from the requirements. legs is sufficient to stand (Fitzpatrick et al., 1994) and that vestibular inputs normally play no part in controlling body sway (Fitzpatrick & McCloskey, 1994). However, there is no PC64 uniquepatternofsomatosensoryinputfromthelegsthat signals the vertical alignment of the body. The implication Effect of b-amyloid injection in hippocampus or olfactory that vestibular inputs are not involved with balance control bulb in an animal model is inconsistent with clinical experience, studies of reflex R. Guevara-Guzman and C. Bernal responses to balance perturbations (Allum & Pfaltz, 1985) and vestibular stimulation (Cathers et al., 2005). Thus, it Fisiologia, Universidad Nacional Autonoma de Mexico, Mexico, seems that the vestibular system has a role in balance con- Distrito Federal, Mexico trol but not one concerned with controlling the extent of Alzheimer disease (AD) is the most common cause of demen- body sway. tia in old age. It is a public health problem in several countries. This study uses the virtual-body method to investigate the pro- β-amyloid is a neurotoxic peptide that forms neuritic plaques prioceptive and vestibular contribution to human standing. in the brain of AD patients. Nevertheless, this impairment in Twelve healthy adults participated in this study that was memory is not the first symptom, as it is preceded by an olfac- approved by the Human Research Ethics Committee of the Uni- tory deficit which also appears in some other neurodegenera- versity of New South Wales. Balance was assessed under three tive diseases that are into dementia. Olfactory impairment conditions; (i) normal standing, (ii) splinted standing to pre- caused by in situ β-amyloid injection has not been assessed in vent rotation of joints above the ankle and (iii) balancing a vir- an animal model, neither the correlation of neuronal impair- tual body (inverted pendulum), which simulated standing but ment in different sites of injection: olfactory bulb (OB), hip- excluded vestibular and graviceptive inputs. In each condition, pocampus (HIPP) and entorrinal cortex. Therefore, the aim of subjects were tested with and without additional weight this research work is to assess if the β-amyloid peptide injected attached to the body or pendulum. Sway, alignment angle and in the olfactory bulb (Group 1) or hippocampus (Group 2) of ankle torque were recorded. the rat (under chloral hydrate (400 mg/kg, i.p.) anaesthesia) While standing normally, the position of the centre of mass produces alteration in the social, olfactory and spatial mem- of the body was approximately over the centre of the perime- ory. To assess olfactory behavior, we carried out different olfac- terofthefeet.Thiswasmaintainedwhena30kgweightwas tory tests, in addition to other behavioral and biochemical tests. Δ ± fastened around the pelvis ( mean: 2.0 4.4 mm). In con- We detected olfactory behavior impairment in the group trast, adding 30 kg to the virtual body with vestibular input injected in the hippocampus in regards to the control group. unavailable caused the centre of mass to shift back towards The olfactory tests showed: a) inability to find a chocolate chunk Δ ± the ankles ( mean: 24.3 1.5 mm; P < 0.001). With only hidden during the search test; b) inability to discriminate somatosensory input available, subjects balanced a lightweight between the two scents in a discriminating test. However, not virtual body over the front of the feet, their matched virtual significant difference in the investigation time during the two body over the centre of the feet and a heavyweight virtual body encounters of the social recognition test was found. A similar over the heels. These positions did not keep constant the cen- increase of lipoperoxidation was showed, not only in the HIPP, tre of foot pressure, muscle force, ankle angle or the excursion but also in the entorrinal cortex and OB in group 2; whereas of sway. group 1 showed higher levels of lipoperoxidation in OB than in It is concluded that vestibular and graviceptive sensory input, the HIPP. although not used to control sway during standing, has a unique We may infer that the β-amyloid injection in HIPP produces role in aligning the body’s centre of mass above the feet, a func- olfactory impairment in the rat throughout oxidative stress tion that cannot be achieved by proprioceptive inputs alone. process in this structure, which extends to the OB and the entor- Allum J & Pfaltz C (1985). Visual and vestibular contributions to pitch rinal cortex. The doses injected in OB did not cause an exten- sway stabilization in the ankle muscles of normals and patients with sive impairment in the olfactory behavior as the one in HIPP. bilateral peripheral vestibular deficits. Exp Brain Res, 58, 82-94. Britton T, Day B, Brown P, Rothwell J, Thompson P & Marsden C GRANTS:IN-216907, 24784-M and SDEI-PTID05.5. (1993). Postural electromyographic responses in the arm and leg Where applicable, the authors confirm that the experiments following galvanic vestibular stimulation in man. Exp Brain Res, 94, described here conform with The Physiological Society ethical 143-151. requirements. Cathers I, Day BL & Fitzpatrick RC (2005). Otolith and canal reflexes in human standing, J Physiol, 563, 229-234.

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Where applicable, the authors confirm that the experiments PC65 described here conform with The Physiological Society ethical requirements. Descending modulation of cool- and cold-evoked spinal nociception by the periaqueductal grey PC66 S. Koutsikou, L. Leith, B. Lumb and R. Apps Physiology & Pharmacology, University of Bristol, Bristol, UK Stimulation of the periaqueductal grey modulates cortical The physiology of cold somatosensation has received much somatosensory evoked potentials elicited by an acute recent interest, and many studies have investigated the noxious stimulus peripheral mechanisms of cool and cold detection. However E.K. Davies1, S. Koutsikou1, B.M. Lumb1 and J.C. Murrell2 little is known of the central processing of cool and cold- evoked responses, nor whether these responses may be mod- 1Department of Physiology and Pharmacology, University of Bristol, ulated by descending control systems that have profound Bristol, UK and 2Department of Clinical Veterinary Science, effects on the processing of other sensory modalities. In par- University of Bristol, Bristol, UK ticular, descending control that originates from the midbrain periaqueductal grey (PAG) is an important determinant of Theperiaqueductalgrey(PAG)playsakeyroleindescending the pain experience. The current study aimed to further control of spinal nociception, and therefore the pain experi- investigate the responses of spinal dorsal horn neurones to ence. It is functionally divided into longitudinal columns cool and cold stimuli in the rat and to determine whether (including the dorsolateral (DL) and ventrolateral (VL) PAG) their activity may be modulated by descending control from which, when activated, produce anti-nociception and distinct the PAG. behavioural responses, characterised as active and passive cop- Extracellular recordings were made from lumbar dorsal horn ing strategies respectively (Lovick and Bandler, 2005). The neurones with receptive fields on the hind limb in alphaxalone- effects of PAG stimulation at the level of the spinal dorsal horn anaesthetised (Alfaxan; 15-30mg.kg-1.hr-1, i.v.) male Wistar have been widely investigated, but concurrent changes in cor- rats (280-300g; n=19). Cells were characterised according to tical activity are unknown. The aim of this study was to inves- their responses to low (brush, tap) and high (pinch) threshold tigate whether PAG stimulation modulates the cortical mechanical stimulation applied to the receptive field of the cell response to an acute noxious stimulus, using somatosensory and classified as class 1 (low threshold, n=3), class 2 (wide evoked potentials (SEPs) as the outcome measure. DL- and VL- dynamic range, n=28) and class 3 (nociceptive-specific, n=6; PAG were stimulated independently to identify whether these Menetrey et al, 1977). Cells were then tested for responsive- regions, known to cause distinct behavioural responses, also ness to thermal stimuli: acetone (‘cool’), ethyl chloride (‘cold’; differentially modulate the cortical response to an acute nox- both 1ml topically) and noxious heat (55∞C water). In class 2 ious stimulus. cells that responded to acetone and/or ethyl chloride, the Eight male Wistar rats (280-320g), anaesthetised with an I.V. effects of activation of the ventrolateral (vl)-PAG were tested. infusion of alfaxalone (~25mg.kg-1.hr-1) were studied. SEPs After 3 control responses to acetone or ethyl chloride (applied were recorded from four active dural electrodes, placed bilat- at 5min intervals), 60-80nl of D,L-homocysteic acid (DLH; 50mM erally over the primary somatosensory cortex (S1) and the ver- in physiological saline saturated with pontamine sky blue dye tex (Vx) (2.5mm caudal, 2.5mm lateral to bregma and 4.5mm to mark injection sites) was pressure injected under stereotaxic caudal, 1mm lateral to bregma, respectively). Ground and ref- guidance into the vlPAG. Test responses to acetone or ethyl erence electrodes were placed over left and right frontal sinuses chloride were measured 10s after DLH injection and 3 further respectively (10mm rostral, 1mm lateral to bregma). SEPs were recovery responses to acetone or ethyl chloride were measured evoked by noxious electrical tail stimulation, and amplitudes at 5 min intervals. of a positive to negative waveform in the 10-30ms range were Of the recorded units, 67% of class 1, 64% of class 2, and 33% of assessed before and after chemical stimulation of VL- or DL-PAG class 3 cells responded to acetone, and 100% of class 1, 68% of using DL-homocysteic acid (DLH; 150nl; 50mM in physiologi- class 2, and 33% of class 3 cells responded to ethyl chloride. In cal saline saturated with pontamine sky blue dye to mark injec- class2cells,activationofthevlPAGbyDLHsignificantlyreduced tion sites). ethyl chloride-evoked responses to 17±7% of control DL-PAG stimulation significantly increased waveform ampli- (mean±S.E.M.;n=8;p<0.05,Kruskal-Wallis).Howeveracetone- tude recorded from both S1 and Vx (to peaks of 118.6±7.5% evoked responses in class 2 cells were not significantly reduced and 115.1±6.1% of baseline respectively, mean±S.E.M., both (92±36%;n=7;p>0.05,Kruskal-Wallis)byactivationofthevlPAG. p<0.05, ANOVA + Tukey’s post-test, n=14, 8) with no signifi- The data show that cold-evoked responses in class 2 dorsal horn cant difference between ipsilateral and contralateral recording neurones, like other sensory modalities, can be modulated by electrodes (p>0.05, t-test, n=7, 4 for S1 and Vx respectively). descending control systems that originate in the PAG. Conversely VL-PAG stimulation did not significantly alter the waveform amplitude recorded from S1 (p>0.05, ANOVA + Menetrey D, Giesler GJ, Jr., Besson JM (1977) An analysis of response properties of spinal cord dorsal horn neurones to nonnoxious and nox- Tukey’s post-test, n=11), but significantly decreased the wave- ious stimuli in the spinal rat. Exp Brain Res 27:15-33 form amplitude recorded from Vx (75.0±6.3% of baseline, mean±S.E.M., p<0.01, ANOVA + Tukey’s post-test, n=6). This work was supported by the BBSRC. These data show that stimulation of VL- and DL-PAG, to mimic activation occurring in response to environmental stressors,

114P Poster Communications differentially modulate cortical responses to acute noxious stim- (2) and/or enhancement of GABA effect currents in cultured uli, which may in turn affect the behavioural response. rat sensory neurons. Lovick T, Bandler R (2005) The organization of the midbrain periaque- Robinson DA, Wei F, Wang GD, Li P, Kim SJ, Vogt SK, Muglia LJ, Zhuo M. ductal grey and the integration of pain behaviours. In: The neurobiol- Oxytocin mediates stress-induced analgesia in adult mice. J Physiol ogy of pain (Hunt S, Koltzenburg M, eds), pp 267-287. 2002; 540:593-606. Yang Q, Wu ZZ, Li X, Li ZW, Wei JB, Hu QS. Modulation by oxytocin of Funded by an MRC-GSK Case Studentship ATP-activated currents in rat dorsal root ganglion neurons. Neu- ropharmacology 2002; 43:910-6 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

PC67 PC68 Oxytocin provokes increase of free intracellular Ca2+ levels in freshly isolated rat sensory neurons Spinorphin Inhibits Membrane Depolarisation-Induced M. Ozcan1, S. Kutlu2, E. Alcin2, B. Yilmaz3, H. Kelestimur2 and Intracellular Calcium Signals in Cultured Rat Dorsal Root A. Ayar4 Ganglion Neurons 1 2 3 2 1Biophysics, Firat University Medical School, Elazig, Turkey, A. Ayar , M. Ozcan , T. Kuzgun and E. Alcin 2 Physiology, Firat University Medical School, Elazig, Turkey, 1 3 Physiology, Karadeniz Technical University, Faculty of Medicine, Physiology,YeditepeUniversityMedicalSchool,Istanbul, 2 4 Trabzon, Turkey, Biophysics, Faculty of Medicine, Firat University, Turkey and Physiology, Karadeniz Technical University, Elazig, Turkey and 3Physiology, Faculty of Medicine, Firat University, Trabzon, Turkey Elazig, Turkey

Although the cellular mechanisms remain unclear, available Spinorphin, an endogenous peptide with antagonist actions evidence indicates that beside its essential role in mam- on enkephalin-degrading enzymes and P2X3 receptors, malian parturition and lactation the nonapeptide hormone presents potential antinociceptive effects. It is known that the oxytocin (OXT) plays an important role in nociceptive mod- primary afferent sensory neurons are functionally heteroge- ulation (1). Evidence has accumulated that free intracellu- neous and small sized subpopulation of cultured DRG neurons lar calcium ([Ca2+]i) plays an important role in signal trans- may serve as a cellular model for studying the peripheral noci- duction to control a wide variety of cellular mechanisms ception. The aim of the present study was to determine the including nociceptive transmission. Previous studies have effects of spinorphin on Ca2+ transients, evoked by high-K+ also shown that OXT receptor is expressed in dorsal root gan- (30 mM), and whether there were differences in spinorphin glia (DRG) and in spinal dorsal horn. Hence, the present study effect among subpopulation of cultured rat dorsal root gan- was undertaken to investigate the possible effects of OXT glion (DRG) neurons. Following enzymatic digestion and on antinociception in freshly isolated rat dorsal root gan- mechanical agitation the DRG neurons were cultured on coated glion (DRG) neurons. coverslips and loaded with 5 uM Fura-2 AM. Standard fura-2 Isolated DRG neurons were plated on coated cover slips fol- ratiometric technique was utilised for quantifying [Ca2+]i lowing mechanical isolation and responses to OXT was stud- responses in individual DRG neurons using fluorescence imag- ied by monitoring changes in [Ca2+]i with a microscopic ing system consisting of CCD camera coupled to an inverted digital image analysis system in fura-2 loaded single neu- microscope with a 40x (1.30 NA) objective. All data were ana- rons. Data were analyzed by using unpaired t test, with a lyzed by using unpaired t test, P <.05 defining statistical signi- 2-tailed P level of <0.05 defining statistical significance. The ficance. Spinorphin, inhibited the Ca2+ transients evoked with majority of the DRG neurons (small, medium and large) 30 mM K+ in concentration dependant manner in a subpopu- responded by increase in [Ca2+]i to extracellular applica- lation of sensory neurons. Spinorphin dose dependently inhib- tion of OXT concentration-dependently. The mean 340/380 ited the HiK+-induced [Ca2+]i responses (1.40±0.09 vs. ± ± nm ratio was 0.80 0.04 (baseline, n=7), 0.92 0.06 (30nM 1.42±0.08, n=15, NS for 10 uM;1.45±0.09 vs. 0.91±0.08 for ± OXT, P<0.05, n=7) and 0.81 0.04 (baseline, n=27), 100 uM spinorphin n=16, P<0.05; and 1.39 ±0.09 vs. 0.84 ±0.08 ± 1.29 0.07 (100nM OXT, P<0.001, n=27), respectively. The for 300 uM spinorphin n=20, P<0.05, respectively) only in stimulatory effect of OXT (100nM) was persistent in Ca2+- small-diameter DRG neurones. Additionally, after application ± free conditions (Baseline: 0.90 0.07 vs. 100nM OXT: of spinorphin a significant percent of small-diameter nocicep- ± 1.06 0.06, n=5). tive DRG neurones did not respond to stimulation by HiK+ We demonstrated first time that OXT evoked a robust (response rate after application of 10, 100 and 300 uM spin- increase of [Ca2+]i in freshly isolated rat DRG neurons. orphin: 95%, 55%, and 54%, respectively) while the percent- Although the physiological significance of the results age of the response was not significantly changed in large- obtained from this study remains to be elucidated, in agree- diameter non-nociceptive DRG neurones. Results from this ment with previous studies our results indicates that OXT study indicates that spinorphin significantly inhibits calcium could modulate the somatosensory transmission (nocicep- signalling, transient changes in free intracellular Ca2+ con- tive or non-nociceptive) via inhibiting ATP receptor function

115P Poster Communications centration which are key for the modulation of cell membrane Wheater, C P, Langan,A M and Dunleavy, P J (2005) Students assessing excitability and neurotransmitter release, in only small-diam- students: case studies on peer assessment, Planet No 15, 13-15 eter nociceptive subpopulation of DRG neurons and this Where applicable, the authors confirm that the experiments endogenous agent may be effective analgesic with potential described here conform with The Physiological Society ethical combination with opioids. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC70

The Virtual Microscope in Histology Teaching: Evaluation of Effectiveness and Student Preference PC69 F.M. MacMillan, S.D. Barnes, J.C. Hamilton, I.S. Steane and P.D. Langton Validity of student peer assessment Physiology and Pharmacology, University of Bristol, Bristol, UK A.J. Al-Modhefer and L.E. Montgomery At the University of Bristol histology is taught alongside phys- Centre for Biomedical Sciences Education, Queens University Belfast, iology and has traditionally been taught in practical classes Belfast, UK using light microscopes (LM) and glass microscope slides of tissue specimens. As part of the ‘The Applied and Integrated Med- The assessment of students by their peers has been posited as ical Sciences Centre for Excellence in Teaching and Learning’ a useful means of class evaluation, giving students an insight (AIMS CETL) we have developed a virtual microscope (VM) to into the marking process, in addition to the mark received. This deliver digital scans of our collection of histological specimens is often appropriate in assessing group work in oral and poster via the internet. Users are able to navigate around the virtual presentation, and is particularly valuable if both product and slides at a range of magnifications on networked computers process are assessed (Race et al, 2005). Van den Berg et al (2006) using a software application ‘Digital Slidebox’ (Slidepath, point out that it is also useful in that it allows the students to Dublin). The VM was introduced for histology teaching within work with colleagues in a way that they will do during their pro- the department from 2006. fessional career. Wheater et al (2005) demonstrated that results Our previous study (MacMillan et al. 2008) on first year veteri- from peer assessment are reproducible and comparable with nary science students, with previous experience of the LM and the equivalent staff results, helping to allay fears that students VM, demonstrated that learning outcomes are not hindered by will ‘over-mark’ or attribute marks based on their personal feel- the use of the VM over the LM and may well be enhanced. ings towards other students. The aim of this study was to com- In the current study we investigated the effectiveness of the pare the marks given by staff and students for the same pieces LM vs VM in a group of first year BSc student volunteers with of work at this institution to see how similar they were. limited previous experience of LM and no experience of using The pieces of work involved were poster presentations in a sec- the VM. ond year Physiology/Pharmacology module, and oral presen- Student volunteers (n=27) were given a prior knowledge test tations given as part of a third year Neuroscience Module. based on identification of structures in two histological images Results are expressed as mean ± standard error, and an unpaired taken from the virtual microscope (spinal cord and lung tissue). t-test was used to compare data. Results were accepted as being Thestudentswerethendividedintotwogroups(n=13and14), statistically significant at the 95% level. with both groups undertaking self-guided tutorials on the spinal Staff marks are significantly higher than those given by students cordandlunghoweveronegroupusedtheLMforthespinalcord in both Neuroscience oral presentations, (n=50, 73.6±0.7 Vs tutorialandtheVMforthelungtutorialandthesecondgroupdid 68.9±0.4), and Physiology/Pharmacology poster presentations, thereverse.Oncompletionofthetutorialsthestudentsre-satthe (n=32, 70±0.6 Vs 67.4±0.6). The results clearly indicate that prior knowledge tests and completed an opinion questionnaire. advanced students tend to ‘under’ mark each others work in The students’ test scores improved following the learning ses- comparison to academic staff. Fears that students maybe exces- sions and the improvement in scores was greater when the stu- sively lenient have proven unfounded. Similar trends have been dents had used the VM for the tutorial rather than the LM. The shown in at least two other studies, both Heywood (2000) and scores were tested for statistical significance using a one way Stefani (1994) found peer grading to be somewhat lower than analysis of variance and Tukey’s post comparison test. There staff grading. This merits further investigation to discover why was a significant improvement in the scores in the group doing this is the case, to enhance the learning process and facilitate the lung tutorial on the LM (p<0.05) and to a greater degree of any adjustments required in the guidance on attributing marks. confidence in the group doing the lung tutorial using the VM Heywood, J (2000) Assessment in Higher Education, London, Jessica (P<0.01). There was also an increase in student scores follow- Kingsley Publishers ing the tutorial on the spinal cord, the increase was significant Race P, Brown S & Smith B (2005) (2nd ed) 500 tips on assessment Lon- for the group using the VM (P<0.01). don: Routledge Falmer The questionnaire results show that the students considered Stefani, L.A.J (1994) Peer, Self and tutor assessment : relative reliabili- the VM to greatly improve the opportunity for group learning ties, Studies in Higher Education, 19 (1) 69-75 and that it was more useful as a learning tool than the LM. Van den Berg I, Admiraal W, Pilot, A (2006) Peer assessment in univer- In summary, the improvement in test scores following the tuto- sity teaching: evaluating seven course designs, Assessment and Eval- rials provides evidence that the VM is a more effective learning uation in Higher Education, Vol.31, No.1, 19-36 tool than the LM. Questionnaire data demonstrates that the

116P Poster Communications students find the VM easier and more enjoyable to use than the their scores in a physiology test delivered before and after the LM when studying histology. teaching session (n=119). The undergraduate students enjoy F.M. MacMillan et al (2008). Proc Physiol Soc 11 PC50 the challenge of designing, delivering and evaluating rigorous, Where applicable, the authors confirm that the experiments ‘stand-alone’ teaching material in the MTU, and devising an described here conform with The Physiological Society ethical appropriate related biometric research project. requirements. Ljustina-Pribic R, Roncevic, N and Milutinovic B (2001) Reference values of ventilatory variables for healthy adolescents aged 15-19 years. Central European Journal for Public Health, 9(1) 46-48. PC71 Wilkinson RT and Allison S (1989) Age and simple reaction time: Decade differences for 5,325 subjects. Journal of Gerontology, 44(2) 29-25. Final Year Undergraduate Teaching Projects Delivered via a Mobile Teaching Unit L.K. Hughes, K. Healey, R.E. Hinton, A.R. Lillie, P. Rickard and Where applicable, the authors confirm that the experiments J.R. Harris described here conform with The Physiological Society ethical requirements. Physiology & Pharmacology, University of Bristol, Bristol, UK Final year projects that enable students to teach physiology to school age pupils develop communication skills that are of widespread value, especially for careers in education, medicine and PC72 communicating science to the general public. Since 2007, we have offered final year projects that allow stu- Validating the Human Patient Simulator (HPS) as an dents to develop an A-level teaching session that takes place educational tool: A comparison of the responses to inside a Mobile Teaching Unit (MTU), a custom-built HGV lorry intravenous administration of adrenoceptor agonists with that expands into a classroom. The benefits of this arrangement human data are that teaching can be delivered in a self-contained unit that does not require resources from the school and allows univer- P. Maskell, E. Lloyd and R.J. Helyer sity-level physiological recording equipment to be easily trans- Department of Physiology and Pharmacology, University of Bristol, ported and set up on the school site. Bristol, UK Project students work in pairs and choose a physiological topic of interest that is relevant to their studies and maps onto the The Human Patient Simulator (HPS) (HPS 337; METI, Sarasota, school curriculum. They then design a 1-hr teaching session Florida) is a high fidelity mannequin controlled by computer using a combination of slides, videos, audioclips, demonstra- software. We have previously shown how the HPS can be used tions and practical activities undertaken by the school pupils. to demonstrate sympathetic and parasympathetic control of Each project culminates in the MTU visiting a local school, where the cardiovascular system. (Maskell et al. 2008). The aim of the project students deliver the teaching session to different this study was to compare the response of the HPS to the sim- pupil groups throughout the school day. ulated intravenous administration of the adrenoceptor ago- Each project must include experimental elements. These nists, noradrenaline, adrenaline and isoprenaline with pub- include collecting biometric data from the pupils, which the lished Human data (Allwood et. al. 1963) in order to validate project students subsequently analyse and set within the con- the HPS as an educational tool for teaching cardiovascular phys- text of relevant scientific literature. The undergraduates are iology. also required to devise ways of quantitatively evaluating the Baseline measurements were made of heart rate (HR), systolic teaching session in terms of pupils’ learning and enjoyment. (SBP), diastolic (DBP), mean arterial (MAP) blood pressure and Two teaching sessions, one each on the respiratory and nerv- peripheral vascular resistance (PVR). The responses were deter- ous systems, have been developed and evaluated to date. mined to the simulated intravenous administration of each of School pupils were supervised by the undergraduates in gen- the three adrenoceptor agonists. They were infused at doses erating data to investigate respectively the correlation between of 10mg/min for 15 minutes. Measurements of the variables height and forced vital capacity (FVC) (Ljustina-Pribic et al, were made at intervals of five seconds. 2001) and between age and reaction times (Wilkinson & Alli- The infusion of noradrenaline resulted in appropriate increases son, 1989) in 16-18 year olds. Subsequent analysis revealed a in SBP, DBP, MAP and PVR but did not produce an appropriate positive correlation between height and FVC (r=0.755; p<0.01; decrease in HR. n=46; Pearson’s correlation) and a negative correlation between The infusion of adrenaline resulted in appropriate changes in age and auditory reaction times (r= -0.443; p<0.001; n=66; HR, SBP, MAP and PVR but did not produce an appropriate Spearman’s rank correlation). Additional reaction time data decrease in DBP. ± presented as mean SEM demonstrated that auditory reaction The infusion of isoprenaline resulted in appropriate changes ± times were faster than visual reaction times by 0.04 0.007 s in HR and PVR but did not produce appropriate changes in SBP, (n=66) p<0.001. DBP or MAP. Feedback collected from pupils and teachers demonstrates a We conclude that the HPS is a potential tool for demon- high level of engagement in the MTU teaching sessions, with strating responses to the adrenoceptor agonists noradren- 91% of pupils stating that they enjoyed the sessions (n=111). aline, adrenaline and isoprenaline, but further work is School pupils also benefit academically, with 94% improving required to improve the software pharmacodynamic and

117P Poster Communications baroreceptor to improve the validity of the HPS as an edu- upon PACO2 under conditions of external mechanical ventila- cational tool. tion with 100% neuromuscular blockade. Maskell, P.D., Brandom, K.; Lloyd, E., & Robinson E. J. (2008). Using a We wish to acknowledge the technical support of Mr Peter human patient simulator to demonstrate the autonomic control of the cardiovascular system. Proc Physiol Soc 11, PC45 Dickens. HPS funding as part of the AIMS CETL was provided by HEFCE. Allwood, M.J., Cobbold, A.F. & Ginsburg J (1963). Peripheral vascular effects of noradrenaline, Isopropylnoradrenaline and dopamine. Brit. Where applicable, the authors confirm that the experiments med. Bull. 19 (2) 132-136. described here conform with The Physiological Society ethical requirements. We wish to acknowledge the HEFCE funding that established and supports the Applied and Integrated Medical Sciences Cen- tre for Excellence in Teaching and Learning (AIMS CETL), through PC74 which this study was carried out.

Where applicable, the authors confirm that the experiments Using a virtual microscope to link structure to function in described here conform with The Physiological Society ethical undergraduate histology teaching requirements. S.D. Barnes, P.M. Headley, J.R. Harris, J.A. Mitchell and P.D. Langton

Physiology and Pharmacology, University of Bristol, Bristol, UK PC73 The virtual microscope (VM) is core project within the Applied Validating the Human Patient Simulator (HPS) as an and Integrated Medical Sciences (AIMS) Centre for Excellence educational tool: determining the relationship between in Teaching and Learning (CETL). In collaboration with Slide- alveolar ventilation and the alveolar partial pressure of path (Dublin) we have created a digital archive of ~300 histo- carbon dioxide logical images by scanning our existing glass-mounted tissue sections. We have previously reported how we have embed- E. Lloyd1,2, G. Coverdale1, A. Thompson1 and R. Helyer1,2 ded the use of the VM within our laboratory teaching sessions (MacMillan et al., 2008). 1Department of Physiology and Pharmacology, University of Bristol, In 2007-08, the VM was used in histology classes for around Bristol, UK and 2AIMS Centre for Excellence in Teaching & Learning, 900 undergraduates, consisting of first and second year med- University of Bristol, Bristol, UK ical, dental and veterinary students. In Bristol undergraduate The inversely proportional relationship between alveolar ven- histology has historically been coordinated and largely staffed tilation (VA) and the alveolar partial pressure of carbon dioxide by the department of Physiology and Pharmacology. It is also (PACO2) is a fundamental principle of respiratory physiology our policy that where possible, academic staff who give a par- and the practice of anaesthesia. PACO2 = Vco2 x K/VA where K ticular series of lectures also run the associated histology prac- is a constant and Vco2 is the rate of production of carbon diox- tical(s). The purpose of this is to facilitate the integrated teach- ide (equation 1). The Human Patient Simulator (HPS) was devel- ing of function and structure. To encourage students to reflect oped by anaesthetists for trainees to learn about the clinical on structure-function relations, we have developed a series of practice of anaesthesia in a safe environment. The aim of this VM-based formative on-line quizzes, using the Digital Slidebox study was to determine how PACO2 varies with alveolar venti- (DSB, Slidepath) software, that test student’s understanding lation in the HPS. of structure-function relationships as well as recognition of cells The manikin was intubated under complete neuromuscular and tissues. Quizzes are scheduled at the beginning or the end blockade and connected to a mechanical ventilator (Siemens of laboratory sessions and we are currently evaluating the 900C Servo Ventilator) set to deliver 21% Oxygen. The minute impact of this difference on measures of student performance. volume (VM) was adjusted by altering the respiratory rate and In quizzes, cells or structures are annotated and labelled alpha- tidal volume and PACO2 was recorded after 20 minutes. Phys- betically as ‘items’ and quizzes constructed to be similar to iological dead space was determined using the Bohr method extended matching questions, e.g.: and used to calculate alveolar ventilation (alveolar ventilation 1. Which of the structures indicated can conduct action poten- = [tidal volume – physiological dead space] x respiratory rate) tials at high speeds? (equation 2). 2. Which item indicates cells which are a source of platelets? The relationship between alveolar ventilation and the PACO2 3. Which item indicates epithelial cells understood to be spe- in the HPS showed good correspondence with the theoretical cialised for antigen uptake? relationship predicted by the alveolar ventilation equation 4. Which item identifies an area of lymphoid tissue undergo- (equation 1). The limitations of the study include the assump- ing B cell clonal expansion? tions that the rate of carbon dioxide production remained con- 5. Which item identifies an area of tissue with an exocrine stant throughout the experiment and that twenty minutes was function? sufficient to achieve a new steady state following a change in Foreachquestiontheremaybeupto15optionslabelledA, minute volume. B, C etc. We conclude that the HPS is a valid educational tool for demon- Staff regularly analyse cohort results and provide further form- strating the effects of hypoventilation and hyperventilation ative feedback that focuses on features that students have dealt with poorly. In addition, some summative assessments that 118P Poster Communications were previously based on light microscopy are now delivered Fitness was positively correlated with volunteered weekly hours in a format based on these formative quizzes. We have found of physical activity. Grades and weekly hours of physical activ- the VM to support learning with increasing grades despite more ity were positively correlated for males (Pearson; p<0.05; R 0.73, challenging examinations and to be highly popular with stu- R2 0.53), but negatively correlated for females (Pearson; dents (MacMillan et al., 2008). The 2008 mid-session exami- p<0.05; R -0.43, R2 0.19). Interestingly, there was a difference nation in first year medicine returned an average of 72% with in response of males and females to the questionnaire state- a range from 20% to 100% (n=229). ment, ‘I exercise to loose weight’, with the median response Our findings support the principle of linking the teaching of being 6 for females and 0 for males (10 = agree completely) structure (histology) with function (physiology). (Mann Whitney U; p<0.001). The negative correlation for the MACMILLAN, FM, et al. (2009). Proc Physiol Soc 11 PC50 questionnaire response of females that they are ‘on top of their school work’ and fitness (Pearson; p<0.05, R-0.45), suggests We acknowledge HEFCE for funding and Mrs Debbie Martin that for females exercise is linked to body image. and Mrs Debi Ford for technical support. These data do not support a universal view that physical exercise and fitness correlate positively with academic achievement. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Yusuf S et al. (2004). Lancet 364: 937–52. requirements. Trudeau F, & Shephard RJ. (2008). Int J Behav Nutr Phys Act. 200 25;5:10. Chomitz VR, et al. (2009). J Sch Health. 79(1): 30-7.

The authors acknowledge the help and support of St. Mary PC75 Redcliffe School, Mr David Gee, Mr Pete Dickens and Mr Mar- tin Thirkettle Correlations between physical activity and academic Where applicable, the authors confirm that the experiments performance amongst A-level students: An Undergraduate described here conform with The Physiological Society ethical Ambassador Scheme research project requirements. P.D. Langton, L. Barnfield, A. Du-Preez and M.H. Randall Physiology and Pharmacology, University of Bristol, Bristol, UK PC76 The Undergraduate Ambassador Scheme (UAS) aims to improve achievement in science, technology, engineering and maths The response to hypoxia: a refined Human Patient Simulator (STEM), increasing the proportion of school leavers studying (HPS) model to demonstrate high altitude physiology STEM subjects in University. UAS students in our department R. Helyer and E. Lloyd also conduct a research project. Obesity is a huge social and economic problem and is set to University of Bristol, Bristol, UK become the largest cause of ‘ill health’ ahead of malnutrition The Human Patient Simulator (HPS; METI, Sarasota, Florida) and infectious diseases in developed countries (Yusuf S, 2004). has a computer driven mechanical lung and gas exchange Contributory factors include relatively low food costs, increas- mechanism, designed to model the human respiratory system. ingly sedentary leisure activities and, for school children, We have previously demonstrated that the HPS is a useful edu- reduced emphasis on competitive ‘team’ sports in schools. The cational tool for demonstrating physiological responses to findings that physical fitness is correlated with academic changes in the environment such as hypoxia, but that the model achievement (Chomitz et al., 2009) and that taking time from requires some adjustment in order to more accurately repre- academic activities to favour physical activity does not reduce sent human data (Helyer et al, 2008). measures of academic outcome (Trudeau and Shephard, 2008) The aim of this study was to refine the response of the HPS to led us to question how physical activity impacts positively on hypoxia to improve the correspondence with human data in academic performance. order to improve the validity of the HPS for teaching high alti- Participating A-level students were allocated numbers used in tude human physiology. all data collection so UAS students remained blinded to the In our previous study we constructed an Oxygen-Carbon Diox- pupils’ identities. Using these numbers and an on-line ques- ide diagram for the HPS that demonstrated a linear relation- tionnaire, data was gathered on average hours of sleep, history ship between PAO2 and PACO2 over the entire range investi- of recent and regular exercise, extracurricular activity and alco- gated (Helyer et al, 2008). In comparison unacclimatised hol consumption as well as the student’s subjective assessment humans show a linear relationship between PAO and PACO of their academic ability/confidence, personal motivation and 2 2 over a PAO2 range of 60-100 mmHg but below this range there contentment with body image. In a separate laboratory ses- is a non-linear relationship (Fenn et al, 1946; Rahn & Otis, 1949; sion, blood pressure, heart rate, body mass index, forced vital O’Brien, 1995). This is due to hyperventilation resulting in a res- capacity and forced expiratory volume in one second were piratory alkalosis when PAO2 decreases below approximately measured. Our measure of cardiovascular fitness (fitness) was 60mmHg (Fenn et al, 1946). recovery of heart rate over a one minute period immediately In this study we found that the relationship between PAO and after intense exercise. Pupils’ academic grades (expressed as a 2 minute volume was linear (between PAO2 = 30 to 100 mmHg) score) were provided by the school, with pupils identified only in the HPS whereas humans show an increase in gain when by their allocated number. PAO2 decreases below 60 mmHg. We changed the gain of the HPS ventilatory response incrementally and this resulted in a

119P Poster Communications more accurate representation of the human data. This pro- (Millar & Williams 1990) using trapezoidal (flat-top) scans lim- duced an improved correspondence between the HPS and ited to a +450 mV positive potential for the electrochemical human O2-CO2 diagrams. Subsequent refinement allowed detection of 5-HT was used. This waveform is highly selective, demonstration of equivalent responses for acclimatised detecting 5-HT at levels of <10 nM. For dopamine or nora- humans as well as appropriate changes in Oxygen saturation, drenaline and ascorbate the detection thresholds are >100 nM arterial pH and heart rate. and >1 μM, respectively. All values are means ±S.E.M. Responses We conclude that the HPS response to hypoxia can be refined were measured as the height of oxidation peaks. to increase its validity as an educational tool to illustrate high Under barbiturates anaesthesia vagal stimulation (20 Hz) altitude physiology. evoked an increase by 6±1 nM (n=5), a decrease by 11±4 nM Helyer R, Coombs A, Cousins A, Dee H, Kermode E, Rogers C & Lloyd E (n=5) and one biphasic effect change in 5-HT levels, while under (2008) The response to hypoxia: a comparison of the Human Patient chloralose increases were mainly observed of 23±5 nM (n=14). Simulator (HPS) with human data. Proc Physiol Soc 11 PC46 In some cases there was no response at 20Hz, however at 50Hz Fenn WO, Rahn H, Otis, AB (1946). A theoretical study of the compo- an increase of 22±8 nM (n=7) was observed, while in others sition of the alveolar air at altitude. Am J Physiol 146, 637 release could be observed at 5 and 10Hz of 29±9 nM (n=3) and Rahn H & Otis AB (1949). Man’s respiratory response to during and 28±6 nM (n=5). Citalopram (100 μg kg-1; i.v.; 20Hz) tended to after acclimatization to high altitude. Am J Physiol 157, 445 cause a decrease in release from 20±7 to 15±5 nM. Increasing O’Brien DM (1995). USAF School of Aerospace Medicine - Flight Sur- the dose had no further effect. Cadium applied topically to geon’s Guide, 4th edition, Chapter 2 the NTS (10-3 M in 100μl; n=1) completely abolished release after 1st stimulus in a train of three. In one experiment using We wish to acknowledge the technical support of Mr Peter chloralose a decrease in 5-HT was observed. Dickens. HPS funding as part of the AIMS CETL was provided The data indicates that vagal afferent stimulation can produce by HEFCE. increases in the level of extracellular 5-HT and this seems to be Where applicable, the authors confirm that the experiments an all or nothing response. described here conform with The Physiological Society ethical Millar J & Williams GV (1990). J Electronanal Chem 282:33-49. requirements. Nosjean A, Compoint C, Buisseret-Delmas C, Orer HS, Merahi N, Puizill- out JJ & Laguzzi R (1990). Neurosci Lett 114:22-26. Ramage AG & Villalón CB (2008). TiPS 29:472-481. PC79 SchaffarN,KesslerJP,BoslerO&JeanA.(1998).Neuroscience26:951-958. Steinbusch HWM (1981). Neuroscience 6:557-618. Using fast cyclic voltammetry, 5-HT increases can be detected in the nucleus tractus solitarius (NTS) in response DO is supported by a BHF grant PG/07/119/24169 to vagal afferent stimulation in anaesthetized rats Where applicable, the authors confirm that the experiments J. Millar2, D. Oskutyte1 and A.G. Ramage1 described here conform with The Physiological Society ethical requirements. 1Neuroscience, Physiology and Pharmacology, UCL, London, UK and 2Medical Education, The London Queen Mary’s School of Medicine and Dentistry, London, UK The NTS is richly innervated by 5-HT terminals (Steinbusch, PC80 1981) originating centrally (Schaffar et al. 1988) and from vagal 5-HT receptors play a role in the mediation of afferent afferents (Nosjean et al. 1990). Vagal afferent activation of NTS 7 transmission within the NTS in anaesthetized rats neurons has been shown to be mediated by 5-HT3 and 5-HT7 receptors (Ramage & Villalón, 2008). This indicates vagal affer- D. Oskutyte and A.G. Ramage ents cause the release of 5-HT within this nucleus. Experiments Neuroscience, Physiology and Pharmacology, UCL, London, UK were carried out to determine if this release could be detected in real time in anaesthetized rats using fast cyclic voltammetry. Central 5-HT pathways acting via 5-HT1A, 5-HT3 and 5-HT7 Sprague-Dawley rats (250-330g) were anaesthetized with iso- receptors play a critical role in the regulation of cardiovascular fluorane (5% in 100% oxygen) and maintained with either reflexes (Ramage & Villalón, 2008). 5-HT1A receptors are sodium pentobarbitone (60 mg kg-1, i.v.) or α-chloralose (120 involved in the reflex regulation of parasympathetic (vagal) mg kg-1, i.v.), neuromuscular blocked (α-bungarotoxin 150 control of the heart and data indicates that the predominant μ -1 g kg , i.v.) and artificially ventilated with O2-enriched air. location of these receptors is within the nucleus ambiguus. In Depth of anaesthesia was assessed by the stability of BP and HR contrast, 5-HT3 receptors are involved in afferent processing following a noxious stimulus and additional anaesthetic was within the nucleus tractus solitarius (NTS) where their activa- given when necessary. NTS was exposed by removing the occip- tion involves the release of glutamate, probably to some extent ital bone. The left vagus nerve was exposed and tied distally from glia. However, the location(s) of 5-HT7 receptors within to the stimulating site. A carbon fibre electrode (tip dia. 7-10 the cardiovascular reflex pathways (Kellett et al. 2005) is μm) was then inserted into the medial NTS and lowered until unknown. The present study was carried out to investigate the evoked potentials could be detected from vagal stimulation effects of the 5 HT7 receptors non-selective agonist 5-carbox- (10-400 μA, 0.2-1.0 ms, 10x threshold). The sites were con- amidotryptamine (5-CT) and the selective antagonist SB firmed histologically. The system was then switched to voltam- 258719 (Forbes et al. 1998) applied ionophoretically on the metry. A modified form of fast differential scan voltammetry ongoing and vagal-evoked activity of NTS neurones.

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Sprague-Dawley rats (250-330g) were anaesthetized with of peripheral sites. How the brain nuclei are involved in facili- sodium pentobarbitone (60 mg kg-1, i.v.), neuromuscular tating the sympathoexcitation occurring after MI and its pro- blocked (α-bungarotoxin 150 μg kg-1, i.v.) and artificially ven- gression to heart failure is unclear. Functional studies indicate tilated with O2-enriched air. NTS neuronal activity was recorded that within the paraventricular nucleus of the hypothalamus as previously described (Wang et al., 1998). Depth of anaes- (PVN), nitric oxide (NO) is responsible for a sympathoinhibitory thesia was assessed by the stability of arterial blood pressure action on sympathetic nerves and alteration of this may be and heart rate following a noxious stimulus and additional responsible for the sympathoexcitation manifest in heart fail- anaesthetic was given when necessary. NTS neurones were ure (Li & Patel, 2003). This investigation sort to correlate PVN identified by orthodromic excitation from stimulation of the neurochemical change after MI to identify which neurones are vagus nerve (10-400 μA, 0.2-1.0 ms, 0.5-1.0 Hz). Cardiopul- involved in the sympathoexcitation. monary afferents were stimulated by right atrial injection of TheUniversityofAucklandAnimalEthicalCommitteeapproved phenylbiguanide (PBG, 12-24 μg kg-1). All values are means ± all experiments. Male Wistar rats were anaesthetised with S.E.M. Comparisons between mean were made with Student’s isoflurothane (4% in 2.5l/min O2). A left intercostal thoraco- paired t-test. P < 0.05 was considered to be significant. tomy was performed to expose the heart, the pericardium was In 18/76 neurons 5-CT (40-100nA; pH 4) significantly increased removedandtheleftcoronaryarteryligated.Intheshamgroup baseline firing rate from a mean of 3.2 ± 0.5 to 5.1 ± 0.5 spikes thechestwasopened,pericardiumremovedwithnoligationof s-1. SB 258719 (100-150nA; pH 4) alone did not affect back- the coronary artery and in control no intervention was per- ground activity. In 9 neurones SB 258719 (100-150nA) signifi- formed.Animalsrecoveredfor3weeks,whentheywerere-anes- cantly blocked the increase in activity evoked by 5-CT (5.0 ± thetised(pentobarbitol60mg/kg)andanechocardiographper- 0.9 to 4.9 ± 0.7 cf. 4.5 ± 0.8 to 6.3 ± 0.8). SB 258719 also signi- formed to assess cardiac function. Following this animals were ficantly decreased vagal-afferents evoked activity from 17 ± 1 humanely killed (pentobarbital, 60mg/kg), perfused-fixed (4% to 8 ± 1.3 spikes 20 sweeps-1 in 20/35 tested. Out of these, 5- paraformaldehyde)withremovalofbrainandspinalcord.Frozen CT excited 3 neurons, inhibited 7 and had no effect on the sections (40μm) were incubated in goat anti c-Fos followed by remaining 10 neurons. In 2 of 3 neurones 5-CT exciation was biotinylated donkey anti goat IgG then strepavidin Alexa Fluor blocked by SB 258719 at the same current that attenuated the 594.SectionswereincubatedinrabbitantinNOSthengoatanti evoked activity. Further cardiopulmonary afferent-evoked activ- rabbit Cy2. The tissue was examined under epifluorescence. ity was also significantly inhibited by SB 258719 in 4 out of 6 Fractional shortening, (measure of left ventricular function) for neurons (66%) tested. 5-CT also had an inhibitory action, and MI was less than half that of control and sham animals (MI: in 9 neurons tested this was unaffected by SB 258719. 19.5±0.9% SE, n=3, Sham: 48.5±5.7% SE n=5, Control: 49.3±6% This data indicates that 5-HT7 receptors within NTS are involved SE n=4). Heart weight for MI was larger than that for control in vagal afferent processing. and sham (MI: 1.8±0.1g SE n=3, Sham: 1.4±0.1g SE n=5, Forbes IT, et al. (1998). J Med Chem 41:655-657 1.3±01g n=4). For sham and control, Fos immunoreactive (FOS- IR) neurones were localised to the parvocellular regions of the Kellett DO, Ramage AG & Jordan D (2005). J Physiol 563:319-31 PVN. For the MI group, FOS-IR neurones were concentrated in Ramage AG & Villalón CB (2008). TiPS 29:472-481. the dorsal cap, a region involved in regulation of renal sympa- Wang Y, Ramage AG & Jordan D (1998). J. Physiol 509:603-694 thetic nerve activity (Deering & Coote, 2000). Neuronal nitric oxide expression within the PVN was consistent across the This work was supported by BHF grant PG/04/123/17935 groups. Where applicable, the authors confirm that the experiments These preliminary findings suggest 3-weeks after MI, neurones described here conform with The Physiological Society ethical in the dorsal cap of the PVN appear to be preferentially acti- requirements. vated. This may represent a state where neurones in the dorsal cap drive sympathetic activity but are still under the inhibitory influence of NO. Li & Patel (2003). Acta. Physiol. Scand. 177: 17-26. PC81 Deering & Coote (2000). Exp. Physiol. 85: 177-186.

Central brain nuclei and the development of This work was funded by BHF & Auckland Medical Research sympathoexcitation after myocardial infarction Foundation. S. Pyner1, M.I. Pinkham2, S. Guild2, S.C. Malpas2, G. Whalley3 Where applicable, the authors confirm that the experiments and C. Barrett2 described here conform with The Physiological Society ethical requirements. 1School Biological & Biomedical Sciences, University of Durham, Durham, UK, 2Physiology, University of Auckland, Auckland, New Zealand and 3Medicine, University of Auckland, Auckland, New Zealand The central nervous system regulates cardiovascular homeostasis through the actions of sympathetic nerves. Sympathetic nerve activity is elevated in cardiovascular disease including myocardial infarction (MI). Sympathetic nerve activity is the output from brain nuclei that receive inputs from a multitude

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PC82 PC83

Sexual intercourse and the sympathetic nervous system Temporal Dispersal of Repolarisation in a Ferret Model of Heart Failure F.D. McBryde1,2, S. Guild2, C.J. Barrett2, D.M. Budgett3 and S.C. Malpas2,3 G. Kirkwood, H.K. Graham and A.W. Trafford 1Department of Physiology & Pharmacology, University of Bristol, Cardiovascular Electrophysiology, University of Manchester, Bristol, UK, 2Department of Physiology, University of Auckland, Manchester, UK Auckland, New Zealand and 3Bioengineering Institute, University Background Heart failure is associated with an increased risk of Auckland, Auckland, New Zealand of ventricular arrhythmias. Increased QT variability has been Sexual intercourse is one of the most natural events in life, yet proposed as a marker of arrhythmia risk (1). We examined QT anecdotally it has also been associated with an increased risk variability in a model of heart failure. of arrhythmia development, myocardial infarction or stroke. Method To induce heart failure, adult male ferrets underwent It has been suggested that elevations in sympathetic nerve ascending aortic banding (n=5) or sham operation (n=3). Oper- activity (SNA) may mediate this increased cardiovascular risk. ations were performed with isoflurane 2-3% inhaled anaesthesia Thus an understanding of sympathetic outflow during coitus and meloxicam analgesia (0.2mg/kg IM). Post-operatively, inter- is liable to be important in recommending the safety of sexual mittent telemetric ECG recording was performed at baseline activity in subjects with underlying cardiovascular disease. We and end stage heart failure. These were analysed to obtain mean have utilised a new telemetry technology to enable recordings RR and QT interval, from which QT Variation Index was calcu- of renal SNA, blood pressure and heart rate in male (n=5)and lated as a normalised ratio of QT to RR variance. Mean beat-to- female rabbits (n=5) before, during and after mating. All pro- beat variation in RR and QT intervals were measured, from cedures were approved by the University of Auckland Animal which Short Term QT Variation Index was calculated as a log Ethics Committee. Rabbits underwent surgery to implant ratio of QT to RR beat-to-beat variation. Statistical analysis was telemetry devices under isoflurane anaesthesia, at least 7 days performed using Student’s T-Test. Results (see summary table) prior to the experiment. Renal SNA was normalised to gan- Mean QT interval increased from experiment start to finish only glionic blockade and the nasopharyngeal reflex response (0- in the heart failure group. No significant change in beat-to-beat 100 normalised units, n.u.). ANOVA statistical analysis was per- variation of RR or QT interval was observed in the heart failure formed (p<0.05). Sexual activity was associated with transient group as a whole. A subgroup within the heart failure group (8-14s) but extreme increases in renal SNA in both male (9±1 was identified in which beat-to-beat RR variation declined from n.u. to 189±32 n.u.) and female (8±1 n.u. to 134±18 n.u.) rab- baseline to end-stage heart failure (n=3, mean change -39.9% bits. This increase was significantly greater than that observed SD 13.4, p<0.05) and this was associated with increased beat- during physical exertion immediately prior to mating (Male to-beat QT variation (mean change +57.8% SD 30.7 p<0.05). 27±6 n.u., Female 47±18 n.u.). Mean arterial pressure and heart This increase was not observed in heart failure subjects with- rate also increased significantly during physical exertion in male out reduced beat-to-beat RR variation (mean change –16% SD (88±4 mmHg vs. 110±12 mmHg; 236±11 bpm vs. 312±18 bpm) 36, ns) or in control subjects (mean change +14.7% SD 23.3, and female rabbits (89±5 mmHg vs. 107±8 mHg; 247±9 bpm ns). Conclusion In this model induction of heart failure causes vs. 307±31 bpm), with further increases observed during mat- QT prolongation but not increased QT temporal dispersal. How- ing (Male 134±9 mmHg, 422±21 bpm; Female 128±11 mmHg, ever, a heart failure subgroup is noted in which reduced beat- 366±15 bpm). These results show sexual activity in healthy to-beat RR variability is associated with increased beat-to-beat adult rabbits to be associated with profound transient increases QT variability. The finding of a distinct group with increased in sympathetic drive to the kidneys, as well as significant temporal heterogeneity of repolarisation is in keeping with find- increases on the workload of the heart as indicated by increases ings from other models (2) and warrants further investigation. in heart rate and arterial pressure. Our study supports the ECG parameters at baseline and experiment end hypothesis that sexual activity in subjects with underlying cardiovascular disease could carry an elevated degree of risk associated with the extreme increase in SNA. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

*b2b - beat to beat variation. **QTVI - QT Variation Index. ***STQTVI - Short Term QT Variation Index. All values expressed as mean +/- St Dev Berger RD, Kasper EK, Baughman KL, Marban E, Calkins H, Tomaselli GF. Beat-to-beat QT interval variability: novel evidence for repolarization lability in ischemic and nonischemic dilated cardiomyopathy. Circula- tion 1997;96:1557-65 Thomsen MB, Truin M, van Opstal JM, Beekman JDM, Volders PGA, Stengl M, Vos MA. Sudden cardiac death in dogs with remodelled hearts is associated with larger beat-to-beat variability of repolarization. Basic Res Cardiol 2005;100: 279-287

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC85 requirements. Anatomical and molecular mapping of rabbit free running Purkinje fibres PC84 A.J. Atkinson, J.O. Tellez, M.R. Boyett and H. Dobrzynski

Superoxide anion-mediated endothelial dysfunction and the Cardiovascular Research Group, University of Manchester, fate of nitrous oxide isoenzymes in the penis of long-term Manchester, UK diabetic rat The cardiac conduction system (CCS) consists of the sinoatrial S. Suresh, E. Prithiviraj and S. Prakash and atrioventricular nodes, the bundle of His, the bundle branches and the Purkinje fibres (PF). The PF form the terminal Department of anatomy, University of Madras, Chennai, India portion of the ventricular conduction system and provide a Oxygen and nitrogen derived free radicals and oxidant plays rapid conduction pathway. They have been shown to have spon- important role in the pathogenesis of diabetic endothelial dys- taneous diastolic depolarization, a more negative plateau function. Present study was aimed to analyze the role of super- potential than ventricular muscle and a susceptibility to the oxide (O2.-) on endothelial impairment and its correlation with production of early afterdepolarizations. The PF have been constitutive nitrous oxide synthetase isoenzymes in the penis linked to a number of ventricular arrhythmias including torsade of the long-term diabetic rat. Wistar albino rats (Rattus norvegi- de pointes arrhythmias associated with long-QT syndrome. cus) were randomly divided into two groups i.e. control The whole-mount immunohistochemical method employing (received 0.1M of citrate buffer) and diabetes (received single middle neurofilament as a marker of the CCS was used to map dose of Streptozotocin at 60 mg/kg in 0.01M citrate buffer the anatomical distribution of the bundle branches and the free through i.p.). At the end of 120th day, animals were sacrificed running left and right Purkinje fibres (LPF and RPF) in the rab- by overdose of anesthesia (thiopentone sodium 40mg/kg b.wt.) bit heart. The left bundle branch (LBB) appeared as a ribbon and immediately penile tissue were dissected collected and like structure composed of many fine fibres (perhaps only one used for the various analyses like protein and mRNA expression myocyte wide) running in parallel on the septal surface. The of iNOS, nNOS, eNOS and MnSOD. Small pieces of tissues were fine fibres formed denser strands before branching into a net- used for histological and immunohistological analyses (iNOS, work of fine fascicles that covered the left ventricular free wall. nNOS, eNOS and MnSOD). In-situ detection O2.- using dihy- The right bundle branch appeared to be narrower than LBB and droethedine was performed on fresh tissue sections. Results ran over the septal surface. It had a number of fascicles branch- showed significant increase in the production of superoxide ing from it along its length. These strands extended across the and significant decrease in the levels of NOS isoensymes (iNOS ventricle and formed a dense network on the right ventricular – p< 0.001, eNOS – p< 0.001 and nNOS – p< 0.001) and Mn SOD free wall. (p< 0.001) in the penis of the diabetic animal. Levels of mRNA Quantitative RT-PCR was used to analyse the expression of var- expression of NOS isoenzymes (iNOS – p< 0.001, eNOS – p< ious transcripts in eight rabbits. ~30 transcripts for ion chan- 0.01 and nNOS – p< 0.001) and MnSOD (p< 0.001) were signi- nels, connexins, Ca2+-handling proteins and cellular markers ficantly deceased in diabetic rat penis. Histological observa- were investigated in the LPF, RPF, right atrium (RA) and left ven- tions showed increase in endothelial thickening of the diabetic tricle (LV). The results show that the PF have an ion channel rat penis. These observations indicates that the elevated levels expression profile distinct from that of the working of O2.- mediates endothelial dysfunction and this increased myocardium. In the LPF and/or RPF, there was a significantly levels of O2.- might be due to the impaired mitochondrial higher expression (versus LV) of Nav1.1, HCN1, HCN4 and NFM antioxidant defense system or/and alterations in the electron mRNAs and a significantly lower expression of Cav1.2, KvLQT1, transport system. This might down-regulate the mRNA expres- ERG, KChIP2, SUR2, Cx45, RYR2, SERCA2a and NCX1 mRNAs. sion of NOS isoenzymes or up-regulate the degradation of these There was also a tendency for a higher expression of Kir3.1, enzymes. Thus understanding the mitochondrial pathophysi- TWIK-1 and Cx40 mRNAs and a lower expression of Kv1.4, ological source of O2.- and mRNA expression of these enzymes Kir2.1, Kir6.2, RYR3 and NCX1 mRNAs in the PF. The expression under hyperglycemic condition would give more insight into in the free running PF was also distinct from that in the RA: in ensuing erectile dysfunction. the LPF and/or RPF there was a significantly higher expression of NFM mRNA and a significantly lower expression of HCN4, Where applicable, the authors confirm that the experiments Kir3.1, KChIP2, Cx45 and ANP mRNAs. There was also a ten- described here conform with The Physiological Society ethical dency for a higher expression of Nav1.1, Kv4.2, Kv4.3, TWIK- requirements. 1, Kir2.1 and minK mRNAs and a lower expression of Navβ1, Kir6.2, SUR2, Cx40 and SERCA2a mRNAs in the PF. We have demonstrated that the free running PF form a complex asymmetrical network in the left and right ventricular chambers of the rabbit heart. We have also shown that the free running PF have a unique pattern of expression of ion channels, connexins, Ca2+ -handling proteins and cellular markers.

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Where applicable, the authors confirm that the experiments enhanced, the ability to respond to physiological challenges described here conform with The Physiological Society ethical was impaired. requirements. Flanagan, ET, Buckley, MM, Aherne, CM, Lainis, F, Sattar, M & Johns, EJ (2008). Exp. Physiol. 93.9, 1058-1064.

This work was in part supported by a Vacations Studentship PC86 awarded to F Lainis by the Health Research Board. Where applicable, the authors confirm that the experiments Thyroxine induced heart hypertrophy: impact on cardiac described here conform with The Physiological Society ethical function requirements. F. Lainis, M.M. Buckley and E.J. Johns Department of Physiology, University College Cork, Cork, Ireland PC87 Cardiac hypertrophy occurs when there is increased workload on the heart and is recognised as the first stage in the pro- Hypoxic Upregulation of the BMP Antagonist Gremlin Blocks gression into heart failure. Recently, this laboratory reported BMP Signalling in Pulmonary Endothelial Cells that cardiac function parameters were elevated in a rat model E. Cahill1, C.M. Costello1, K. Howell1, M.O. Leonard1, of hypertrophy induced using isoprenaline and caffeine and by 2 2 1 chronic thyroxine administration (Flanagan et al. 2008). The M. Southwood , N.W. Morrell and P. McLoughlin aim of this study was to determine how different exposure 1Conway Institute, University College Dublin, Dublin, Ireland and times to thyroxine impacted on basal cardiac function param- 2University of Cambridge, Cambridge, UK eters and on the ability of the heart to respond to a β-adren- Pulmonary hypertension (PH) is a common complication of ergic challenge. chronic hypoxic lung diseases. Recently we reported that grem- Groups of male Wister rats (250-270g) were given normal diet lin, a BMP antagonist, was selectively upregulated in hypoxic tap water to drink (n=9) or received daily i.p. injections of thy- human pulmonary microvascular endothelial cells in vitro. Given roxine (1mg/kg) for 7 (n=9) or 14 (n=9) days. On the day of the important role of bone morphogenetic proteins (BMP) in study, anaesthesia was induced using 1ml ip chlo- normal pulmonary vascular homeostasis, we postulated that ralose/urethane (16.5/250 mg/ml) and cannulae were inserted upregulation of gremlin was an important pathogenetic mech- into a femoral artery and vein to measure mean arterial pres- anism contributing to the development of hypoxic PH. sure (MAP) and heart rate (HR) and to infuse saline (0.9g/l NaCl) To examine BMP signalling in an in vivo mouse model of hypoxic at 3ml/h, respectively. A micro-tip pressure transducer catheter PH, mice (male SPF C57BL/6) were exposed to environmental was introduced into the left ventricle via the right carotid artery hypoxia (FiO2 0.10) and killed under anesthesia (sodium pen- (Flanagan et al 2008) to allow computation of cardiac index (CI) tobarbitone, i.p.) for RNA or protein analysis. We demonstrated and dP/dtmax. Following 1-2 h recovery, basal measurements selective upregulation of gremlin in the hypoxic lung by RT-PCR were taken over a 3 min period; thereafter, isoprenaline, 0.75mg (n=8) and immunohistochemistry (n=5). The gremlin target was given i.v over 40s, and further recordings of the haemo- proteins BMP-2, -4 and -7 were basally expressed in the mouse dynamic variables were taken as MAP recovered from its nadir. lung (n=8) but hypoxia only caused upregulation of BMP-2 Data, means ± S.E.M. were considered significant when P<0.05 mRNA. However, BMP-2 protein was down-regulated in hypoxia (one way ANOVA). (n=6), together with reduced Smad1/5/8 phosphorylation (n=6) In the control group, basal levels of MAP, HR, CI and dP/dtmax and reduced expression of the BMP target gene, Id1 (n=8). were 123±5mHg, 351±8 s-1 and 11.10 ±0.90 mmHg s-1 10-3 Recombinant gremlin blocked BMP-2-stimulated wound heal- and these values were comparable to the basal values recorded ing in cultured human microvascular endothelial cells and also in rats treated with thyroxine for 7 days. In rats treated with blocked BMP-2-induced Smad1/5/8 phosphorylation and Id1 thyroxine for fourteen days, MAP was comparable at 131±10 expression in these cells. Conditioned medium from hypoxic mmHg, but HR, CI and dP/dtmax were higher (all P<0.05) at endothelial cells also blocked BMP signalling and BMP stimu- 408 ± 18 b/min, 167±7 s-1 and 13.75±0.67 mmHg s-1 10-3, lated endothelial wound healing. Finally, gremlin protein was respectively. Administration of isoprenaline i.v. in the control upregulated in the pulmonary vascular endothelium of patients rats transiently decreased MAP, increased HR by 14% and CI by with IPAH (n=4) when compared with controls (n=4) as demon- 15% (both P<0.05), but minimally changed dP/dtmax. In the strated by immunohistochemistry on lung sections. group given thyroxine for 14 days, isoprenaline i.v. caused signi- Thesefindingsdemonstratelung-selectiveupregulationofgrem- ficant reductions (both P<0.05) in CI and dP/dtmax of some 9 lininpulmonaryhypertensionandsuggestanautocrinerolefor and 15% respectively. gremlininmodifyingBMP-inducedendothelialresponses.Grem- Together, these data demonstrated that as the exposure to thy- lin may represent a previously unrecognized mechanism that roxine was prolonged, heart size was increased and baseline plays an important role in the development of PH. cardiac function in terms of CI and dP/dtmax was raised. By contrast, a stimulatory challenge with isoprenaline, which Health Research Board and UCD Ad Astra Scholarship enhanced cardiac function in normal rats, was blunted follow- Where applicable, the authors confirm that the experiments ing the two weeks of thyroxine treatment and these parame- described here conform with The Physiological Society ethical ters were decreased. The findings would suggest that in car- requirements. diac hypertrophy that although basal heart function was

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Where applicable, the authors confirm that the experiments PC88 described here conform with The Physiological Society ethical requirements. Upregulation of ACh release in mouse synapses after activation of L-type Ca2+ channels is due to release of stored calcium PC89 A. Gaydukov and O.P. Balezina Human And Animal Physiology, Moscow State University, Effects of tumour necrosis factor-a and glutamate Moscow, Russia pretreatment on glutamate-induced calcium influx in rat organotypic hippocampal cultures It is well known that ACh secretion in mature mammalian motor synapses is triggered by Ca2+ influx into nerve terminals mainly O. Watters and J.J. O’Connor 2+ through voltage-dependent Ca channels of the P/Q-type [1]. UCD School of Biomolecular and Biomedical Science, UCD Conway 2+ Nerve terminals possess also Ca channels of L-type - normally Institute, University College Dublin, Belfield, Dublin 4, Ireland “silent”, but when activated, they provide additional Ca2+ influx which leads to increase of quantal content of single evoked end- Glutamate-induced excitotoxicity contributes to neuronal dam- plate potentials [2,3]. In many excitable cells, Ca2+ influx age during a cerebral ischaemic event such as stroke. Physio- through L-type Ca2+ channels activates ryanodine receptors logical levels of glutamate and proinflammatory cytokines such (RyRs) of calcium stores, thus elevating intracellular concen- as tumour necrosis factor-α (TNFα) play a role in the regulation tration of calcium. So, the aim of this study was to check the of synaptic plasticity within the hippocampus. During a stroke possible functional coupling between activation of L-type Ca2+ pathophysiological levels may cause dysregulation of these channels, subsequent release of stored calcium through RyRs processes, enhancing vulnerability of these cells to an ischaemic and evoked mediator secretion. The cut in vitro neuromuscu- insult. Previous studies suggest that a mild transient ischaemic lar preparation of mouse left hemidiaphragm and intracellular attack (TIA) within 72 h of a stroke may result in attenuation microelectrode recordings were used in order to analyse rhyth- of its clinical severity (Castillo et al. (2003)). We have developed mically (50Hz) evoked endplate potentials (EPPs) and sponta- an in vitro model of TIA using organotypic hippocampal cul- neous miniature endplate potentials. Statistical analysis was tures (modified technique of Stoppini et al. (1991)). Hip- performed using a Mann-Whitney test; all data are presented pocampal slices (400 μm) were cultured from male Wistar rats as mean±S.E.M. Pharmacological disinhibition of L-type Ca2+ at post-natal day 7 (humanely killed - decapitated). At 6 days channels (directly - by their agonist S(-) BAY K 8644 (1 microM), in vitro (DIV) cultures were pretreated for 30 min with 30 μM or indirectly – by blocker of Ca2+-activated K+ channels –iberi- glutamate or 5 ng/ml TNFα. They were then placed in fresh otoxin (100 nM)) was found to greatly potentiate EPP ampli- media for 24 h (recovery period). As Ca2+ is a well-established tudes (due to increase of quantal content of each single EPP in mediator of glutamate-induced excitotoxicity, we investigated the train up to 41±8% (p<0.05, n=25) or 34±10%(p<0.05, n=25), whether glutamate/TNFα preconditioning altered glutamate- respectively) in the train during rhythmic synaptic activity – up induced Ca2+ influx. At 7 DIV pretreated cultures were loaded to 10 mV. Furthermore, these drugs additionally augment the with a Ca2+ indicator dye, Fluo-4 (3 μM) and imaged with Zeiss facilitation phase up to 10-12% in the beginning of EPPs train. laser scanning confocal microscope. Relative changes in fluo- This upregulation of mediator secretion was completely pre- rescence of Ca2+-bound dye correlates with relative changes in 2+ μ vented by preapplied nifedipine (10 microM) or verapamil (5 [Ca ]i. After 20 s of baseline recording, 30 M glutamate was microM) - blockers of L-type Ca2+ channels (they weren’t able applied and the Ca2+ response was recorded for 70 s. Data analy- to affect amplitude or quantal content of EPPs by themselves). sis was carried out with Zeiss Image Examiner software and sta- Blockade of intraterminal RyRs with ryanodine (2 microM) tistics were compiled using one-way ANOVA followed by Bon- diminished the facilitation in EPPs train during rhythmic activ- ferroni post-test. Results are expressed as mean±S.E.M. ity: from 110±1.5 in control to 103,3±1.7 (p<0.05, n=32). More- Pretreatment with 5 ng/ml TNFα/30 μM glutamate resulted in over, the described effects of S(-) BAY K 8644 and iberiotoxin a reduction in glutamate-induced Ca2+ influx after 24 h (TNFα; were totally prevented by primarily application of ryanodine. 1293±28, n=3340, glutamate; 1088±23, n=2439, Vs. control; The obtained data indicate that potentiation of mediator release 2679±48, n=3524, p<0.001). Changes in baseline Ca2+ levels after involving of L-type Ca2+ channels in activity is not due to due to pretreatments were analysed using a ratiometric Ca2+ increased Ca2+ influx through these channels itself, but is medi- dye, Indo-1 (5 μM). Preliminary data suggest that ated by activation of presynaptic RyRs and mobilization of TNFα/glutamate pretreatment significantly lowered baseline 2+ stored calcium. So, activation of RyRs is a necessary step to facil- [Ca ]i compared to control. Thus, an acute mild α 2+ itate rhythmically evoked transmitter release in mouse motor TNF /glutamate exposure may alter both basal [Ca ]i within synapses after increased external Ca2+ influx through L-type the cell and its responsiveness to glutamate-induced Ca2+ influx. Ca2+ channels. Pretreatment with 5 ng/ml TNFα in the presence of the mGluR5 μ Katz E. et al. Br J Pharmacol (1997) 121: 1531-40. metabotrophic glutamate receptor antagonist, MPEP (10 M), significantly enhanced TNFα’s preconditioning effect (1125±21, Urbano F. et al. Pflugers Arch (2001) 441 (6): 824-31. n=2298, p<0.05). The NMDA receptor antagonist, D-AP5 (100 Flink M., Atchison W. J Pharmacol Exp Ther (2003) 305 (2): 646-52. μM) did not significantly alter the preconditioning effect of glu- ± The work is supported by RFBR grant # 07-04-00920-a. tamate when co-applied (1072 27, n=1764, p>0.05). We conclude that TNFα’s preconditioning effects may be TNFR and

125P Poster Communications mGluR5 receptor mediated, whereas NMDAR activation dur- Gordon,G.R., Choi,H.B., Rungta,R.L., Ellis-Davies,G.C., and ing glutamate preconditioning may be less important. MacVicar,B.A. (2008). Brain metabolism dictates the polarity of astrocyte control over arterioles. Nature. 456, 745-749. Castillo J, Moro MA, Blanco M, Leira R, Serena J, Lizasoain I, Dávalos A. (2003) The release of tumor necrosis factor-alpha is associated with Funded by Fondecyt 10070046. ischemic tolerance in human stroke. Ann Neurol. 54(6): 811-9. Stoppini L, Buchs PA, Muller D. (1991) A simple method for organotypic Where applicable, the authors confirm that the experiments cultures of nervous tissue. J Neurosci Methods. 37(2): 173-82. described here conform with The Physiological Society ethical requirements. Science Foundation Ireland. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC91 requirements. Effects of Chronic and Acute Simvastatin Treatment on Synaptic Transmission in Hippocampal Slices from C57Black PC90 6J Mice C.P. Metais and C.E. Herron A novel method to measure the rate of glycolysis in single cells reveals fast tuning of astrocytic glycolysis by K+ but not School of Biomolecular and Biomedical Sciences, University College by glutamate Dublin, Dublin, Ireland C.X. Bittner1, A. Loaiza1, I. Ruminot1, V. Larenas1, T. Sotelo- Statins are agents commonly used in the clinic to treat hyper- Hitschfeld1, H. Moldenhauer1, W.B. Frommer2 and L.F. Barros1 cholesterolemia, however little is known regarding their effects on neuronal function. Chronic treatment with simvastatin was 1 Center for Scientific Studies, Valdivia, Region de Los Rios, Chile and shown to improve learning and memory in behavioural tasks 2 Department of Plant Biology, Carnegie Institution for Science, (Li et al., 2006). The aim of this research was to investigate acute Stanford, CA, USA and chronic effects of simvastatin on synaptic transmission, Neuronal activation demands metabolic energy that must be paired pulse facilitation (PPF) and long-term potentiation (LTP) supplied within seconds. The study of the mechanisms cater- in the CA1 region of the hippocampus. Transverse hippocam- μ ing for such demand has been hampered by lack of techniques pal slices (400 m thick) were prepared from C57 black6J mice. capable of measuring metabolic flux with the required resolu- EPSPs were recorded in the CA1 region. Paired pulse stimuli tion. A Fluorescence Resonance Energy Transfer (FRET) - based were applied at an interval of 50ms. Fibre volleys were evoked microscopy method is presented here which offers much in the presence of DNQX (2,3-dihydroxy-6-nitro-7-sulfamoyl- improved spatiotemporal resolution over existing methods like benzo[f]quinoxaline-2,3-dione) by stimulating the alveus and 2-deoxyglucose autoradiography and NMR spectroscopy. recording in the CA1 cell body region. Baseline stimuli were Applied to mouse astrocytes in culture and in brain slices, the applied at a frequency of 0.033 Hz, (0.1ms) and recordings method showed that glycolysis can be activated within sec- made for 20min prior to application of simvastatin or LTP induc- onds by physiological concentrations of extracellular K+ . No tion. LTP was induced using 100 or 200 Hz. 100 Hz protocol: activation was observed in response to glutamate, widely Two stimulus trains at 100Hz for one second applied 30s apart. regarded as responsible for neuronal modulation of astrocytic 200 Hz protocol: Two sets of ten trains of ten stimuli at 200Hz, metabolism. The effect of K+ on glucose metabolism was read- 2s inter-train interval; 30s between sets. Control LTP (100Hz) ily reversible, required Na+/K+ ATPase pump activation and was measured at 60min in slices from 8week old animals (young) ± mediated by depolarization-induced alkalinization (DIA), a phe- measured 169.0 11.2% (n=13), while that induced using ± nomenon of hitherto unknown 200Hz measured 166.9 6.5% (n=10). Acute application of sim- μ physiological significance. A rapid activation of astrocytic gly- vastatin (35 M) caused a significant increase in the EPSP slope ± colysis was also observed in brain slices exposed to exogenous to 154.4 12.3%, (p<0.05, n=12) measured at 35 min com- ± μ K+ and during electrical stimulation. These results expose a pared to vehicle (105.3 3.6% n=4) whereas 10 M simvastatin ± novel phenomenon linking synaptic activity to timely local pro- did not increase the EPSP slope (118 6%; n=4) significantly. ± ± duction of lactate by astrocytes and ascribe astrocytic pH a cen- Simvastatin also decreased the PPF ratio (1.47 0.06 to 1.30 tral role in neurometabolic coupling and neurovascular cou- 0.06; p<0.01, n=12) at 35min. LTP (100 or 200Hz) measured pling. This technique to measure glycolysis in single cells, in from baseline prior to induction in the presence of simvastatin μ real time and reversibly, may also be used to investigate the (35 M) was decreased significantly compared to control (133.2 ± ± transport and metabolism of glucose in other cell types. 4.8% n=8 and 132.9 10.9%, respectively n=6). Following Barros,L.F., Bittner,C.X., Loaiza,A., and Porras,O.H. (2007). A quanti- chronic simvastatin treatment (0.04%, in the diet for 6 months), tative overview of glucose dynamics in the gliovascular unit. Glia. LTP (100Hz) induced in slices from 18 month old animals meas- 55,1222-1237. ured 158.3 ± 6.9% (n= 7) and was not significantly different to Pellerin,L., Bouzier-Sore,A.K., Aubert,A., Serres,S., Merle,M., Costa- levels recorded in slices from 8 or 16 month old animals (177.8 lat,R., and Magistretti,P.J. (2007). ± 9.0%, n=7 and 175.2 ± 16%, n=8 respectively). In slices from μ Activity-dependent regulation of energy metabolism by astrocytes: an young animals treated acutely with simvastatin (35 M), fibre ± update. Glia. 55, 1251-1262. volley amplitude increased to 137.3 5.7% (n=9, p< 0.001) compared to vehicle control 105.3 ± 7.3% (n=4). Fibre volleys recorded in slices from chronically treated animals increased

126P Poster Communications to 108.7 ± 7% following acute simvastatin (35μM) application 1. Butovsky, O., Bukshpan, S., Kunis, G., Jung, S., Schwartz, M. Microglia which was not significantly different from vehicle control. Acute can be induced by IFN-gamma or IL-4 to express neural or dendritic-like simvastatin appears to increase neurotransmitter release, pos- markers. Mol. Cell. Neurosci., 2007; May 1;: 17560122. sibly by increasing fibre volley amplitude while chronic treat- The authors acknowledge grant support from the Health ment has no effect on LTP. Research Board. Li,L.,Cao, D.,Kim, H.,Lester, R. and Fukuchi, K (2006)Simvastatin Enhances Learning and Memory Where applicable, the authors confirm that the experiments Independent of Amyloid Load in Mice.Ann Neurol60:729–739 described here conform with The Physiological Society ethical requirements. Irish Health Research Board and Marie Curie EST network Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC93 requirements. Influence of slow and fast Ca2+ buffers on ACh secretion activated by several Ca2+ inputs in mouse motor nerve PC92 terminals O. Skiteva, V. Lapteva and O. Balezina Evidence that NK cells are present in the rat brain and have an age-related association with microglial activation Human and animal physiology, Moscow State University, Moscow, Russia K.J. Murphy and M.A. Lynch In mouse motor nerve terminals activation of Ca2+ channels Physiology, Trinity College Institute of Neuroscience, Dublin, Ireland and ryanodine receptors creates Ca2+ signals that are differ- Ageing is the most significant contributory factor in the patho- ent in dynamic and modality. Pattern of these Ca2+ signals, lead- genesis of neurodegenerative diseases such as Alzheimer’s dis- ing to individual changes in mediator secretion is unclear. An ease, and inflammatory changes in the brain contribute to the effective approach to solve this problem is the use of Ca2+ deficits associated with aging and neurodegenerative disor- buffers to discriminate Ca2+ signals created by different Ca2+ ders. Fundamental to these changes is an increase in microglial searches. The aim of this research was to study changes in medi- activation, the immune cells of the brain, which are the primary ator release caused by activation of Ca2+ channels or ryanodine source of pro-inflammatory cytokines. Microglial activation receptors in conjunction with different Ca2+ buffers. has been shown to be induced by IFN-γ (1), a macrophage-acti- Experiments were carried out on the dissected neuromuscu- vating factor, but the cell source of IFN-γ in the central nerv- lar preparation of mouse diaphragm. Evoked endplate poten- ous system is not known. IFN-γ is produced by Natural Killer tials (EPPs) were registered in response to unitary (0,3 Hz) nerve (NK) cells, which are traditionally found in the peripheral nerv- stimulation using standard microelectrode technique and then ous system, and the finding that IFN-γconcentration is increased quantal content (QC) of unitary EPPs was analyzed. We used in the brain of aged rats suggests a possible infiltration of NK intracellular Ca2+ buffers BAPTA-AM and EGTA-AM (50 microM), cells into the CNS. BAY K 8644 (1 microM), caffeine (2.5 mM). In this study young (2-3 month old), middle-aged (14-15 Activation of presynaptic (P/Q type) Ca2+ channels in response month old) and aged (18-25 month old) male Wistar rats (n=6 to nerve stimulation leads to generation of EPPs with QC of per group) were anaesthetised (i.p.) with urethane, and intrac- 21.32±0.4 (n=93). The L-type Ca2+ channel activator BAY K 8644 ardially perfused with saline. Dissociated cells were prepared (1 microM) showed a 18.7±4.72% increase of QC of EPPs (n=16, from one half of the brain and assessed, using flow cytometry, p<0,05, t-test). Application of ryanodine receptors agonist caf- for the presence of NK cells. Cortical and hippocampal tissue feine (2.5 mM) created a 12.9±4.32% increase of EPPs QC in taken from the other half of the brain was assessed for IFN-γ comparison to control level (n=33, p<0.05, t-test). mRNA, and expression of markers of microglial activation, Loading terminal with fast Ca2+ buffer BAPTA-AM (50 microM) MHCII, CD11b, TLR2 and TLR4. did not affect QC, but slow buffer EGTA-AM (50 microM) led to The data demonstrate that NK cells are present in the rat brain a 13±5.96% decrease of this parameter (n=15, p<0,05, t-test). and that the number of cells positive for NK cell markers, BAPTA-AM didn’t affect effects of BAY K 8644, but EGTA-AM CD161a and NKp30, was significantly greater in brain tissue prevented the increase of QC of EPPs induced by BAY K 8644. prepared from aged, compared with middle aged, rats (p<0.05; In this case QC was 83.94±5.96% compared to control (n=15, Student’s t-test). This finding was associated with a significant p<0,05, t-test). age-related increase in cortical and hippocampal mRNA expres- Both Ca2+ buffers prevented the increase of EPPs QC initiated sion of MHC II (p<0.05; 1-way ANOVA) and CD11b (p<0.001; by caffeine (2.5 mM). It’s important to note that in EGTA-loaded Student’s t-test). These results were also coupled with age- terminals there was no significant difference in QC between related increases in hippocampal IFN-γ, TLR2 and TLR4 mRNA control and caffeine effect, while EGTA-AM itself decreased this (p<0.05; Student’s t-test). parameter. These findings provide the first evidence for the presence of So, we found, that the increase of acetylcholine secretion after NK cells in the aged rat brain, and suggest a possible role activation of different calcium inputs can be influenced by Ca2+ for NK cells in the activation of microglia during age-related buffers, but demonstrates individual patterns of sensitivity to neuroinflammation. EGTA and BAPTA. Selective action of EGTA but not BAPTA preventing QC increase created by activation of P/Q or L-type chan-

127P Poster Communications nels allows us to suggest that activation of Ca2+ channels leads ites to form linear chain-like assemblies following placement to slow and local elevation of Ca2+ concentration in nerve ter- in a magnetic field. minals. In contrary, similar efficacy of both Ca2+ buffers in pre- The results confirm the potential use of these novel nanocom- venting caffeine effects gives evidence that activation of ryan- posites as bimodal contrast agents. odine receptors is associated with tonic increase of intracellular Huh YM, Jun YW, Song HT, Kim S, Choi JS, Lee JH, et al. In vivo magnetic Ca22+ level which influences high sensitive Ca2+ sensors, con- resonance detection of cancer by using multifunctional magnetic trolling mediator secretion. nanocrystals. J Am Chem Soc. 2005 Sep 7;127(35):12387-91. The work is supported by RFBR grant # 07-04-00920-a The authors acknowledge grant support from the Health Research Board and Science Foundation Ireland. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

PC93A PC93B

In vitro evaluation of magnetic-fluorescent nanocomposites Platelet L-Arginine-Nitric Oxide Pathway in Bipolar Disorder in mouse mixed glial culture P.F. de Souza 1, V. Pinto1, C. Elie2, M. Moss1, C. Matsuura1, O. da 3 2 1 2 J.J. Gallagher , R. Tekoriute , C.M. Kerskens , Y.K. Gun’ko and Silva1, T. Brunini1 and M. Antônio Cláudio1 L.A. Marina3 1Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil 1Institute of Neuroscience, Trinity College, Dublin, Ireland, and 2Instituto de Psiquiatris da Universidade do Rio de Janeiro, 2Department of Chemistry, Trinity College, Dublin, Ireland and Rio de Janeiro, Brazil 3Department of Physiology, Trinity College, Dublin, Ireland The bipolar disorder (BD) is a humor disorder, according to DMS- Magnetic-fluorescent nanocomposites enable the capabilities IV (Diagnostic and Statistic Manual of Mental Disease). This dis- of both magnetic resonance imaging (MRI) and fluorescence- order affects approximately 1.5% of the population and it is based technologies, such as confocal microscopy, to be characterized by the presence of mania and depression exploited in single experiments(1). Here we report the in vitro episodes. Similarly to other psychiatric disorders, such as evaluation of a novel Rhodamine B functionalised polyelec- depression, anxiety and schizophrenia, the BD is an important trolyte stabilised magnetic-fluorescent nanocomposite. cardiovascular risk factor. However, the exact mechanisms Mixed glial cultures from neonatal mouse cortices were pre- underlying this relationship remain unknown. Recent studies pared, following decapitation in accordance with local ethical suggest the involvement of L-arginine-nitric oxide pathway on guidelines. Following mixed glia incubation in the presence of the pathophysiology of BD. nanocomposite (5 μM) for 2 hours, nanocomposite uptake was Nitric oxide (NO) is a lipophylic gas which is produced by dif- assessed using confocal fluorescence microscopy and light ferent blood cells. L-arginine, it’s precursor, is transported to microscopy. Live cell imaging data was acquired to investi- platelets by y+L carrier, and activates the enzyme NO synthase gate the method of nanocomposite internalization. Cell via- (NOS), which produces NO and L-citrulline. NO has many phys- bility was measured in a mixed glia sample using the MTT assay, iological functions, including: vasodilatation, neurotransmis- following incubation with nanocomposites (1, 5, 10, 20 μM) sion and platelet aggregation inhibition. Despite intracellular for 2, 18, 24, and 48 hours. The concentration of pro-inflam- concentration of L-arginine is above the Km of NOS, the extra- matory cytokines, IL-1β and IL-6, of mixed glia following a 2 cellular transport of L-arginine is necessary for NO production. hour incubation in the presence of the nanocomposites (1, 5, The aim of this study was to investigate L-arginine transport 10, 20 μM) was assessed using ELISA. MRI phantoms contain- and NOS activity in platelet of bipolar patients. ing varying numbers of labelled cells in 0.5% Agarose were pre- Nine patients with BD and nine healthy volunteers were pared. T2 and T2* relaxation times of these phantoms were included in this study. Extracellular L-arginine transport into calculatedfromimagestakenona7TBrukerspectrometer platelet was measured by kinetic methods, using crescent con- using multi-spin-echo (TE=2.09-59.09ms) and multi-gradient- centrations of [3H] L-arginine; and NOS activity was evaluated echo (TE=10-200ms) sequences respectively. Tranmission elec- by the conversion of [3H]L-arginine on [3H]L-citrulline. Data tron microscopy (TEM) and confocal microscopy were used to were presented by mean and standard error. The student t test assess nanocomposite behaviour following placement in a mag- was used for statistical analysis and statistic difference was con- netic field. sidered when p<0.05. Confocal and light microscopy confirmed the internalization of Total L-arginine transport (pmol L-arginine/108 cells/min) was ± the nanocomposites. Live cell imaging data suggests both similar in bipolar patients (Vmax total=95 31) and healthy con- ± + phagocytosis and endocytosis as methods of nanocomposite trols (Vmax total=86 19). L-arginine transport via system y L + ± internalization. No reduction was seen in cell viability. There did not differ between bipolar patients (Vmax y L=83 59), com- β + ± was no increase in the concentrations of IL-1 or IL-6 following pared with healthy controls (Vmax y L=43 10). NOS activity a 2 hour incubation. The contribution to MR contrast was cal- (pmol L-citrulline/109 cells) was reduced on bipolar patients culated using γΔB ∝ (1/T2*) – (1/T2) and was linear (Pearson (0.059±0.018) compared with controls (1.138±0.027) Correlation, R = 0.9967, p<0.001). TEM and confocal (p=0.04292). These results suggest that NO production by NOS microscopy demonstrated the tendancy of the nanocompos- is reduced in bipolar patients. This lower production of NO can

128P Poster Communications be contributing for the higher cardiovascular risk factor seen in patients with BD. Our next step is to determine platelet func- PC94 tion, to see if platelet aggregation is increased in these patients. This would increase the incidence of thrombotic events in bipo- Modulation of pancreatic alpha-cell function by leptin lar patients. involves effects on electrical activity, calcium signalling and exocytosis Financial Support: FAPERJ S. Soriano1, E. Tuduri1, L. Marroqui1, A.B. Ropero1, T.M. Batista2, Where applicable, the authors confirm that the experiments S. Piquer4, M. Lopez-Boado3, E. Carneiro2, R. Gomis4, A. Nadal1 described here conform with The Physiological Society ethical and I. Quesada1 requirements. 1Instituto Bioingenieria and CIBERDEM, Universidad Miguel Hernandez de Elche, Elche, Alicante, Spain, 2Physiology and Biophysics, Instituto Nacional Pesquisa en Obesidade e Diabetes, campinas, Brazil, 3Institut de Malalties Digestives i Metabòliques. PC93C Hospital Clinic Barcelona, Barcelona, Spain and 4Endocrinology and Diabetes Unit, IDIBAPS- Fundació Clinic, Hospital Clinic, Control of spike timing by GABA(A) receptor-mediated Barcelona, Spain inhibitory synaptic input during theta frequency oscillation in rat CA1 pyramidal neurons Leptin inhibits insulin secretion by direct effects on the pancreatic beta-cell (Kieffer et al. 1997), and thus it can modulate J. Kwag and O. Paulsen glucose homeostasis. Although alpha-cells and glucagon secretion also play a critical function in the control of glycaemia, the Department of Physiology, Anatomy and Genetics, University of effect of leptin on these islet cells has not been examined. In Oxford, Oxford, UK the present work, we have studied whether alpha-cells are Extensive experimental evidence suggests that carefully con- directly regulated by this hormone. The existence of several trolled spike times relative to theta-frequency network oscil- leptin receptor isoforms (a-e subtypes) was observed by PCR lations play an important role in hippocampal memory proin the glucagon-producing alpha-TC1-9 cell line (n=3). The b cessing. Although excitatory phase response properties have isoform, the main one involved in leptin signalling, was demon- been well characterized, how inhibitory inputs could control strated in alpha-TC1-9 cells by western blot (n=3) as well as in spike timing is less well studied. Here we report the control of alpha-cells from OF1 mice and human by immunocytochem- spike timing during theta oscillation by inhibitory input and istry (n=3). The functional role of leptin was first analyzed by characterize the spike timing characteristics in spike time patch-clamp in both alpha-TC1-9 cells (n=8) and mouse alpha- response curves (STRC). Using whole-cell patch-clamp record- cells (n=3). At 0.5 mM glucose, these cells exhibit electrical ings from CA1 pyramidal cells in vitro and with dynamic clamp activity characterized by sodium and calcium action potentials to simulate theta-frequency oscillation (5 Hz), we show that (Gromada et al. 2004). In all the cells tested, leptin (6.25 nM) GABA(A) receptor-mediated IPSPs can not only delay but also hyperpolarized the membrane potential, inhibiting the elec- advance the postsynaptic spike time depending on the timing trical activity induced by 0.5 mM glucose. Additionally, 6.25 of the inhibitory input relative to the oscillation. The maximum nM leptin produced an inhibitory effect on the calcium signals spike time delay was 23.2 ± 2.9 ms and the maximum spike in alpha-TC1-9 cells as well as in mouse and human alpha-cells time advancement was -4.1 ± 1.4 ms (n = 5). Dynamic clamp- (n=13, n=16, n=12, respectively). Similar effects on calcium sig- simulated artificial IPSP mimicking GABAergic input to the soma nalling were observed at 0.625 nM leptin. Since alpha-cell exo- could also both delay and advance the spike time. The maxi- cytosis is calcium-dependent, we analyzed the effect of 6.25 mum spike time delay was 32.0 ± 2.1 ms and the maximum nM leptin on glucagon secretion by radioimmunoassay. At this spike time advancement was -7.7 ± 0.8 ms (n = 8). The intrin- concentration, leptin reduced glucagon secretion at 0.5 mM sic mechanism underlying spike time advancement with IPSP glucose by almost 30% (n=8; p<0.05). However, in the presence is due to h-channels since the application of 10 μM ZD7288, an of the PI3-kinase inhibitor wortmanine, leptin did not produce Ih channel blocker, completely abolished such advancement (n any effect, indicating that this pathway is involved. Our results = 9). These results suggest that spike timing during theta-fre- indicate that leptin participates in glucose homeostasis not only quency oscillation can be bidirectionally controlled by inhibitory via beta-cell actions but also through its inhibitory effect on synaptic inputs and that the spike time advancement caused alpha-cells. by IPSPs is due to h-channels intrinsic to the CA1 pyramidal neu- Kieffer et al. (1997) Diabetes 46, 1087-1093. ronal membrane. Gromada et al. (2004) Diabetes 53 (Suppl 3), S181-S189. This work was supported by Biotechnology and Biological Sci- Where applicable, the authors confirm that the experiments ences Research Council (UK) [grant number BB/D015758/1], described here conform with The Physiological Society ethical University of Oxford Clarendon Scholarship (UK) and Kwan- requirements. jeong Educational Foundation Scholarship (Korea). Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

129P Poster Communications

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Adaptation of CellProfiler software for high throughput Ornithine decarboxylase, key enzyme for proliferation, in measurement of cell size in phase contrast images lymphocytes of omeprazole-treated rats at different stages of stress induced gastric lesions D.C. Campbell and A. Collins O.V. Bogdanova, L.I. Kot and L.I. Ostapchenko Queen’s University, Belfast, UK Cell division and apoptosis are normal processes that are nec- Biochemistry, Taras Shevchenko Kyiv National University, Kyiv, Kyiv, essary for human health when kept in proper balance. Upset- Ukraine ting this balance can lead to a variety of diseases. Change in cell Neurogenic stress is precipitating factor for peptic ulcers. The volume is a fundamental and probably essential feature of both role of immune cells in that process is not completely studied. cell division and apoptosis, so it is important to understand The procedures in this study follow the guidelines of the local mechanisms of cell volume control. Particularly in the case of ethics committee. We used Wistar rats (6 per group) for provo- apoptosis, this mechanism is not well understood. Studying cation of neurogenic gastric ulcers (1). Following this, a group cell volume control requires an efficient method of measuring of animals was treated each day with antiulcer drug Omepra- cell size in populations of cells. The method of choice is usually zole (OME) with dose of 0.8 mg/kg body weight. Animals were flow cytometry, which involves expensive equipment and high sacrificed by decapitation immediately and each day after stress running costs that may tend to prohibit speculative pilot stud- until gastric lesions were present. Lymphocytes from spleen ies. We aimed to develop a cheap and easy method for meas- and thymus were isolated. Activity of ornitine decarboxylase uring cell size that could generate pilot data to be followed up (ODC), key regulator of polyamine synthesis, was measured in with more sophisticated methods. CellProfiler (1) is a software triplicates(2) to estimate proliferative ability of the cells. All of package that was designed for the automated analysis of fluo- the data were expressed as mean+/-S.E.M. The significance was rescence images, which feature high contrast between labelled calculated using One-Way ANOVA and t-test in STATISTICA 5.0. cells and the background, enabling the software to identify cells A decrease in ODC activity in spleen lymphocytes was shown in the image. On the other hand, phase contrast images tend on days 2, 4 and 5 after stress (table 1). More ODC inhibition not to have a high contrast between the cell interior and the was observed in OME treated animals during 1-5 days after ulcer background, but show an edge at the boundary of the cell. Cell- provocation. OME injections to unstressed animals resulted in Profiler has an edge finder module that we have exploited in 1.5 fold ODC activation on day 2, but 7- to 11- fold inhibition developing a pipeline that can automatically measure the sizes on the last two days. of cells in a large number of phase contrast images. The pipeline In thymocytes a 2.8- fold decrease in ODC activity after stress also incorporates upper and lower size cutoffs to rule out cell was followed by an increase on days 3 and 4 (table 2). OME treat- clusters and debris. Phase contrast images were taken of Jurkat ment resulted in ODC hyperactivation on the first and the last cells in culture with a Nikon Diaphot inverted microscope fit- two days. In unstressed animals treated with OME, 2.4- and 5.2- ted with a Nikon D-40 digital camera. Images were saved as jpg fold increases in this parameter were observed on the first two files for analysis by CellProfiler. The pipeline was validated days. These data reveal different changes in proliferative against manual analysis using Scion Image software (Scion Cor- processes in spleen and thymus lymphocytes during stress ulcer poration, Frederick, MD, USA). The jpg files were converted to recovery and OME treatment. tif files for Scion Image. In 5 images of well separated Jurkat cells Table 1. ODC activity in spleen lymphocytes, μmol putrescine×mg-1×min-1 in culture, manual analysis identified 236 cells with profile areas of (mean ± s.d.) 6608 ± 2466 pixels, while CellProfiler identified 185 cells with profile areas of 6190 ± 3175 pixels. Although Cell- Profiler missed some cells, this result indicates that it could be a useful tool for analysing changes in the size distribution of cell populations imaged with phase-contrast microscopy. * - P less then 0.05, n=6 Carpenter AE et al. (2006). Genome Biol 7, R100. Table 2. ODC activity in thymus lymphocytes, μmol putrescine×mg-1×min-1 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

* - P less then 0.05, n=6 Biswas K. et al. (2003) J Biol Chem. 278, 10993-11001. Ngo TT et al (1987) Anal Biochem. 160, 290-293.

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

130P Poster Communications

Where applicable, the authors confirm that the experiments PC97 described here conform with The Physiological Society ethical requirements. Adult rat mesenchymal stem cell chondrogenesis: a role for the cannabinoid system? K.K. McKayed1,2 and V.A. Campbell1,2 PC98 1Physiology, Trinity College Dublin, Dublin, Ireland and 2Trinity Insulin stimulates vascular endothelial growth factor Centre for Bioengineering, Trinity College Dublin, Dublin, Ireland receptor 2 production via RAS pathway in human retinal pigment epithelial cells Mesenchymal stem cells (MSCs) are multipotent progenitor cells which give rise to specialised skeletal cells, including chon- P.C. Kothary, D.J. Rocke and M.A. Del Monte drocytes, osteocytes and adipocytes. Under the appropriate Ophthalmology and Visual Sciences, University of Michigan, Ann conditions, these cells can be manipulated in vitro for the devel- Arbor, MI, USA opment of new tissue such as cartilage. Thus, MSCs pose as an Intensive insulin treatment causes a transient worsening of dia- ideal cell source for use in skeletal tissue engineering. When betic retinopathy (PDR) (1). Vascular endothelial growth fac- isolated from a donor source, the cells retain their multipo- tor (VEGF) upregulation and pathological human retinal pig- tent potential in vitro and can differentiate along a specific lin- ment epithelial (hRPE) cell proliferation have been implicated eage, depending on the local environment in which they reside. in PDR (2,3). Since very little is know about VEGF receptors, This study focused on elucidating the stimuli that enhance MSC we investigated the role of the RAS signaling pathway in VEGF differentiation along the chondrogenic route. Hypoxic signalling receptor2 (VEGFR2) production in cultured hRPE cells. through hypoxia inducible factor-1α (HIF-1α) has been shown Cultured hRPE cells were stimulated with insulin and insulin + to promote chondrogenesis [1] and non-hypoxic stimulation mevastatin (5μM), an inhibitor of post translation prenylation of HIF-1α is currently under investigation. The endogenous of the RAS molecule. The effect of insulin and mevastatin on cannabinoid system has recently been implicated in skeletal cellular proliferation and cell viability were assessed by meas- physiology [2]. Thus, the link between cannabinoids and chon- uring 3H-thymidine incorporation (3H-thy) and direct cell drogenesis was examined in this study, including their possi- counting using the trypan exclusion method (T) respectively. ble role as non-hypoxic regulators of HIF-1α. VEGFR2 synthesis was measured by immuno-precipitation of MSCs obtained from the femora and tibiae of 3 month old Wis- 14C-methionine-VEGFR2 and immunocytochemical methods. tar rats were cultured with chondrogenic growth factors Data were analyzed by student t-test. p < 0.05 was considered (100nM dexamethasone, 100ng/ml, transforming growth fac- as significant difference. tor-β, 50mM ascorbic acid-2-phosphate), Δ9-tetrahydro- Insulin stimulated hRPE proliferation and cell viabilty in a dose cannabinol (Δ9-THC, 5μM) or URB597 (1μM) for 3 weeks. dependent manner as measured by 3H-thy and T. The addi- URB597 inhibits the fatty acid amide hydrolase enzyme that tion of mevastatin inhibited insulin-stimulated hRPE prolifera- degrades anandamide and functions in enhancing endo- tion as measured by 3H-thy incorporation in hRPE cells (1033.28 cannabinoid tone. Cells were examined for proteoglycan dep- ±67.86 vs 154.65±18.39, CPM ±SEM, N=10, p<0.05). Insulin osition and expression of collagen II, as markers of chondroge- also stimulated 14C-methionine-VEGFR2 synthesis in hRPE cells nesis. Histological analysis of proteoglycans revealed a in a dose dependent manner as measured by immunoprecipi- significant increase from 5.35±0.52 (mean±SEM, arbitrary units) tation assay. The addition of mevastatin inhibited insulin-stim- in control cells to 10.03±1.37 in cells treated with chondrogenic ulated VEGFR2 synthesis (1337.38 ±78.47vs 138.12±9.23, CPM growth factors for 3 weeks (P<0.05, ANOVA, n=6). A similar ±SEM, N=6, p<0.05) as measured by 14C-methionine-VEGFR2 increase was observed from 4.58±0.34 in control cells to immunoprecipitation. This was qualitatively confirmed by 6.24±0.80 in Δ9-THC treated cells and to 6.70±0.38 in URB597 immunocytochemical staining using anti-VEGFR2 and rho- treated cells (P<0.05, ANOVA, n=6). Immunocytochemistry has damine-conjugated anti-rabbit IgG. shown that 3 week treatment with chondrogenic growth fac- Insulin is a mitogen for hRPE cells and stimulates intracellular tors, Δ9-THC or URB597 induced an increase in collagen II VEGFR2 synthesis. Both hRPE cell proliferation and VEGFR2 syn- immunoreactivity (n=6). The HIF-1α inhibitor, Vitexin (20μM), thesis were inhibited by mevastatin. This suggests that RAS sig- downregulated HIF-1α expression and attenuated the expres- naling plays a key role in regulation of VEGFR2 synthesis and sion of collagen II, as assessed by immunocytochemistry (n=6). secondarily insulin stimulated hRPE proliferation. Our data sug- In conclusion, this study has demonstrated a regulation of MSC gest that an inhibitor of the RAS pathway may be of therapeu- chondrogenesis by cannabinoids, under normoxic conditions tic value in treating PDR. via a pathway that involves, in part, HIF-1α. Optimisation of the 1. Henricsson, M, et al. Progression of retinopathy after change of treat- in vitro conditions for chondrogenic differentiation of MSCs will ment from oral antihyperglycemic agents to insulin in patients with have a significant impact in treating musculoskeletal defects NIDDM. Diabetes Care 1995. 18:1571-1576. in the ever advancing field of skeletal tissue engineering. 2. Aiello, LP, et al. Vascular endothelial growth factor in ocular fluid of 1. Kanichai M, Ferguson D, Prendergast PJ, Campbell V (2008). patients with diabetic retinopathy and other retinal disorders. N Engl Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: a J Med 1994. 331:1480-1487. role for AKT and hypoxia-inducible factor (HIF)-1. J Cell Physiol 216(3), 708-15. 3. Baffert, F, et al. Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling. Am J Physiol Heart 2. Idris AI, van t’Hof RJ, Greig IR, Ridge SA, Baker D, Ross RA & Circ Physiol 290: H547-H559, 2006. Ralston SH (2005). Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat Med 11, 774-779. Supported by an endowment from the Skillman Foundation. 131P Poster Communications

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC100 requirements. The role of nitric oxide in the diurnal variation in excitation- contraction coupling in ventricular myocytes PC99 H.E. Collins and G. Rodrigo

Calcium Handling in Superior Cervical Ganglia is enhanced Cardiovascular Sciences, University of Leicester, Leicester, UK in Prehypertensive Spontaneously Hypertensive rat Over 10% of the rat cardiac genes exhibit diurnal variations, and D. Li, C. Lee, K. Buckler and D.J. Paterson cycling of metabolic genes link metabolic activity and oxygen Department of Physiology, Anatomy and Genetics, University of consumption with peak power. We have demonstrated that Oxford, Oxford, UK key elements of E-C coupling in rat ventricular myocytes show strong diurnal rhythms, including their response to the non- Hypertension is associated with noradrenergic hyperactivity specific β-adrenoreceptor agonist isoproterenol, which stimu- and oxidative stress(1). We hypothesized that Ca2+ signalling lates β1, 2 and 3-receptors. As β3-adrenoreceptor stimulation in postganglionic sympathetic neurons is enhanced in sponta- involves activation of nitric oxide (NO)-dependent pathways neously hypertensive rats (SHR). To test this, we measured through coupling to nitric oxide synthase (NOS), we looked at depolarisation (high [K+]0) evoked Ca2+ influx in sympathetic the involvement of NOS in the diurnal variation of E-C coupling neurons from hypertensive and normotensive Wistar-Kyoto (Massion & Balligand, 2003; Seddon et al., 2007). (WKY) rats. Rats were humanely killed by an approved Home Adult male Wistar rats were housed with a 12 hour light/dark Office schedule 1 method. Superior cervical ganglia (SCG) were cycle. Hearts were excised from animals at two opposing time- enzymatically isolated from age and gender matched SHR and points of ZT3 and ZT15, where ZT0 refers to “lights on”, and WKY rats and plated onto poly-D-lysine/ laminin coated 6 mm single left ventricular myocytes were isolated by enzymatic cover slips and cultured 2-3 days in N2 medium(2). Intracellu- 2+ digestion. Measurements of [Ca ]i were made using Fura-2 lar Ca2+ concentration ([Ca2+]i) was measured with ratio imag- and L-type Ca2+ channel density was determined electrophys- ing using fura-2(3). Neurons were briefly perfused with Tyrode’s iologically using the whole cell, patch clamp technique. We solution containing 100 mM KCl to induce membrane depo- looked at the modulating effect of the non-specific NOS larization, and the resultant transient increase in [Ca2+]i was ω 2+ inhibitor N -Nitro-L-arginine (L-NNA) on systolic [Ca ]i and L- determined. In neonatal (4-7 days) rats, [Ca2+]i transients were type Ca2+-current density at rest and during β-adrenergic stim- significantly higher in the SHR (2.65 ±0.11 μM, n=14) compared ulation with isoproterenol. L-NNA (500μM) had no significant with the WKY rats (1.88 ± 0.12 μM, n=9, p<0.001, unpaired t- 2+ 2+ effect on the basal systolic [Ca ]i and L-type Ca -current test). In young pre-hypertensive (4-5 weeks) rats, [Ca2+]i tran- recorded from ZT3 or ZT15 myocytes in normal Tyrode. How- sients were higher in the SHR (male: 2.51 ± 0.34 μM, n=14; ever, following stimulation with 5nM isoproterenol, L-NNA had female: 1.98 ± 0.13 μM, n=23) compared with the gender 2+ 2+ a stimulatory effect on both systolic [Ca ]i and L-type Ca - matched WKY rats (male: 1.81 ± 0.30 μM, n=14, p<0.05, current density. This stimulation was greater in ZT15 compared unpaired t-test; female: 1.66 ± 0.10 μM, n=24, p=0.07). In adult 2+ ± to ZT3 myocytes, with an increase in systolic [Ca ]i of 32.6 (16-18 weeks) rats the [Ca2+]i transients were also significantly 7.5% (n=29) in ZT3 myocytes vs. 68.8 ± 9.9% (n=46) in ZT15 increased in the SHR (male: 3.70 ± 0.21 μM, n=11; female: 2.93 myocytes (S.E.M; students t-test, P<0.05) and an increase in ± 0.22 μM, n=13) compared with the gender matched adult current density of 2.7 ± 2.4% (n=12) in ZT3 myocytes vs. 15.4 WKY rats (male: 2.46 ± 0.17 μM, n=11, p<0.01, unpaired t-test; ± 2.6% (n=13) in ZT15 myocytes (S.E.M; students t-test, P<0.01). female: 1.83 ± 0.14μM, n=19, p<0.001, unpaired t-test). Inter- Quantitative RT-PCR analysis of snap frozen left ventricular tis- estingly, there were no gender differences in young SHR and sue revealed no significant difference in expression levels of the WKY rats, whereas in adult SHR and WKY, [Ca2+]i transients L-Type Ca2+-channel (cacna1c) or eNOS between time points. were significantly higher in male than female (both p<0.05, However, we detected a significant 5-fold increase in nNOS unpaired t-test). These results demonstrate that increased Ca2+ mRNA levels in ZT15 over ZT3 myocytes (S.E.M; n=4, students transients were observed in postganglionic sympathetic neu- t-test on ΔCt values, P<0.05). rons from newborn pre-hypertensive SHR when compared with Our data shows that the diurnal variation in the sensitivity of the WKY. The overall responses are consistent with the hypoth- E-C coupling of rat ventricular myocytes to stimulation with esis that enhanced Ca2+ signalling may contribute to the nora- isoproterenol, and this appears to involve NOS. drenergic hyperactivity that precedes hypertension itself. Li D et al.(2007). Hypertension 50(1),69-74. HECollinshasaDepartmentofCardiovascularSciencesstudentship He Y & Baas PW(2003). Methods Cell Biol 71,17-35. Where applicable, the authors confirm that the experiments Wang L et al.(2007). J Mol Cell Cardiol 43(6),717-725. described here conform with The Physiological Society ethical requirements. This work was supported by British Heart Foundation and Well- come Trust. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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Where applicable, the authors confirm that the experiments PC102 described here conform with The Physiological Society ethical requirements. Mitochondrial Oxidative Responses To Increased Work Intensity In Rabbit Ventricular Myocytes Assessed By Intrinsic Fluorescence Methods PC103 I.A. Ghouri, O.J. Kemi and G.L. Smith Institute of Biomedical and Life Sciences, University of Glasgow, The effect of increased frequency of field stimulation on Glasgow, UK cardiomyocytes isolated from female apolipoprotein E knockout mice fed normal or Western style high-fat diet Cardiomyocytes are intrinsically fluorescent and spectroscopic analysis of rabbit ventricular myocytes indicated that the major- J. Hawi2, L. Hua1, S.L. Passey1, C.L. Jackson1, G. Angelini1 and ity of this fluorescence arises from the metabolic coenzymes M. Suleiman1 nicotinamide adenine dinucleotide (NADH) in the reduced state 1Clinical Science at South Bristol, Bristol Heart Institute, University and flavin adenine dinucleotide (FAD) in the oxidised state. The of Bristol, Bristol, UK and 2Faculty of Medicine, University of mitochondrial redox state of single cells was assessed using the Balamand, El-Koura, Lebanon mitochondrial inhibitors p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) and cyanide. Treatment with 2μM Apolipoprotein E knockout (apoE-/-) mice fed a high-fat, West- FCCP established a completely oxidised state, resulting in NADH ern-style diet for 6 months are grossly hypercholesterolaemic fluorescence decreasing to a minimum and FAD fluorescence and develop atherosclerotic lesions in the brachiocephalic artery increasing to a maximum. Treatment with cyanide (2mM) and aorta [1, 2]. In contrast to their male counterparts, female established a completely reduced cellular state, generating apoE-/- mice on high fat diet do not show evidence of occlusive maximal NADH and minimal FAD fluorescence. coronary lesions or myocardial infarction. The aim of this work The mitochondrial redox state of single rod-shaped myocytes was to investigate whether high-fat diet alters the character- was examined with confocal microscopy. NADH was excited at istics of Ca2+ transients of myocytes from these female mice 405nm and FAD at 488nm. From this, the average NADH redox during field stimulation at different frequencies. state was calculated as 0.58±0.03 (n=22) and the average redox Female apoE-/- mice at 8 weeks old were fed either a high fat, state of FAD was calculated as 0.17±0.01 (n=22). Similar exper- Western-type diet (21% fat; 0.15% cholesterol) or were main- iments using 2-photon excitation fluorescence microscopy tained on normal rodent diet for approximately 6 months. Car- (exciting at 720 and 750nm) revealed a comparable value for diomyocytes were isolated as described previously [3]. Car- NADH redox state of 0.57±0.04 (n=20), although FAD fluores- diomyocytes were also isolated from hearts of female wild type cence could not be clearly detected with this method. C57Bl/6 mice fed a standard rodent diet for comparison. Ven- Measurements of intrinsic fluorescence were then utilised in tricular myocytes were superfused with normal Tyrode solu- order to assess the mitochondrial redox response of cardiac tion in a chamber on the stage of an inverted microscope. Sin- cells to increased energy demand. Isolated cardiomyocytes gle myocytes were chosen based on the criteria that they were were field stimulated and fractional shortening simultaneously rod-shaped with clear cross striations and well-defined edges, recorded with epifluorescence measurements of NADH and and were able to contract in response to field stimulation with FAD. Cells were paced at 0.5Hz and the stimulation frequency no spontaneous contractions. Myocytes were loaded with 5 step increased to 1Hz, 2Hz and 3Hz in order to increase work μM of the fluorescent indicator Fura-2. Cardiomyocytes were intensity and energy demand. NADH was excited at 340nm and stimulated for 2 min at 0.2Hz followed by increasing the fre- FAD was excited at 430nm. Step increasing stimulation frequency of stimulation every 30 seconds to 0.5, 1 & 2 Hz and quency resulted in a decrease in NADH fluorescence and an Ca2+ transients (Fura ratio) were measured using photometry. increase in FAD fluorescence, indicating oxidation of the cell At 0.2Hz, wild type cardiomyocytes had larger transient ampli- environment. The magnitude of response was dependent on tude, faster time to peak and time to decay compared to both stimulation frequency. The greatest response was obtained groups of apoE-/- cardiomyocytes. The Ca2+ transient ampli- when pacing was increased from 0.5Hz to 3Hz, NADH fluores- tude at 0.2 Hz was significantly smaller in apoE-/- cardiomy- cence decreased by 11.16±1.42% and FAD increased by ocytes fed high-fat diet compared to those fed normal diet 11.74±1.34% (n=22). Reducing work intensity back to 0.5Hz (ratio: 0.41 ±0.03 vs. 0.65 ±0.08, p<0.05, t-test). Data shown pacing resulted in immediate recovery of metabolite fluores- are the mean ± S.E. for n=45 cells from 6 mice for apoE-/- high cence. fat, n=24 cells from 3 mice for apoE-/- normal diet and n=16 cells In conclusion, the majority of intrinsic fluorescence from iso- from 3 C57Bl/6 wild-type mice. As stimulation frequency lated heart cells could be attributed to the metabolic coen- increased, the amplitude of the calcium transients decreased, zymes NADH and FAD. Metabolic inhibition enabled modula- with myocytes from apoE-/- high fat hearts continuing to show tion of the oxidative status of these enzymes, allowing for smaller transient amplitudes than myocytes from apoE-/- hearts calculation of relative redox state. Simultaneous measurements fed a standard diet. Times to peak and time to decay of the of redox status and fractional shortening in field-stimulated calcium transients decreased as the stimulation rate increased, cells have demonstrated that rabbit ventricular myocytes and was similar for both groups of cardiomyocytes. become oxidised when work rates are increased, suggesting a This work demonstrates that apoE-/- cardiomyocytes have transient mismatch between energy supply and demand. altered calcium handling characteristics and that these alterations are accentuatedby high fat diet feeding,indicatingimpor-

133P Poster Communications tant differences in myocyte function in the hearts of athero- diomyocytes, myofibrillar loss, vacuolisation and large clusters scleroticfemalemiceintheabsenceofcoronaryarterydisease. of cells showing end stage apoptosis. Morphometric analysis Coleman, R., et al., A mouse model for human atherosclerosis: long- undertaken in 25 fields for each parameter indicated a mod- term histopathological study of lesion development in the aortic arch est reactive hypertrophy, reductions in capillary area fraction, of apolipoprotein E-deficient (E0) mice. Acta Histochem, 2006. 108(6): significant reduction in capillary to myofiber ratio (1.03±2.6 vs. p. 415-24. 0.74±5 ratio units) and a significant increment in collagen area Williams, H., et al., Characteristics of intact and ruptured atheroscle- fraction at 15 weeks of treatment (12.75±0.33%) compared rotic plaques in brachiocephalic arteries of apolipoprotein E knockout to age-matched controls (5.14±18%) and 6-8 week STZ-treated mice. Arterioscler Thromb Vasc Biol, 2002. 22(5): p. 788-92. rats (7.99±0.91%). (P<0.05, students t test).The results indicate Chase, A., et al., Coronary artery disease progression is associated with that STZ-induced type 1 diabetes mellitus results in the devel- increased resistance of hearts and myocytes to cardiac insults. Crit Care opment of a cardiomyopathic phenotype characterised by func- Med, 2007. 35(10): p. 2344-51. tional abnormalities manifesting as impaired Ca2+ homeosta- This work was supported by the British Heart Foundation sis and contractility that appear to precede the development of histopathological changes. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

PC104 PC105 Temporal Characteristics of Phenotypic Alteration and its Functional Correlate in the STZ-induced Diabetic Rat Heart: Modified electrode placement changes wave amplitudes but Preliminary Observations not ST height on the 12 lead electrocardiograms of healthy subjects A. D’Souza, N.M. Woods and J. Singh J.P.Sheppard1, T. Barker2, T.H. Clutton-Brock3, M.P.Frenneaux2 School of Forensic and Investigative Science, University of Central and M.J. Parkes1 Lancashire, Preston, Lancashire, UK 1 The current article represents a set of preliminary observations School of Sport & Exercise Sciences, University of Birmingham, Birmingham, UK, 2Department of Cardiovascular Medicine, in the investigation of the mechanisms underlying type-1 dia- 3 betic heart disease with primary reference to alterations in his- University of Birmingham, Birmingham, UK and Department of tological features of the myocardium and assessments of con- Anaesthesia and Intensive Care, University of Birmingham, tractile function in streptozotocin (STZ)-treated type 1 diabetic Birmingham, UK male Wistar rats compared to age-matched controls. 6-8 weeks Wilson et al.,1934 argued that modifying the placement of ECG following STZ-administration (40 mg/kg), ventricular action electrodes on the limbs should have no effect on ECG wave potentials were measured in the isolated, spontaneously beat- amplitudes unless they are moved onto the torso. Despite this, 2+ ing Langendorff perfused rat heart. Contraction and [Ca ]i limb electrodes are routinely placed on the torso during exer- transients were measured in electrically stimulated ventricular cise stress testing or when it is inconvenient or impossible to myocytes by a Video Edge Detection system.At time points of position them on the extremities. Under these conditions, mon- 6-8 and 15 weeks,isolated ventricular tissue was processed for itoring the ECG, and in particular the ST segment is of great quantitative assessment of fibrosis, measurements of capillary importance when diagnosing suspected myocardial density, myocyte diameter and myofiber to capillary ratio using ischemia/infarction. There remains no agreement on exactly routine staining techniques. Measurements of ventricular how such electrode modification affects the 12 lead ECG (Mason action potential indicated that heart rate (calculated in & Likar, 1966; Takuma et al., 1995). beats/minute) was significantly reduced (P<0.05, students t We have therefore made 12 lead ECG measurements in 18 test) in the STZ-treated rats (174±13 bpm, n=6) compared to healthy subjects using both standard and modified electrode controls (238±12 bpm, n=5).The amplitude of contraction (as placements with the arm electrodes placed on the “anterior a percentage of resting cell length) was significantly greater acromial region” and the leg electrodes on the “anterior supe- (P<0.05, students independent samples t test) in STZ-induced rior iliac spine” (Takuma et al., 1995). Data was collected with diabetic myocytes (5.3±0.23% n=35) compared to age matched a customised data collection system that averages ~120 heart controls (3.67±0.5%, n=33). The amplitude and time to peak beats per subject and performs automated offline analysis of of Ca2+ transients were not significantly altered (P>0.05, stu- the ECG waveform. Mean ± sem wave amplitudes were com- dents t test) in the STZ treated myocytes whereas decay of the pared using a two tailed paired T-test with the Bonferroni cor- Ca2+ transient was significantly longer (P<0.05, students t test) rection for multiple comparisons. in myocytes from STZ-treated (67±3 milliseconds, n=16) com- As expected, mean R and T heights in the precordial leads were pared to control rats (52.8 ± 12 milliseconds, n=20). Visual not significantly different using the modified electrode place- analysis of stained sections did not reveal any major abnor- ment (all < 0.01mV). R height and T height changed signifi- malities at 6-8 weeks following STZ-administration in control cantly by at least 0.06 ± 0.01mV in all the limb leads (except for vs. STZ-treated groups compared to the marked structural alter- lead aVR). In a clinical setting, the amplitude changes in leads ations evident in the 15 week STZ-treated right and left ven- III and aVF could be considered as potentially misleading (i.e., tricle sections presenting as focal scarring, hypertrophied car- ≥ 0.1mV). Modification produced a rightward shift of ~15∞ in

134P Poster Communications both QRS and T axes, but this would only be clinically impor- Isometric peak torque for right knee extension was 311.1±57.1 tant in patients with a borderline axis in the standard ECG. In N.m and 300.6±57.0 N.m measured in concentric contraction. no leads did clinically important ST depression or elevation Flexion peak torque was 178.6±28.1 N.m (isometric) and occur (i.e., none had elevated or depressed J / ST(80) points by 175.4±31.1 N.m (concentric). Bland-Altman plots indicated ≥ 0.1mV) and the largest change in ST height was only 0.03 ± that the limits of agreement between the two methods were 0.03mV (V3). +82.1 and -61.2 N.m for right knee extension and +50.0 to - Moving the limb electrodes of a 12 lead ECG to the modified 43.5 N.m for flexion, suggesting poor agreement between the positions for exercise stress testing and emergency monitor- two methods. ing (when the limbs are inaccessible) does produce measura- Angle of peak torque for right knee extension was 105.9±7.3∞ ble and reproducible changes in ECG wave amplitudes in healthy when measured isometrically and 111.5±6.8∞ measured con- subjects. It does not however, produce a misleading ST seg- centrically (significantly different, P<0.01, t-test). The same ment change. Modified electrode placement therefore appears values for flexion were 157.3±4.6∞ and 159.5±5.6∞ respec- acceptable for ST segment monitoring under these commonly tively. Figure 1 illustrates the normalised torque-angle rela- used conditions. tionships for knee extension using both methods. There was a Mason RE & Likar I (1966). American Heart Journal 71, 196-205. significant method-angle interaction (P<0.01) for both knee Takuma K, et al., (1995). American Journal of Emergency Medicine 13, flexion and extension, suggesting a lack of agreement between 514-517. the methods. Wilson FN, et al., (1934). American Heart Journal 9, 447-458. These results suggest that dynamic measurement of angle- torque relationship at this angular velocity does not closely Where applicable, the authors confirm that the experiments agree with isometric measurement. described here conform with The Physiological Society ethical requirements.

PC106

Comparison of dynamic and isometric assessments of muscle angle-torque relationships in human knee extensors and flexors A.E. Donnelly, C.A. Cuddihy, S.T. O’Brien, K.D. Higgins, C.N. Mason and A. Shafat Department of Physical Education and Sport Sciences, University of Limerick, Limerick, Ireland The assessment of length-tension relationship in human mus- Figure 1. Torque-angle relationship for right knee extension. Values are ± cle is cumbersome, and is usually derived from isometric meas- mean SD, * = P<0.05 difference between points, ANOVA with 95% confidence intervals. urements of the angle-torque relationship. Slow dynamic con- Proske U, Morgan DL, Brockett CL, Percival P (2004) Clin. Exp. Pharm tractions may offer an efficient and accurate alternative (Proske Physiol 31: 546-550. et al., 2004). The aim of this study was to compare these two methods for measurement of torque-angle relationship in Where applicable, the authors confirm that the experiments human knee extensors and flexors. described here conform with The Physiological Society ethical Twenty-two male Gaelic football players (aged 21.9±1.4 yrs, requirements. height 184.5±6.0 cm, body mass 85.7±7.7 kg, all data mean±SD) participated in this study after giving written informed consent. Both dynamic concentric and isometric measurements of left and right legs were made in a single test- PC107 ing period one week after a familiarisation session. Volunteers were seated on an isokinetic dynamometer with knee range 50 min. of aerobic exercise with a caloric expenditure of ~540 of motion set from 80∞ to 160∞ (where 180∞ is full extension). Kcal. modifies short-term leptin response to CHO intake For each leg, a single maximal dynamic contraction at an angu- A.J. Lopez-Davila and A. Fernandez-Ramirez lar velocity of 0.52rad.s-1 (30o/sec) was performed for both Department of Physiology, University of Costa Rica, San Pedro, San knee extension and flexion. Volunteers then performed two Jose, Costa Rica sets of isometric maximal voluntary contractions of the knee extensors and flexors every 10 degrees through the range of Acute effect of aerobic exercise on serum leptin concentration motion with 30 seconds rest between contractions. Angle of has been subject of inconsistent results. A recent review con- peak torque was extracted using both methods and compared cluded that, in order to modify serum leptin concentration a using t-tests. Torque-angle relationship were normalised for single session of aerobic exercise must either be at least longer peak torque in each individual and the two methods were com- that 60 min. or consume more than 800 Kcal (Bouassida et al pared using repeated measures ANOVA; Bland-Altman plots 2008). The present study was designed to assess the effects of were used to assess agreement between the two methods. 50 min. of aerobic exercise with a caloric expenditure of ~540

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Kcal. on short-term serum leptin levels in fasting subjects and of Helsinki (revised in 2000). The hospital ethic committee following carbohydrate (CHO) intake. Ten healthy and physi- approved the protocol and we obtained informed consent from cally-active males were randomly assigned to four testing con- all subjects before sampling took place. ditions: a) FR: fasting-rest b) FE: fasting-exercise; c) BR: break- Bypass patients had lower numbers of CD34+/CD144+ EPCs, fast-rest; d) BE: breakfast-exercise. Blood samples were compared to valvular (0.2239±0.032% vs 0.4370±0.08%, collected every 30 minutes during the 8-hour testing condi- p=0.0187), and lower numbers of CD34+/CD144+/CD3- EPCs tions for analysis of plasma leptin, insulin and blood glucose. (0.3309±0.0.054% vs 0.7786±0.1922%, p=0.026). However, Data were analyzed by two way repeated measures ANOVA and cultured EPCs from healthy donors exhibited a lower apoptotic simple effects follow up. During all testing conditions, signifi- rate after 24, 48 or 72h incubation with 1% plasma from bypass cant reductions in the serum leptin levels were observed (p < patients, compared to valvular (0.9998 vs 2.038, 0.9971 vs 0.05). Our data show that: 1. Aerobic exercise did not induce 1.951 y 0.998 vs 1.599, respectively), being this effect pre- direct alterations of serum leptin levels neither in fasting nor in vented by TGFbeta blockers SIS3 and SB431542. Incubation fed subjects since no significant differences were detected when with 1% plasma from bypass patients induced higher expres- comparing BR vs. BE and FR vs. FE conditions. 2. Exercise dimin- sion of CD105 and CD144, (measured by Western blot in at least ished postprandial glucose and insulin concentrations: the BE two different experiments, using plasma from three different condition elicited increases in glucose and insulin levels, but patients each one). these were significantly lower than for the BR condition (p < Atherosclerotic patients possess a lower pre-surgical number 0.05). 3. A single meal (439 Kcal.) with a high CHO content of EPCs, compared to valvular. EPC dysfunction in culture is not induced an increase of serum leptin concentration: starting at observed in EPCs from healthy donors incubated with plasma 14:00 hours (five hours after CHO intake), serum leptin in the from atherosclerotic patients. The widely described in vitro EPC BR condition was significantly higher than in the FR condition dysfunction in atherosclerotic patients may be due to intrinsic (p < 0.05). 4. Aerobic exercise abolished the observed CHO defects in these cells from the bone marrow. effect on serum leptin levels since no significant differences were found when comparing FR and BE conditions. Thus, 50 Where applicable, the authors confirm that the experiments min of aerobic exercise with a caloric expenditure of ~540 Kcal. described here conform with The Physiological Society ethical is sufficient to modify leptin response to feeding. requirements. Bouassida A, Chamari K, Zaouali M, Feki Y, Zbidi A, Tabka Z. Review on leptin and adiponectin responses and adaptations to acute and chronic exercise. Br J Sports Med. 2008 Oct 16 (Epub ahead of print). PC109 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Leg vascular conductance kinetics in older versus younger requirements. men during constant load calf plantar flexion exercise

H.N. Reilly1, M. Egana1 and S. Green2 PC108 1Physiology, Trinity College, Dublin, Ireland and 2Physiology, University of Otago, Dunedin, New Zealand Low endothelial progenitor cell number in peripheral blood of atherosclerotic patients is not due to plasma-induced in Older men exhibit a preserved vascular conductance (VC) vitro dysfunction response to incremental leg extension exercise compared to active young men1. However, the age-related effects on the S. Redondo1,2, A. Gonzalez-Rocafort3, M. Carnero3, F.Reguillo3, rate at which the leg vascular conductance response increases E. Rodriguez3, J. Navarro-Dorado1, M. Ramajo1 and T. Tejerina1 at the onset of constant load exercise (VC kinetic response) has not yet been examined. 1Pharmacology, Universidad Complutense, MADRID, NC, Spain, To examine the vascular conductance kinetics response during 2Hemathology, Hospital Clinico San Carlos, Madrid, Spain and calf constant-load plantar-flexion exercise performed at dif- 3Cardiac Surgery, Hospital Clinico San Carlos, Madrid, Spain ferent intensities in older and younger men We aimed to study the number of endothelial progenitor cells Thirteen older (60-84 years) and thirteen younger (20-30 years) (EPCs) in atherosclerotic patients who underwent bypass graft- sedentary men were tested. Ethical approval was obtained from ing, or valve replacement. Plasma from these patients was the Trinity College Dublin Faculty Research Ethics Committee. added to cultured EPCs from healthy blood donors in order to Subjects performed three constant load exercise bouts (6 min try to reproduce in vitro what found in vivo. long) of intermittent calf plantar flexion exercise (6s duty cycle: EPC number was assessed by flow cytometry, as percentage 2 s contraction, 4 s relaxation) at an intensity of 30% maximum of CD34+/CD144+/CD3-, CD34+/KDR+/CD3- and voluntary contraction (MVC) on a custom-built calf ergometer CD14+/CD105+/CD3- mononuclear cells. 44 bypass patients at a tilt of 67 degrees. This was then repeated at an intensity of and 43 valvular patients were chosen. For EPC cultures, 45% MVC. Calf BF was measured contraction by contraction mononuclear cells were obtained from healthy blood donors using venous occlusion plethysmography. Kinetic analysis was and separated using ficoll. Cells were plated on fibronectin and performed by fitting a biexponential function to the mean (3 incubated with MV2 medium. Apoptosis was assessed by DNA bouts) of the vascular conductance (BF/MAP) data. Student t- fragmentation. Protein expression was measured by Western tests were used to detect differences between groups. Results blot. The study was conducted according to the Declaration are depicted as mean ± SD.

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The time constant of the fast component was significantly measures changed over time (P<0.05) except for torque at shorter in the young (30% ; 19.3±16s: 45%; 37.5 ±13.20) com- 0.87rad.sec-1. Soreness (arbitrary scale) increased to a maxi- pared to the older (30% ;82.2 ± 8.6s :45%; 54.3±48.7) group mum at 24 hours of 34.8±13.4 (CWI) and at 48 hours 37.7±14.9 but the rest of the VC kinetic parameters, the mean response (Control), and 20:50 Hertz ratio declined to a minimum of time and the end exercise amplitude at 360s were not differ- 0.65±0.09 (CWI) and 0.65±0.08 (Control) at 24 hours. There ent between both groups. was a significant effect for differences between treatment This study showed that the rate at which vascular conductance groups only for the isometric MVC measure (P<0.05, see Fig- responses increase during constant load static calf exercise is ure 1). faster in younger men compared to older men. Muscle soreness and reduction in muscle force suggest that Parker BA, Smithmyer SL, Pelberg JA. Sex-specific influence of aging on the resistance training protocol induced delayed onset mus- exercising leg blood flow. J. Appl. Physiol 2007; 104: 655-664. cle soreness and damage. Contrast water therapy had a significant effect on the recovery of isometric force, but not on other Where applicable, the authors confirm that the experiments markers. described here conform with The Physiological Society ethical requirements.

PC110

The effects of alternating hot and cold water immersion on recovery of muscle function after resistance training in humans

L.A. Algar, K.M. Doyle, L.P. Conan, L.M. O’Sullivan and A.E. Donnelly

Physical Education and Sports Sciences, University of Limerick, Limerick, Ireland

Eccentricmusclecontractionsmayresultintemporaryrepairable Figure 1. Normalised isometric MVC force. Pre values (0) were 820.6±217.4 damage to human muscle. This is associated with muscle sore- Newtons for CWI, and 751.1±217.4 Newtons for Control. * = P<0.05 (ANOVA ness and a prolonged decline in muscle force production. Con- with 95% confidence intervals). trast Water Immersion (CWI) has been reported to accelerate Vaile J et al., (2008) Eur J Appl Physiol 102:447–455 recovery of muscle function after intense exercise (Vaile et al., Where applicable, the authors confirm that the experiments 2007).CWIinvolvesrepeatedalternateimmersioninhotandcold described here conform with The Physiological Society ethical water. The aim of this study was to evaluate the effects of CWI requirements. treatment on muscle force recovery after resistance training. Thirty-five active human volunteers (mean±S.D.; height 173.9±7.4centimetres,bodymass72±9.6kilograms,age22±2.6 years) participated in this study. The volunteers were randomly PC111 assignedtoeitheraCWIgroup(n=18)ortepidwaterimmersion control group (n=17). Each volunteer performed a familiarisa- The effect of caffeine ingestion on antigen-stimulated tionsession,apre-testsessiontorecordbaselinemeasures,a45 human T cell activation following prolonged cycling minute resistance training protocol, and four post-testing ses- D. Fletcher, P. Bowry, M. Noon and N. Bishop sions at 24, 48, 72 and 96 hours after exercise. Measures were: School of Sport and Exercise Sciences, Loughborough University, kneeextensorisometricMaximumVoluntaryContraction(MVC) Loughborough, UK force and electrically stimulated force at 20 and 50 Hertz measured at an internal knee angle of 120 degrees, concentric and Caffeine is consumed by many athletes for its known ergogenic eccentric torque measured during knee extension at 0.87 and properties. However despite the high intention by athletes to 1.74rad.s-1, and muscle soreness measured by questionnaire. use caffeine (Chester & Wojek, 2008), little research has con- TheCWIgroupcompleted4cyclesofa1minuteimmersionat centrated on its effects on human immune cell function fol- 9±1∞C alternated with 4 minutes of immersion at 39±1∞C. The lowing high intensity exercise. A study by Bishop et al. (2005) controlgroupcompleted20minutesimmersedinwaterata demonstrated that caffeine ingestion 60 min before a 90 min ± ∞ temperature of 29 1 C. Subjects were seated in the tanks, cycle at 70% VO2peak increased the natural state of activation immersed to the level of the anterior superior iliac spine. Treat- of CD4+ and CD8+ cells in vivo before and after exercise. How- mentswereadministeredimmediatelyafterresistancetraining ever, this does not necessarily suggest enhanced effector func- and 24, 48 and 72 hours later, after post tests were completed. tion when faced with an antigenic challenge. Therefore, the aim A repeated measures ANOVA was used to compare results of of the present study was to investigate the effect of caffeine the two treatment groups over five time points and 95% con- ingestion on human T cell (CD4+ and CD8+) function following fidence intervals were used to compare individual points. All

137P Poster Communications prolonged, high intensity cycling in response to antigen stimulation, as assessed by the early activation molecule CD69. PC112 Following University Ethical Committee approval, 8 healthy Interaction between pH -sensing histidine residues in male endurance trained cyclists (mean ± SD.; age 22 ± 2 years, o proton-gated, two-pore domain K+ channel TASK-3 VO 62.9 ± 3.2 ml.kg-1.min-1) volunteered to participate in 2peak 1 1 1,2 1 the study. Subjects performed two exercise trials in a single- L. Zúñiga , L. Cid , F.V. Sepúlveda and M.I. Niemeyer blind randomised crossover design. Each trial was performed 1Centro de Estudios Científicos, Valdivia, Chile and 2CIN, Centro de following an overnight fast and 60 h abstention from caffeine Ingeniería de la Innovación asociado al CECS, Valdivia, Chile containing foods and beverages. Each trial comprised 90 min TASK-3 and TASK-2 are two-pore domain, background K+ chan- cycling on a stationary ergometer at 70% VO 60 min after 2peak nels gated by extracellular pH. TASK-3 dependence upon pH ingesting 6 mg.kg-1 body mass of either caffeine (CAF) or dex- o is cooperative with K of 5.9 ± 0.05 and n of 2.1 ± 0.09 (n = 8; trose placebo (PLA) in capsule form. During the trials, subjects d H all errors quoted as SEM). The pH -sensor in TASK-3 is histi- consumed 2 ml.kg-1 body mass of water at 15 min intervals and o dine 98 located at the extracellular entrance of the pore and a further 5 ml.kg-1 body mass 5 min post-exercise. Venous blood TASK-3-H98N mutant is pH -independent (Rajan et al., 2000; samples were obtained before supplementation, pre-exercise, o Kim et al., 2000). The pH -gating of TASK-2 occurs with a pK immediately post-exercise and 1 h post-exercise. The trials were o 1/2 of 8.0, is not cooperative and is mediated by neutralization of performed at least 7 days apart. Human T cells were stimulated arginine 224, so that mutant TASK-2-R224A is pH -independ- with the Pediacel (5 in 1) vaccine at a dose of 1:4000 and 1:8000 o ent (Niemeyer et al., 2007). To find out whether both pH -sen- (optimum and sub-optimum, respectively) and incubated for o sors occurring in these dimeric channels are required in the gat- 20 h at 37 ∞C, 5% CO . Subsequent expression of CD4, CD8 and 2 ing process we have covalently linked the C-terminus of one CD69 was analysed by flow cytometry. Results were expressed channel with the N-terminus of the following to form con- relative to the pre-supplement value and analysed using a two- catenated structures containing either a normal set of pH -sen- factor (time x trial) repeated measures ANOVA. Post hoc t tests o sors (WT-WT), mixed structures containing one able and one with Holm-Bonferroni adjustment were applied where appro- neutralised pH -sensor (WT-Mut, Mut-WT) or two pH -sensing priate. Statistical significance was accepted at P<0.05. o o disabled channels (Mut-Mut). Analysis was done by transient Caffeine ingestion did not affect 1 h post-exercise concentra- expression into HEK-293 cells and patch-clamp. Gating by pH tions of total lymphocytes or percentage of CD4+ and CD8+ cells. o of TASK-3 WT-WT concatenated channels (n=8) or Mut-Mut However, at 1 h post-exercise geometric mean fluorescence (Mut = TASK-3-H98N, n=4) structures was similar to that of their intensity (density of expression) for CD69 on stimulated CD4+ non-concatenated equivalent channels. In contrast, the mixed and CD8+ cells was significantly decreased on CAF compared WT-Mut and Mut-WT followed pH in a non-cooperative man- with PLA (CD4+; PLA, 91.97% ± 20.23%; CAF, 65.94% ± 20.70%; o ner and required more acidification for inhibition to occur. The CD8+: PLA, 99.77% ± 24.13%; CAF, 79.65% ± 11.85%, P<0.05, respective pK values for TASK-3 WT-Mut and Mut-WT were n=8). These findings suggest that caffeine ingestion before exer- 1/2 4.9 ± 0.04 (n=5) and 5.1 ± 0.12 (n=6). Assuming that N98 mim- cise inhibited antigen-stimulated CD69 expression on CD4+ and + ics a neutral form of H98, we have used the K1/2 of these mixed CD8 cells 1 h after high intensity prolonged cycling. -5 constructs (10 M) as Kd2 in a fit to the data of a simplified Bishop NC et al. (2005). Eur. J. Appl. Physiol., 93, 606-613 model for the gating of TASK-3 by protons in which there are Chester N & Wojek N (2008). Int. J. Sports Med., 29, 524-528 two closed states with two or one charged H98 residues and an open state with both H98s neutralised. This analysis applied Where applicable, the authors confirm that the experiments to the WT-WT data gave a value for K = 3.26*10-7 M consis- described here conform with The Physiological Society ethical d1 tent with the idea that neutralisation of a first H98 sensor of requirements. TASK-3 affects the pKa of the second making it 30-times more susceptible H+ loss. WT-WT TASK-2 concatenated constructs behaved similarly to the non-concatenated channels with pK1/2 8.2 ± 0.15 (n=4), whilst Mut-Mut (Mut = TASK-2-R224A) TASK- 2 constructs lacked pHo-dependence. The mixed WT-Mut and Mut-WT TASK-2 constructs had pHo-dependences (respective ± ± pK1/2 values of 7.8 0.05, n=6 and 7.9 0.10, n=6) very similar to that of WT and WT-WT channels. Our data show that both pHo-sensors of TASK-3 and TASK-2 channels need to be neutralised for channel opening. There appears to be no interaction between sensors in TASK-2 channels accounting for the lack of cooperativity in the pHo effect. In TASK-3 however, the data indicate that neutralisation of one sensor profoundly affects the ability of the second sensor to become neutral thus accounting for cooperative, high-gain response to pHo. Kim Y et al (2000) J Biol Chem 275, 9340-9347 Niemeyer MI et al (2007) Proc Natl Acad Sci USA 104, 666-671

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Rajan S et al (2000) J Biol Chem 275, 16650-16657 enables us to evaluate and then ensure the model structures’ Supported by Fondecyt 1090478 and 1061069. ability to predict the current during any other clamp. Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

PC113 PC114

Tools for optimising experiments and developing predictive Protein Kinase A phosphorylation of hK2P3.1 and hK2P9.1 models: an example for ion channel kinetics carboxy-termini enables channel function G.R. Mirams, D. Noble and M. Fink D. Elliott3, P. Eyers2 and I. O’Kelly1 Department of Physiology, Anatomy & Genetics, University of 1Human Genetics, University of Southampton, Southampton, Oxford, Oxford, Oxon., UK Hampshire, UK, 2YCR Institute for Cancer Studies, University of 3 “The application of mathematics to natural phenomena is the Sheffield, Sheffield, UK and The Faculty of Biological Sciences, aim of all science, because the expression of the laws of phe- University of Leeds, Leeds, UK nomena should always be mathematical” (Bernard, 1865). The Export of newly synthesised hK2P3.1 (TASK1) and hK2P9.1 need for mathematical tools has long been acknowledged by (TASK3) channels from the endoplasmic reticulum (ER) and physiologists, in order to integrate data from past experiments delivery to the cell surface is subject to quality control mecha- with the testing of possible hypotheses about underlying mech- nisms. ER retrieval motifs that bind βCOP have been demon- anisms, and thus to guide future experimental design. strated on both the N- and C- termini of both channels (O’Kelly A synergy between the wet-lab and modelling approaches et al., 2002; O’Kelly & Goldstein, 2008; Zuzarte et al., 2009) enabled Hodgkin-Huxley, Noble and DiFrancesco to unravel the while a C-terminal motif (MKRRSSV.) recruits 14-3-3 which over- interaction of ion currents in many different cell types. The com- comes βCOP binding and enables forward transport of the chan- putational revolution led to a separation between “mathe- nels to the membrane (O’Kelly et al., 2002; Rajan et al., 2002; matical modellers” and “physiologists”, reducing communi- O’Kelly & Goldstein, 2008). 14-3-3 binding to this motif is phos- cation accordingly. A major criticism of current mathematical phorylation dependent. Previous studies have shown that while modelling in cardiac electrophysiology is that most of the there are two potential phosphorylation sites within this motif, results only replicate what is already known experimentally, 14-3-3 binding is dependent on phosphorylation of the termi- even though (or because) the mathematical models have nal serine (S393 for hK2P3.1 or S373 for hK2P9.1; O’Kelly et al., become evermore complex. 2002; Rajan et al., 2002). To date the kinase responsible for this Weproposeasetoftoolstoensurevalidatedandpredictivemod- phosphorylation is unidentified. This study presents evidence els(appliedheretoion-channelmodelsandvoltageclampexper- that phosphorylation of the terminal serine by protein kinase iments).Thesetoolsinvestigatetherelationbetweenexperimental A (PKA) is required for K2P3.1 and K2P9.1 functional expression. data,itsinformationcontent,andmodelstructure.Currently,mul- In vitro kinase assays demonstrate that alanine substitution of tiple voltage clamp step experiments are done to determine the S393 (hK2P3.1) resulted in a significant reduction in phospho- kineticsofionchannels(activation,deactivation,inactivationand rylation by PKA. When expressed in Xenopus oocytes, mutant reactivation); most often these are undertaken in different cells, channels in which the consensus PKA site was ablated (S393A and the results have to be normalised for analysis. The full set of for K2P3.1 or S373A for K2P9.1) showed a marked reduction in protocols produces redundant data and we show a much shorter current (4.0 ± 0.37 μA and 0.59 ± 0.16 μA at 60mV for WT ± μ ± protocol (length <15 s) that suffices to evaluate models’ parame- hK2P3.1 and S373A respectively and 1.35 0.31 A and 0.54 μ ters. We compare the information content of the optimized step 0.11 A at 60mV WT K2P9.1 and S373A respectively). Oocytes protocol to action-potential and ramp clamp protocols. injected with the protein kinase A peptide inhibitor (PKI; 1 nM) The optimization of experiments is a necessary step for provid- 24 hours post WT hK2P3.1 RNA injection and incubated for a ingpredictivemodels,asthereisalackofknowledgeofhowmuch, further 16 hours failed to produce currents significantly differ- and which kind of, experimental data suffices for a unique fit of a ent from that of water injected oocytes (0.23 ± 0.05 μA and 0.5 ± μ model’s parameters. As a result many models are fitted using all 0.21 A for WT hK2P3.1 plus PKI injected and water injected the available data, which can be inconsistent and leaves no data cells respectively at 60 mV). Cells injected with RNA but not PKI formodelvalidation.Amodelsimplymatchingtheexperimental gave acid sensitive currents similar to those seen previously. datadoesnotimplya“goodfit”ifthemodelisoverparameterised. Similarly hK2P9.1 injected oocytes treated with PKI also resulted We analyse simulated experimental protocols and models to in loss of WT currents (0.3 ± 0.07 μA and 1.36 ± 0.26 μA for WT establish whether there is sufficient information to fit all of the with and without PKI injection respectively at 60 mV). This data models’parametersuniquely.Thisprovidesaseparationbetween demonstrates that PKA phosphorylation of hK2P3.1 or hK2P9.1 the ‘training’ data used to fit the model parameters and ‘valida- on their terminal serine is required for functional expression tion’ data used to evaluate the predictive power of the model. of these channels. This study of each protocol’s parameter-information content O’Kelly I et al. (2002). Cell 111, 577-588. allows us to design optimised patch-clamps for voltage gated O’Kelly I & Goldstein SA (2008). Traffic 9, 72-8. ion channels. Fitting model parameters to such protocols Rajan S et al. (2002). J Physiol 545, 13-26

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Zuzarte M et al. (2009). J Physiol 587, 929-52. a parameter of the ICa,L channel inactivation time kinetics We thank the BBSRC for the support of this (pZ1) in the Zhang case. work(BB/E014453/2) Conclusions: This MI analysis shows that although there is a common set of parameters regulating model responses, there Where applicable, the authors confirm that the experiments are subtle differences. Often different models rely upon distinct described here conform with The Physiological Society ethical mechanisms to reproduce various desired responses and MI requirements. provides a global stastical measure for identification of the same. MI can further assist in parameter estimation during model development. PC115

A Systems Biology Computational Comparision of Two Caridac Pacemaker Cell Models S. Kharche1, L. Ming2 and H. Zhang1 1School of Physics and Astronomy, University of Manchester, Manchester, UK and 2School of Medicine, University of Manchester, Manchester, UK Aim: Biophysical cardiac cell models consist of coupled ordi- nary differential equations with many regulatory biophysical parameters (> 100). Model responses are differentially regulated by such model specific parameters. This study quantitatively compares the underlying prametric regulation of model responses among two rabbit pacemaker models using a systems biology approach with a novel global sensitivity mutual information index (MI). Methods: The rabbit pacemaker models developed by Kurata et al. [1] (Kurata) and Zhang et al. [2] (Zhang) were parameterically analysed in this study. Model responses in terms of action potential (AP) features were defined as maximum diastolic potential (MDP), over shoot potential (OS) andAPduration(APD90) [3]. Independent parameters associated with channel conductances, gating steady states, time constants and intracellular ionic homeostasis in the models were identified with 194 in the Kurata case, and 176 in Zhang case. The global information-theoretic sensitivity MI index has been developed in a previous study [4]. It constitutes deterministic quantification of the correlation between modelling parameters and model responses. Through multiple evaluations of the model (> 105) for randomly selected configurations of parameters, distirbutions of samples of model responses based on the perturbed parameter sets were obtained. With the large sampling, MI gave a quantitative statistical correlation between individual parameters and model responses. With such quantification, model parameters with respect to each model response were ranked from most relevant (MI = 1) to unin- fluential (MI ~ 0). This enabled models comparision at parametric level. Results: Standard AP profiles from the models are shown in Fig. 1A. Due to the differing parametric regulation of the models, the two AP profiles are different. This is reflected in (A) Standard AP profiles from Kurata (solid line) and Zhang (dashed line) the differential parametric responses as seen in Fig 1B. Rel- models. (B) Left column show relative MI data for Kurata and right column ative MI shows that MDP is mostly regulated by a compo- for Zhang. Top panels show data for MDP, middle for OS and bottom for APD . Parameters that maximally influence the respective model responses nent of the I channel activation time kinetics (p ) in the 90 Ca,L K1 are shown. See text for details. Kurata case, whilst IK,r channel conductance (gKr)inthe Kurata Y et al. (2002). Am. J. Physiol. 283, H2074–H2101. Zhang case. Reversal potential of ICa,L channel (ECa,L)regulates the OS in both models. APD90 is also regulated by dif- Zhang H et al. (2000). Am. J. Physiol. 279, H397–H421. ferentially in the two models with a parameter of the ICa,L Wilders et al. (1991). Biophys J 60 1202–1216. channel activation time kinetics (pK2) in the Kurata case and Lüdtke N et al. (2008). J. R. Soc. Interface 5(19), 223–235.

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This work was supported by Wellcome Trust (UK) project grant channels, in the regulation of PA contractility and also provide (WT/081809/Z06/Z). indirect evidence for possible involvement of mitochondria in Where applicable, the authors confirm that the experiments contraction mediated by inhibition of KV channels. described here conform with The Physiological Society ethical Archer S & Michelakis E (2002). News Physio Sci 17, 131-137. requirements. Smirnov S V et al. (2002). J Physiol 538, 867-878.

University of Bath ORS scheme PC116 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical + Role of voltage-gated K channels in rat pulmonary arteries: requirements. The tale of two channels M.M. Rahman1, G.A. Knock2 and S.V. Smirnov1 1Pharmacy and Pharmacology, University of Bath, Bath, Avon, UK PC117 and 2Division of Asthma Allergy and Lung Biology, King’s College London, London, UK IP3 rescues heterotetrameric canonical transient receptor potential 6/7 (TRPC6/C7) channel activity from inhibition by Voltage-gated K+ (K ) channels are the predominant K+ chan- V phosphatidylinositol-4,5-bisphosphate (PIP2) in rabbit nels expressed in pulmonary vasculature and have been vascular myocytes thought to play an important role in regulation of vessel excitability and contribute to hypoxic pulmonary vasocon- M. Ju, S.N. Saleh, A.P. Albert and W.A. Large striction (Archer & Michelakis, 2002). Although KV channel cur- Division of Basic Medical Sciences, St George’s University of London, rents were well characterised in isolated pulmonary arterial London, UK myocytes, their relative contribution to contraction of intact We have shown that synergism between the diacylglycerol ana- pulmonary arteries remains incompletely understood. The main logue OAG and IP on TRPC6-like channel activity in portal vein aim of this work was to investigate the contribution of the K 3 V myocytes although the mechanism governing the increase in channels in small intrapulmonary arteries (PAs) isolated from activity is not known (Albert & Large, 2003). In recent work male Wister rats (200-250 gm) using small vessel wire myog- we proposed that endogenous PIP produces a marked raphy and a specific inhibitor of K channels 4-aminopyridine 2 V inhibitory action on homotetrameric TRPC6 activity in mesen- (4-AP). Small mesenteric arteries (MAs) were used as a repre- teric artery myocytes (Albert et al, 2008). The aims of the pres- sentative of the systemic circulation. In PAs, application of 4- ent work are to investigate whether PIP also inhibits TRPC6- AP (0.5-10 mM) caused contraction typically only at 5 or 10 2 like activity in portal vein myocytes and, to study if removal of mM. Membrane depolarisation with 20 mM KCl significantly an inhibitory action of PIP by DAG and/or IP mediate open- potentiated (p < 0.001, t-test) the effect of 4-AP, probably by 2 3 ing of these channels. causing greater activation of the K channels. Pre-treatment of V In freshly dispersed single portal vein myocytes, single cation PAs with the selective inhibitor of background KCNQ channels channel currents were recorded at room temperature using the linopirdine (10 μM) similarly significantly potentiated (p < 0.004, inside-out patch configuration of the patch clamp technique t-test) concentration-dependent 4-AP induced contraction (Albert et al, 2008). Mean values are of n cells ± S.E.M. Statisti- causing a marked contraction even at 0.5 mM 4-AP, the con- cal analysis was carried out using unpaired and paired Student’s centration which block ~50% of K currents in single cells V t-test with the level of significance set at P<0.05. (Smirnov et al. 2000). Potentiation of 4-AP-induced contrac- To test the effects of PIP on OAG-evoked activity we compared tion by linopirdine and 20 mM KCl was significantly less in MAs. 2 control cells with cells pre-treated with 20 μM wortmannin for These results suggest that K and KCNQ channels are impor- V 30 min to deplete PIP levels. Mean open probability of OAG tant regulators of the resting cell membrane potential in PAs. 2 (10 μM)-induced TRPC6-like activity was significantly increased 10 mM 4-AP-induced contraction of PAs developed in the pres- from 0.03 ± 0.01 (n=9) in control cells to 2.53 ± 0.37 (n=48) in ence of 10 μM linopirdine was only partly blocked by the cells pre-treated with wortmannin at -50 mV. Application of inhibitor of L-type Ca2+ channels diltiazem (10 μM, 35±4%, n=6) PIP inhibited OAG-evoked activity with an IC value of 0.74 or by the selective Rho kinase inhibitor Y-27632 (10 μM, 2 50 μM at -50 mV. Inhibition of OAG-induced activity by 10 μM PIP 37±14%, n=6). In MAs, the effects of diltiazem (94±2%, n=6) 2 was significantly rescued by over 50 % by co-application of 10 and Y-27632 (66±12%, n=6) on the 4-AP-induced contraction μM IP (n=5,). In addition the rescuing action of IP was not recorded under identical condition was significantly greater 3 3 affected by IP receptor blocker heparin (1 mgml-1, n=5). Nora- than in PAs (p < 0.001, t-test). The effects of Y-27632 is likely 3 drenaline-evoked channel activity was inhibited by anti-TRPC6 to reflect the inhibition of the basal level of the enzyme activ- (88 ± 9 %, n=7) and anti-TRPC7 antibodies 85 ± 13 % (n=10) but ity, because the pre-treatment of arteries with 4-AP (10 mM) not by other anti-TRPC antibodies. did not change levels of phosphorylated regulatory myosin These results show that endogenous PIP has a pronounced phosphatase target subunit (MYPT-1). In alpha-toxin perme- 2 inhibitory action on TRPC6-like activity in portal vein myocytes abilized tissues, pre-treatment with 3 μM carbonyl cyanide-p- which can be removed by interactions with IP . Moreover these trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial 3 data suggest that these channels are likely to be composed of uncoupler, significantly reduced 4-AP contraction in PAs and TRPC6/TRPC7 heterotetramers and that TRPC7 proteins medi- not in MAs. In conclusion, our results demonstrate the involve- ate interactions between PIP and IP . ment of at least two types of K+ channels, the K and KCNQ 2 3 V Albert & Large (2003) J Physiol 552, 789-95

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Albert et al (2008) J Physiol 586, 3087-9 the 3’UTR of Kir2.1. These studies are intended to form the basis of future studies aimed at demonstrating down-regulation of This work was supported by The Wellcome Trust and The British I by specific miRNAs. Heart Foundation K1 Pogwizd SM et al. (2001). Circ Res 88, 1159-1167. Where applicable, the authors confirm that the experiments Ikeda S et al. (2007). Physiol Genomics 31, 367-373. described here conform with The Physiological Society ethical Thum T et al. (2007). Circulation 116, 258-267. requirements. Cheng Y et al. (2007). Am J Pathol 170, 1831-1840. Yang B et al. (2007). Nat Med 13, 486-491. PC118 D. Goldoni holds a studentship from The Harold McCauley fund Targeting of an inward rectifier K+ channel 3’ untranslated for Cardiovascular Research. region by microRNA up-regulated in heart disease The authors thank Dr. Youyou Zhao. D. Goldoni and A. Collins Where applicable, the authors confirm that the experiments Queen’s University, Belfast, UK described here conform with The Physiological Society ethical requirements. MicroRNAs (miRNAs) are a class of noncoding small RNAs modulating gene expression. By annealing to the 3’ terminal untranslated region (3’UTR) of the target mRNA, they lead to translational repression. Kir2.1 is a member of the gene subfamily coding for inward rec- PC119 + tifier potassium channels. Kir2.1 passes inwardly rectifying K current (IK1) which plays an important role in repolarising and Cholesterol depletion does not prevent actin-based stabilizing the membrane potential in cardiac myocytes; indeed activation of non-voltage-gated sodium channels down-regulation of inward rectifier current occurs in cardiomyopathy and is proarrhythmic (1). A. Sudarikova, Y.A. Negulyaev and E.A. Morachevskaya Because the causes of I reduction are still unknown and K1 InstituteofCytology,RussianAcademyofSciences,St.Petersburg,Russia because a variation in expression of miRNAs has been shown in cardiac hypertrophy and heart failure (2-4) we hypothesize that Cholesterol is a major lipid component of the plasma mem- miRNAs are involved in the down-regulation of Kir2.1 and IK1. branes, and it plays an important role in lipid organization, lat- We aim to demonstrate the relationship between miRNAs and eral heterogeneity and dynamics of plasma membrane. Lipid down-regulation of Kir2.1 by identifying miRNAs that are up- rafts, cholesterol-rich membrane domains, are assumed to play regulated in cardiomyopathy (2-4) and predicted to target the an essential role in the interactions between cell membrane Kir2.1 3’UTR (TargetScan; Miranda; PicTar), transfecting them and cortical cytoskeleton (Nebl et al., 2002). As we have shown into HEK293 cells and assaying for functional targeting using earlier, the activity of non-voltage-gated sodium channels in a luciferase reporter with putative target sites in the 3’UTR. K562 human leukaemia cells is critically dependent on F-actin miR-1 was chosen as control and to validate the method as it organization (Negulyaev et al., 2000; Shumilina et al., 2003). has been shown to target Kir2.1 (5). Our present work was aimed to investigate possible role of lipid Two luciferase reporter plasmids were constructed from pMIR- environment in the functions of sodium channels and their cou- REPORT (Applied Biosystems); one with the Kir2.1 3’UTR miR- pling with cortical cytoskeleton in K562 cells. The patch-clamp 1 target site (pLuc2.1-1), and the other with predicted miR- method was employed to examine the effect of methyl-β- 132, miR-212 and miR-320 target sites (pLuc2.1-3). cyclodextrin (MbCD), an agent that removes membrane cho- MiRNA expression plasmids were produced in the siRNA expres- lesterol, on sodium currents. Single channel currents were sion vector pSM30 (provided by Dr. G. Du, UTHSC, Houston) by recorded from cell-attached and inside-out patches essentially inserting oligonucleotide cassettes of mature miRNA as described earlier (Morachevskaya et al., 2007). After incu- sequences. bation with MbCD (2.5 or 5 mM) an activation of sodium chan- HEK293 cells were transfected with a luciferase reporter plas- nels in response to cytochalasin B or D was observed in mem- β mid, miRNA vector and a -galactosidase plasmid as normal- brane fragments (inside-out mode) as well as in native cells izer. After 48 hours the Dual-Light® assay (Applied Biosystems) (cell-attached mode). Unitary conductance of the channels in was performed. cholesterol-depleted cells (11.6±0.1 pS, n=8) was similar to The method has been optimized with miR-1 and pLuc2.1-1. those in control cells (11.9±0.7 pS, n=7). Inside-out experiments β Luciferase/ -galactosidase was 40-44% lower in cells transfected with the use of globular actin have indicated that filament with miR-1 vs pSM30 with no insert (p<0.01, n=3, t-test, assembly on cytoplasmic membrane side causes an inactiva- repeated three times with different plasmid ratios). tion of sodium channels. In sum, we conclude that cholesterol miR-212 down-regulated functional expression of pLuc2.1-3 depletion did not affect essentially the biophysical character- in comparison with miR-1, which is a negative control for istics, sodium channel activation and inactivation coupled with β pLuc2.1-3. Luciferase/ -galactosidase was 72% lower in cells F-actin rearrangement. Our electrophysiological data imply transfected with miR-212 vs miR-1 (p<0.05, n=3, t-test). that there is no association of non-voltage-gated sodium chan- In summary, we have demonstrated miRNA targeting using the nels with cholesterol-rich membrane microdomains. pSM30 vector and provided evidence for a miR-212 target in Morachevskaya E. A. et al. (2007). J. Cell Biol. Int. 31: 374-381.

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Nebl T et al. (2002). J Biol Chem. 277, 43399-43409. 3. Testing the predictions: Lastly, the correspondence between Negulyaev Y. A. et al. (2000). J. Biol. Chem. 275: 40933-40937. the predictions derived from the model and the values initially used to describe the experimental recordings was tested. Shumilina E. V. et al. (2003). Mol. Biol. Cell 14: 1709-1716. This study provided evidence that the response of TRPM8 channel to voltage variations can be described in terms of a model This work was supported by RFBR 08-04-00574. that includes channel transitions involving 2 to 4 open states Where applicable, the authors confirm that the experiments and 4 to 6 closed states. The most likely transitions that are described here conform with The Physiological Society ethical voltage regulated have also been identified. requirements. Voets T et al. (2007). Handb Exp Pharmacol 179, 329-344 Colquhoun D and Sigworth FJ (1995). In Single-Channel Recording, 2nd edition, ed. Sakmann B and Neher E, 483-587 PC120 Rothberg BS and Magleby KL (1998). J Gen Physiol 111, 751-780 Magleby KL and Song L (1992). Proc R Soc Lond B Biol Sci 249, 133-142 Modelling TRPM8 single channel gating J.A. Fernandez, J.G. McGeown, C.N. Scholfield and A.V. Zholos We thank European Social Fund for support, Prof. N. Prevarskaya for the HEK293/TRPM8 cells, and Prof. K.L. Magleby for help Queen’s University of Belfast, Belfast, UK with data analysis. Single channel patch clamp recording allows modelling of ion Where applicable, the authors confirm that the experiments channel behaviour. Thus, a single kinetic model can formally described here conform with The Physiological Society ethical describe the complex interactions during poly-modal activa- requirements. tion of the channels. For TRPM8, activation depends on temperature, voltage and chemical signalling (Voets et al., 2007), and such modelling provides the most comprehensive approach PC121 to the study of the channel. Here we show the methods used to derive the kinetic signature of the TRPM8 channel from a set Effects of systemic arterial hypertension on nitric oxide of single channel recordings. synthase activity and expression, arginase activity and lipid Single channel (cell-attached patch mode) currents were peroxidation in human platelets recorded in HEK-293 cells stably expressing the TRPM8 chan- 1 1 1 1 nel. Data was recorded at room temperature and different volt- M.B. Moss , M.A. Siqueira , C.G. dos Santos , W.V. Mury , 1 2 1 1 ages (from 40 to 140 mV). Currents were filtered (low pass V.B. Moss , G.E. Mann , T.M. Brunini and A. Mendes Ribeiro Bessel) at 2 kHz and sampled at 10 kHz. Single channel transi- 1Farmacologia e Psicobiologia, Universidade do Estado do Rio de tions were identified by the half-amplitude threshold crossing Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil and 2Cardiovascular criteria and time-course fitting (Colquhoun and Sigworth, Division, King’s College, London, UK 1995). Recordings were made from 7 different patches with at Introduction: Systemic arterial hypertension (SAH) is a major least 12000 openings in each recording (all values were independent risk factor for cardiovascular disease. The phys- ± expressed in means S.E.M.). iopathology of SAH is multifactorial, complex and remains to Three steps were used to derive a kinetic model from single be elucidated. Nitric oxide (NO) is an important regulator of channel recordings: vascular and haemostatic functions1. The cationic amino acid 1. The data: Dwell time 1D histograms were constructed from L-arginine serves as the substrate for NO synthases (NOS) and transitions >0.16 ms using the square root of the number of arginase, an enzyme of the urea cycle. Our group has previously events per bin with a constant bin width expressed on a loga- reported an inhibition of L-arginine transport in erythrocytes rithmic time axis (ms, 20bins/decade). These histograms were and platelets in hypertension associated with enhanced platelet fitted with a sum of exponentials using pClamp 9. The appro- aggregation2,3. Oxidative stress is involved in the pathophysi- priate number of exponentials was assessed both by visual ology of hypertension4. The aim of the present study was to inspection as well as a statistical estimation of the ‘goodness investigate the L-arginine-NO pathway, urea cycle and oxida- of fit’ (Maximum Likelihood and Least Squares Errors). These tive stress in platelets in SAH patients. methods pointed to the need of 2 to 4 open states (mean open Methods: 11 hypertensive patients and 13 healthy controls ± ± time ranged from 0.2 0.02 to 8.95 0.35 ms) and 4 to 6 closed matched for age and sex were included in the study. Basal NOS ± ± states (mean closed time ranged from 0.11 0.01 to 671.04 activity was measured by the conversion of L-[3H]-arginine into 140.24 ms) to fully describe the data. L-[3H]-citrulline. Inducible and endothelial NOS expressions 2. Modelling: There are two Freeware software packages for were accessed by Western Blotting. Arginase activity was ana- Markov analysis of single channel data: HJCFIT lyzed in platelet lysate through the conversion of [C14]-L-argi- (http://www.ucl.ac.uk/) and QuB nine into [C14]-urea. The level of plasma lipid peroxidation was (http://www.qub.buffalo.edu/). They were used to fit single evaluated on the basis of thiobarbituric acid reactive substances channel sequences to different models and solve for the most (TBARS). Mann Whitney test was used for statistical analysis likely rate constants. In order to discriminate between models, and values were considered significantly different when p<0,05. dwell time 2D distributions (Rothberg and Magleby, 1998) and Results: We provided clear evidence that the NOS activity dependency and dependency difference plots (Magleby and (pmol/108 cells) was diminished in hypertensive patients com- Song, 1992) were also used. pared to controls (0.09 ± 0.02 vs 0.19 ± 0.03). Platelets from SAH patients exhibited a compensatory over expression of

143P Poster Communications inducible NOS (iNOS), but not endothelial NOS (eNOS), whilst This study presents in vivo effects of PPARβ activation on mus- intraplatelet arginase activity (pmol urea/mg protein/2h) was cle morphology on wild-type mice, and describes the mecha- not affected (2.1 ± 0.1 vs 2.8 ± 0.5). There was no difference nism mediating these effects. between TBARS levels (pmol/mg protein) in platelets from SAH Adult C57BL6 mice received daily subcutaneous injections of patients (40 ± 19) and those detected in platelets from controls a PPARβ specific activator, or the calcineurin inhibitor cyclo- (21.8 ± 5). sporine A (CsA) or a combination of both. After different times Conclusion: In conclusion, the thrombotic state observed in (from 2 to 96 hours), tibialis anterior muscles were harvested hypertensive patients could be explained in part by the inacti- immediately after death and used for histological and molec- vation of the L-arginine-NO pathway in platelets. Higher expres- ular analyses. sion of iNOS in platelets might be a compensatory mechanism PPARβ activation leads to a muscle remodelling, character- against platelet activation in hypertension. A better under- ized by increases of oxidative fiber and capillary numbers, and standing of the mechanisms that contribute to platelet dys- a decrease of fiber diameter, completed within 2 days of treat- function in hypertension could provide important tools for the ment. This muscle remodelling is accompanied by a quick and development of new therapies to minimize the risk of cardio- transient upregulation of myogenic and angiogenic markers. vascular complications in these patients. Both myogenic and angiogenic responses are dependent upon Moncada S, Higgs EA. The discovery of nitric oxide and its role on vas- calcineurin activity as remodelling is completely blocked by cular biology. Br J pharmacol 2006, 176(Pt 1):213-254 CsA administration. de Meirelles LR, Mendes-Ribeiro AC, Santoro MM, Mendes MA, da Silva In conclusion, these data indicate that PPARβ activation is asso- MN, Mann GE, Brunini TM. Inhibitory effects of endogenous L-argi- ciated with a calcineurin-dependent effect on muscle mor- nine analogues on nitric oxide synthesis in platelets: role in platelet phology, that enhances the oxidative phenotype. hyperaggregability in hypertension. Clin Exp Pharmacol Physiol. Holst D, Luquet S, Nogueira V, Kristiansen K, Leverve X, Grimaldi PA. 2007;34(12):1267-71 Nutritional regulation and role of peroxisome proliferator-activated Moss MB, Brunini TM, Soares De Moura R, Novaes Malagris LE, Roberts receptor delta in fatty acid catabolism in skeletal muscle. Biochim Bio- NB, Ellory JC, Mann GE, Mendes Ribeiro AC. Diminished L-arginine phys Acta 2003;1633(1):43-50. bioavailability in hypertension. Clin Sci (Lond). 2004;107(4):391-7 Luquet S, Lopez-Soriano J, Holst D, Fredenrich A, Melki J, Rassoulzade- Touyz R.M. Reactive oxygen species, vascular oxidative stress and redox gan M, et al. Peroxisome proliferator-activated receptor delta con- signaling in hypertension. What is the clinical significance? Hyperten- trols muscle development and oxidative capability. Faseb J sion. 2004;44:248–252 2003;17(15):2299-301. Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, Bayuga-Ocampo CR, Financial Support: FAPERJ et al. Regulation of muscle fiber type and running endurance by Where applicable, the authors confirm that the experiments PPARdelta. PLoS Biol 2004;2(10):e294. described here conform with The Physiological Society ethical Oliver WR, Jr., Shenk JL, Snaith MR, Russell CS, Plunket KD, Bodkin NL, requirements. et al. A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport. Proc Natl Acad Sci U S A 2001;98(9):5306-11. Tanaka T, Yamamoto J, Iwasaki S, Asaba H, Hamura H, Ikeda Y, et al. PC122 Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome. Proc Natl Acad Sci U S A 2003;100(26):15924-9. Pharmacological activation of Peroxisome-Proliferator Activated Receptor b promotes rapid and calcineurin- Where applicable, the authors confirm that the experiments dependent fiber remodelling and angiogenesis in mouse described here conform with The Physiological Society ethical skeletal muscle requirements. C. Gaudel1, C. Schwartz2, C. Giordano2 and P.A. Grimaldi2 1School of Biomolecular and Biomedical Science, University College PC123 Dublin, Dublin, Ireland and 2U907, Faculté de Médecine, Institut National de la Santé et de la Recherche Médicale, Nice, France Comparative effects of estradiol and oxidized LDL on Sedentary lifestyle is associated with the metabolic syndrome asymmetric dimethylarginine production in human and promoting the oxidative capability of skeletal muscle has endothelial cells cultured from umbilical arterial and veins been proposed as a therapeutic approach to counteract meta- S. Novella2,1, E. Monsalve1, A. Laguna-Fernandez2, P.J.Oviedo2, bolic disturbances. In cultured myotubes, overexpression of a A. Sobrino2 and C. Hermenegildo1,2 nuclear receptor involved in the regulation of muscle develop- 1 2 ment and metabolism (peroxisome proliferator-activated Physiology, University of Valencia, Valencia, Spain and Research receptor β,orPPARβ ), enhances fatty acid catabolism (1). Mus- Unit, Hosp. Clinico Universitario, Valencia, Spain cle-specific overexpression of PPARβ in mice promotes an Asymmetric dimethylarginine (ADMA) is an endogenous increase in the number of oxidative myofibers and augments inhibitor of nitric oxide (NO) synthase produced by endothelial resistance against diet-induced obesity (2, 3). Recently, admin- cells. ADMA levels are mainly regulated by the activity of istration of PPARβ agonists has been reported to improve the dimethylarginine dimethylaminohydrolases (DDAH-I and metabolic phenotype of obese and insulin-resistant animals DDAH-II). Endothelial release of ADMA is increased in the pres- (4, 5). ence of oxidized LDL cholesterol (oxLDL), whereas estrogens stimulate endothelial NO production by increasing NO syn-

144P Poster Communications thase. These results have been performed in endothelial cells from venous origin, mainly in cultured human umbilical vein PC125 endothelial cells (HUVEC). Our aim was to compare the estradiol effects on changes induced by oxLDL on DDAH expression Glycolysis inhibition reverses contractile end electrical and ADMA production in cultured human umbilical artery responses to hypoxia in guinea pigs and rats pulmonary endothelial cells (HUAEC) with results obtained in HUVEC. artery and aorta mediated by endothelium through Primary HUAEC and HUVEC were exposed to 100 μg/mL of myoendothelial gap junction oxLDL, with or without 1 nM estradiol for 24 hours. ADMA was I.V. Kizub1, O. Bondarenko2 and A.I. Soloviev1 measured in culture supernatants by HPLC. DDAH-I and II mRNA expression and protein content was quantified by real time PCR 1Experimental therapeutics, Institute of Pharmacology and and immunoblotting, respectively. Toxicology of AMS of Ukraine, Kiev, Ukraine and 2Bogomoletz ADMA production remained unaltered in HUAEC exposed to Institute of Physiology of NAS of Ukraine, Kiev, Ukraine estradiol, whereas in HUVEC estradiol decreased ADMA pro- A growing mass of data strongly suggest that a number of vas- duction by 57% (p<0.001). In both cell types, oxLDL significantly cular disorders induced by oxygen lack, including hypoxic pul- increased ADMA production by ~50% (p<0.01). Finally, com- monary vasoconstriction (HPV), are associated with abnor- bined exposure to oxLDL plus estradiol completely abolished malities of endothelial function but underlined mechanisms the increased production of ADMA induced by oxLDL (p<0.05 still remain unclear. The goal of this study was to shed light on vs. oxLDL alone). the possible myoendothelial gap junctions contribution to the DDAH-I protein expression remained unchanged after all treat- mechanisms responsible for HPV, and to clarify the phenome- ments. Exposure of HUVEC to oxLDL, reduced DDAH-II expres- non of paradoxical HPV inversion to relaxation under selective sion by 20% (p<0.01) at both the mRNA as well as the protein glycolysis inhibition. Membrane potentials were recorded from levels, which in turn increased ADMA levels. Estradiol alone endothelial cells (EC) of guinea pig main pulmonary arteries increased DDAH-II mRNA and protein expression up to 170 % (MPA) and aorta using whole-cell patch-clamp technique in cur- (p<0.01). In cells exposed to estradiol in combination with rent clamp mode. The vascular tone was measured on isolated oxLDL, DDAH-II protein levels were the same as those for estra- rat aorta and MPA using contractile recording technique. All diol alone. animals were anesthetized with sodium pentobarbital (50 Nevertheless, in HUAEC the effects were quite different. Estra- mg/kg ip). Hypoxia (pO -10 mmHg) caused rapid hyperpolar- diol alone did not modify DDAH-II expression, whereas oxLDL 2 ization in aorta endothelial cells from -24.2±4.1 to -37.4±2.1 increased DDAH-II expression by 50% (p<0.05), probably as a mV (n=6). In contrast to this, EC in MPA were depolarized under consequence of increased ADMA levels. To check that possi- hypoxic condition from -43.3± 3.2 to -30.6±2.2 mV (n=5). Selec- bility, DDAH activity was measured. DDAH activity was reduced tive glycolysis inhibition with 10 μM iodoacetic acid (IAA) in by oxLDL by 50 % (p<0.05). Estradiol alone did not modify DDAH combination with 10 mM sodium pyruvate led to inversion of activity, but restored the reduced activity caused by oxLDL these electrical responses in endothelial cells in both types of (p<0.01 vs. oxLDL), thus resulting in decreased ADMA levels. vessels, i.e. hypoxia hyperpolarized EC from MPA from -8.5±1.6 Therefore, estradiol counteracts oxLDL-induced ADMA pro- to -30.4±3.6 mV (n=6) and depolarized EC from aorta from - duction in HUVEC, whereas in HUAEC, estradiol counteracts the 28.1±2.4 to -18.2±3.1 mV (n=6). In contractile recording exper- raise of ADMA levels and the reduced DDAH activity induced iments hypoxia elicited constriction in MPA with peak ampli- by oxLDL, whereas the increased DDAH-II expression seems to tude 45.5±5.3% (n=11) and aorta relaxation to 64.3±6.6% (n=8) be a consequence of increased ADMA levels. of norepinephrine-induced pretone (300 nM). 10 μM IAA Min. Ciencia e Innovación, ISCIII (FIS 06/0589, FIS 08/0634, RED reversed HPV to dilatation with amplitude 67.6±7.1% (n=11) HERACLES RD06/0009); Cons. Sanidad, GV (AP 09/2007, AP while relaxant hypoxia-induced responses in aorta were with- 121/08). PJO is a post-doc, and AS is a FPI fellow (BFPI 06/145), out changes and consisted 73.4±3.8% (n=8). Gap junction from Cons. Educación, Generalitat Valenciana, Spain. inhibitor, 18β-glycyrrhetinic acid (18β-GA, 30 μM), significantly decreased HPV amplitude to 7.6±5.8% (n=16) and led to inver- Where applicable, the authors confirm that the experiments sion of hypoxia-induced aorta dilatation to constriction with described here conform with The Physiological Society ethical amplitude 18.8±7.6% (n=10). After the treatment with 18β-GA requirements. selective glycolysis inhibition lost its paradoxical effect on HPV in MPA and hypoxia-induced dilatation in aorta (8.4±2.8% and 7.1±11.2%, respectively). It is likely that one yet unknown glycolysis-controlled mechanism in endothelial cells appears to be a common step for vascular contractility regulation under hypoxia, and that may underlie paradoxical responses of both pulmonary and systemic arterial smooth muscle to hypoxia. Our data suggest also that HPV may be due of depolarization of endothelial cells, which might be conducted from endothelium to smooth muscle cells via myoendothelial gap junctions. This study was supported by The Physiological Society “Centre of Excellence Award Scheme”, 2008.

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC127 requirements. Nitric oxide production and bioavailabity in red blood cells from patients with Anorexia nervosa 1 1 3 2 1 PC126 N.R. Pereira , M. Moss , C. de Assumpção , G. Mann , T. Brunini and A. Mendes Ribeiro1,3 Estrogen receptor alpha mediates estradiol-stimulated 1Farmacologia e Psicobiologia, Universidade do Estado do Rio de endothelial prostacyclin production Janeiro, Rio de Janeiro, Brazil, 2Cardiovascular Division, King’s College London, London, UK and 3Departamento de Ciências A. Sobrino2, P.J. Oviedo2, S. Novella2,1, A. Laguna-Fernandez2, Fisiológicas, Universidade Federal do Estado do Rio de Janeiro, Rio A. Cano3 and C. Hermenegildo1,2 de Janeiro, Brazil 1 2 Physiology, University of Valencia, Valencia, Spain, Research Unit, Anorexia nervosa (AN) is an eating disorder that leads to a 3 Hosp. Clinico Universitario, Valencia, Spain and Pediatrics, marked loss of weight and important clinical complications. Obstetrics & Ginaecology, University of Valencia, Valencia, Spain Nitricoxide(NO)isagasinvolvedinvascularhomeostasis.Its Clinical and experimental data support the consideration of synthesis occurs through the conversion of the semi-essential endothelium as a target for estradiol and other estrogenic com- cationic amino acid L-arginine into L-citrulline and NO, by the pounds. Estradiol acts in the endothelium to promote vasodi- actionoftheenzymeNOsynthase(NOS)1.Arginaseisanenzyme latation through release of several compounds, including syn- involved in urea cycle that competes with NOS by L-arginine. thesis of prostanoids, products of arachidonic acid metabolism. Bioavailability of NO depends not only on its production, but Two main prostanoids play an essential role in vascular physi- also on its degradation by reactive oxygen species (ROS). ology: thromboxane A2 (TXA2), which exhibits a proaggregant The aim of this study was to investigate the NO production, and vasoconstrictor profile, and prostacyclin (PGI2), a potent urea cycle and oxidative stress markers as a possible indicative vasodilator. The different role of both types of estrogen recep- of bioavailability of NO in red blood cells from patients with AN. tors (ERα and ERβ) on the balance PGI2/TXA2 has not been EightfemalepatientswithANfromtheNúcleodeEstudose studied. Our aim were to uncover whether estradiol enhance Saúde dos Adolescentes (NESA) / UERJ and eight age and sex- basal production of PGI2 or TXA2 in cultured human umbilical matched healthy volunteers participated in this study. The vein endothelial cells (HUVEC), to analyze the enzymatic mech- Pedro Ernesto Hospital Ethical Committee approved this study, anisms involved and to value the different role of both types and informed consent was obtained from each participant. of ER. BasalNOSactivitywasdeterminedbytheconversionofL-[3H]- HUVEC were exposed to estradiol, selective ERα (PPT) or ERβ arginine into L-[3H]-citrulline. Arginase activity in erythrocytes (DPN) agonists and antagonists (inespecific: ICI182780; spe- was determined by the conversion of L-[14C]-arginine to [14C]- cific for ERα: MPP) for 24 hours. PGI2 and TXA2 production was urea. As an index of lipid peroxidation, the thiobarbituric acid- measured by ELISA. Production and expression of phopholi- reactive substance formation (TBARS) was evaluated during pases, cyclooxygenases (COX-1 and COX-2), prostacyclin syn- an acid-heating reaction, as previously described by Draper et thase and thromboxane synthase were analyzed by Western al.2.Superoxidedismutase(SOD)activitywasassayedbymeas- blot and quantitative RT-PCR. uring the inhibition of adrenaline auto-oxidation at 480 nm. Estradiol (1-100 nM) dose-dependent increased PGI2 produc- Statistical significance was assessed using Student t-Test, with tion, up to 50 % (p < 0.001 vs. control), without affecting TXA2 p <0.05. production. COX-1 and prostacyclin synthase protein and gene The results showed a diminished NOS activity in patients with expression were increased by 20 and 50 % respectively after AN compared with controls (3.8 ± 0.4 vs. 8.2 ± 1.4 exposure to estradiol (p < 0.001 vs. control), whereas COX-2, pmol/108cells/min; n=8). An increased activity of arginase was phospholipases and thromboxane synthase expression demonstrated in patients with AN in relation to controls (0.09 remained unaltered. All these effects were meditated through ± 0.02 vs. 0.01 ± 0.00 pmol urea/mg of protein/2h; n=8). An ERα, since were produced not only in the presence of estradiol unchanged formation of TBARS (0.001 ± 0.000 vs. 0.003 ± 0.001 but also in the presence of 10 nM PPT, and completely abol- nMol/mg of protein; n=8) associated with elevated SOD activ- ished in the presence of 1 μM MPP. ity (0.42 ± 0.07 vs. 0.12 ± 0.02 U/mg of protein; n=8) was found In conclusion, estradiol, acting through ERα, up-regulates COX- in AN patients in comparison to controls 1 and prostacyclin synthase expression, thus directing Our results demonstrated that in red blood cells from patients prostanoid balance towards increased PGI2 production. with AN the NO production is diminished and it can be in part attributedtoanincreasedarginaseactivity.Theactivityofantiox- Supported by Ministerio de Ciencia e Innovación, ISCIII (FIS idant enzyme SOD was elevated in AN while TBARS concentra- 06/0589, FIS 08/0634, RED HERACLES RD06/0009); Consellería tion was unaffected. The final correlation between oxidative de Sanidad, GV (AP 09/2007, AP 121/08). PJO holds a post-doc stress and NO bioavailability needs to be further investigated. position, and AS is a FPI fellow (BFPI 06/145), both from Con- Moncada S, Higgs EA. The discovery of nitric oxide and its role in vas- sellería de Educación, Generalitat Valenciana, Spain. cular biology. Br J Pharmacol 2006; 147: S193-S201. Where applicable, the authors confirm that the experiments Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A com- described here conform with The Physiological Society ethical parative evaluation of thiobarbituric acid methods for the determina- requirements. tion of malondialdehyde in biological materials. Free Radic. Biol. Med. 1993; 15: 353–363.

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Financial Support: FAPERJ Where applicable, the authors confirm that the experiments Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical described here conform with The Physiological Society ethical requirements. requirements.

PC129 PC128 Alteration of temporal calcium signalling in human Upregulation of Platelet L-Arginine-Nitric Oxide Pathway pulmonary artery endothelial cells by double-stranded RNA May Contribute to the Hypotensive Effect of Exercise Training Z. Balint1, D. Zabini1, V. Konya2, A. Heinemann2 and C. Matsuura1, T.M. Brunini1, M. Moss1, R. Zeitoune1, A. Olschewski1 A.B. Garcia2, J.J. Carvalho2 and A. Mendes-Ribeiro1 1Experimental Anaesthesiology, Department of Anaesthesiology, 1 Departamento de Farmacologia e Psicobiologia, Universidade do Medical University of Graz, Graz, Austria and 2Institute of Estado do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil and Experimental and Clinical Pharmacology, Medical University of 2 Departamento de Histologia e Embriologia, Universidade do Graz, Graz, Austria Estado do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil Spatial and temporal calcium oscillations regulate multiple sig- Introduction Previous studies realized by our group showed nalling pathways in endothelial cells and they are essential for that a reduction in nitric oxide (NO) bioavailability is associated proper endothelial cell function. When the normal pathways with a greater platelet activation in hypertension, which may for interaction break down, as can occur in disease states, contribute to the higher incidence of thrombotic events of this uncontrolled or asynchronous behaviour can occur. Increased disease. Aerobic exercise cause important changes in vascular levels of circulating RNA may result from excessive cell damage function, which contributes to blood pressure attenuation and or cancer. We investigated the effect of double-stranded RNA hypertension outcomes. The aim of the present paper was to (dsRNA) on calcium homeostasis, gene expression, trans- investigate the effects of aerobic training on L-arginine trans- endothelial electric resistance and proliferation of human pul- port and NO synthase (NOS) activity, and also on lipid peroxi- monary artery endothelial cells (hPAECs). Cultured human dation in platelets of spontaneously hypertensive rats (SHR). PAECs from 10 donors were purchased from Lonza Basel, Methods 16 male SHR and 16 Wistar Kyoto rats (12 weeks old) Switzerland and used in passages 4 to 8. Fura-2/am loaded were divided in two groups each (n=8): exercise (EX) and seden- hPAECs were used to investigate calcium changes of 24h incu- tary (SED). Exercise training was realized on a treadmill (5 d/wk; bation with Poly I:C (synthetic dsRNA), dsRNA, natural RNA or 60 min/d; velocity progressively increased up to 16 m/s) dur- control solution by means of live-cell imaging. As a standard ing 20 weeks. The animals were anesthetized with sodium stimulus, 100μM histamine or 15μM BHQ (a selective inhibitor thiopental (40 mg/kg)injected intraperitoneally and blood was of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)) was collected from the abdominal aorta. Platelets were obtained used in the presence and absence of extracellular calcium. Pro- by centrifugation and the following experiments were per- liferation tests were performed on Poly I:C, dsRNA or BHQ stim- 3 formed: L-arginine transport (by incubation with L-[ H]-argi- ulated cells, by means of 3H-thymidine incorporation. Gene 3 nine), NOS activity (by the conversion of L-[ H]-arginine in L- expression of SERCA and toll-like-receptor 3 after dsRNA stim- 3 [ H]-citrulline, and lipid peroxidation (by the production of ulation was analysed by quantitative RT-PCR. The cellular bar- thiobarbituric acid reactive species, TBARS). SBP was measured rier properties of hPAECs as a read-out for cell function were weekly by tail cuff plethysmography. Data were compared with assessed by measuring changes in the trans-endothelial elec- a one-way ANOVA, and significance level was set at 5 %. tric resistance. Results SBP was significantly higher in SHR compared to WKY The calcium response to histamine showed a significantly pro- ± ± at the baseline (186 8 vs. 132 11 mm Hg). After 20 wk of longed duration after dsRNA incubation (from 86 ± 3s to 131 ± training, SBP was significantly lower in SHR/EX (138 8 mm ± 1s, n= 58 and n=77 respectively; p<0.01) but no increase in ± Hg) than in SHR/SED (214 9 mm Hg) and SBP did not differ the basal level of calcium. There was no difference in the BHQ- from WKY. There was a significant increase in platelet L-argi- induced Ca2+ response of the cells, pointing to an inhibitory 9 nine transport (pmol L-arginine/10 cells/min) in both WKY effect of dsRNA on SERCA. Both BHQ and dsRNA inhibited ± ± (SED: 0.196 0.054 vs. EX: 0.531 0.052) and SHR (SED: 0.346 hPAECs proliferation dose-dependently (IC50=60 ± 1 μM for ± ± 0.076 vs. EX: 0.600 0.049). NOS activity (pmol L-cit- BHQ and IC50=2.9 μg/ml for dsRNA). The quantitative RT-PCR 8 rulline/10 cells) was significantly increased in SHR/EX (0.072 showed that SERCA was 3-fold down-regulated by the dsRNA ± ± 0.007) compared to SHR/SED (0.038 0.007), but no changes incubation. The electrical resistance of the monolayer was were observed in WKY. Exercise did not affect and TBARS pro- decreased by 50% after 24h dsRNA treatment (n=4). duction in neither WKY nor SHR group. In conclusion, it is tempting to hypothesize that the inhibition Conclusion These results confirm the importance of regular and down-regulation of sarco/endoplasmic reticulum Ca2+- aerobic exercise as a non-pharmacological tool in hypertension ATPase by double-stranded RNA in human pulmonary endothe- management. An increase in NO bioavailability, through an lial cells contributes to the endothelial dysfunction. upregulation of platelet L-arginine-nitric oxide pathway, may contribute to the hypotensive effect of aerobic exercise train- This study received financial support from the European Com- ing in hypertensive animals. mission under the 6th Framework Programme (Contract No: LSHM-CT-2005-018725, PULMOTENSION) and from the Med- FAPERJ ical University of Graz (PhD Program Molecular Medicine).

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PC130 PC131

Impaired activation of the Nrf2-Keap1 defence pathway in Role of actin/myosin interaction in control of agonist induced endothelial cells in gestational diabetes Ca signaling in endothelium of intact rat tail artery X. Cheng1, A.D. Silva2, R. Hider2, R.C. Siow1 and G.E. Mann1 S. Mumtaz and T. Burdyga 1Cardiovascular Division, King’s College London, London, UK and Physiology, University of Liverpool, Liverpool, Merseyside, UK 2 PharmaceuticalScienceDivision,King’sCollegeLondon,London,UK There is strong evidence that Ca release/ Ca entry coupling Gestational diabetes (GDM), a pregnancy associated disease mechanism plays a crucial role in control of Ca signaling in affecting about 7% of pregnancies worldwide, is associated with endothelial cells. However the mechanism controlling sustained an increased risk of type 2 diabetes and endothelial dysfunc- Ca entry is not well understood. Studies in cultured endothe- tion in later life (Buchanan et al., 2005). Under physiological lial cells have shown that activation of cytoskeletal proteins and conditions, reactive oxygen species (ROS) serve as important several protein kinases [MLCK, PKC, MAP Kinase] may result in signalling molecules (Autreaux et al., 2007), but an overpro- Ca entry thereby regulating the barrier function of endothe- duction of ROS and/or impaired antioxidant gene expression lium [1,2,3]. However, the functional role of cytoskeletal pro- will compromise cellular defences against oxidative stress (Gao teins in intact endothelium were not elucidated. Therefore we & Mann, 2009). In the present study, we have examined the have investigated the role of actin and myosin interactions in effects of advanced glycation end-products on expression of control of agonist induced Ca signaling in intact endothelium endothelial nitric oxide synthase (eNOS) and heme oxygenase- of conduit arteries. Rats were humanely killed under CO2 anaes- 1 (HO-1), an antioxidant enzyme, regulated by the redox sen- thesia; their tail removed from the ventral grove, cleaned of fat sitive transcription factor NF-E2-related factor 2 (Nrf2) (Siow and loaded with Fluo-4 AM (Molecular Probes, 15um) with et al., 1999), in human umbilical vein endothelial cells (HUVEC) pluronic. Confocal imaging was done using Nipkow disc based isolated from normal and GDM pregnancies. confocal imaging system (Ultraview Perkin Elmer, UK). Mini- HUVEC were cultured in M199 containing 20% FCS, and prior mum of 3 animals were used in each set of experiments. We toexperimentsweredeprivedofserum(1%FCS)for4handthen have found that stimulation of intact endothelial cells by car- challenged for 3-12h with AGE modified recombinant human bachol (0.1μM, 1μM, 10μM) produced activation of a Ca tran- serum albumin (AGE-HSA, 25-200μg/mL) or HSA. AGE-HSA (25 sient which consisted of two components: initial fast - depend- and50μg/mL)increasedeNOSexpression1.5-fold(arbitraryden- ent on Ca release and subsequent, sustained dependent on Ca sity: 25μg/mL: 0.37 ± 0.05 vs 0.61 ± 0.06, 50μg/mL: 0.36 ± 0.17 entry. Sustained component of CCh induced Ca transient was vs 0.62 ± 0.08, means ± S.E., n=3) in normal HUVEC (Fig. 1A). 41 ± 0.7 of the peak taken for 100% (n= 372cells, 7vessels). The Notably,eNOSexpressionwassimilarinAGE-HSAorHSA-treated sustained component was superimposed by Ca oscillations. The cells from GDM pregnancies. Although AGE-HSA also increased frequency of oscillation ranged from 0.05 to 0.3 Hz (n=372cells, HO-1 expression after 6 h in normal HUVEC (Fig. 1B), HO-1 pro- 7vessels). Removal of extracellular Ca or SKF-96365 teinlevelswerenotincreasedinendothelialcellsfromGDMpreg- (50μM)abolished Ca oscillations and reduced initial and sus- nancies. As GDM is associated with impaired endothelial func- tained component induced by CCh. Inhibition of calmodulin by tion,ourfindingsprovideinsightintothemolecularmechanisms W-7 (10μM) and MLCK by Wortmanin (10μM) strongly inhib- underlying altered redox signalling in gestational diabetes. ited Ca oscillations, reduced the initial fast component of CCh induced Ca transient to 55.3±3% and 57.2±1.8% (n=52cells, 3vessels) and sustained component to 39.9±2.1% and 33±1.2%(n=52cells, 3vessels) of the peak, respectively. Inhibi- tion of MLCP by calyculin A (1μM) abolished Ca oscillations and reduced the initial fast and subsequent sustained component to 52.6±2.9% and 16.9±2.5%(n=26cells, 3vessels) respectively. Inhibitors of myosin 11 and actin polymerization, blebbistatin (50μM) or cytochalasin D (20μM) also blocked Ca oscillations Fig. 1. Effects of AGE-HSA on eNOS and HO-1 expression in fetal endothe- and reduced the initial fast component of CCh induced Ca tran- lial cells derived from normal pregnancies. Densitometric analysis of ± ± immunoblots three different HUVEC cultures. Values denote means ± S.E.M., sient to 51.4 3.9% and 42.8 2.9%(n=37cells, 3vessels) and sus- ** p<0.01 (Student’s t-test) tained component to 32.4±2.8% and 20.1±1.9%(n=37cells, Autreaux B.D. et al. (2007). Nature 8, 813-824. 3vessels)of the peak, respectively. These results suggest that store-operated Ca entry plays an important role in control of Buchanan T.A. et al. (2005). J. Clin. Invest. 115, 485-491. Ca oscillations in intact endothelial cells of conduit arteries and Gao L. & Mann G.E. (2009). Cardiovasc. Res. 82, 9-20. that protein Siow R.C.M. et at. (1999). Cardiovasc. Res. 41, 385-394. phosphorylation/dephosphorylation and interactions between actin and myosin fibers are playing crucial role in this process. SupportedbyChinaScholarshipCouncilandEUCOSTACTIONB35 Watanabe H et al. (1996)Biochem and Biophy Res Comm 225, 777-784. Mehta D et al. (2003)J Biol Chem 29;278(35):33492-500.

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Goeckeler MZ et al. (2008) Am J Physiol Cell Physiol 295:C994-C1006. in TG fibres could be due to a lower sensitivity of titin filament 2+ Tojyo Y et al. (1995) Jpn J Pharmacol 69, 381-389. to Ca . Holda RJ et al. (1997) FEBS Letters 403, 191-196. Bagni MA et al. (1994). J Physiol 481, 273-278. Bagni MA et al. (2002). Biophys J 82, 3118-3127. Higher Education Commission Pakistan. Bagni MA et al. (2004). Am J Physiol Cell Physiol 286, C1353-C1357. British Heart Foundation. Musarò A et al. (2001). Nat Genet 27, 195-200. Where applicable, the authors confirm that the experiments This work was supported by MIUR, Telethon and Fondazione described here conform with The Physiological Society ethical Cassa di Risparmio di Pistoia e Pescia (Italy). requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC132

A calcium-dependent non-crossbridge stiffness in single intact skeletal muscle fibres from wild-type and transgenic PC133 MLC/mIGF1 mouse B. Colombini1,3, G. Benelli1,3, M. Nocella1,3, A. Musarò2,3, Reversal of the myosin power stroke induced by fast G. Cecchi1,3 and M. Bagni1,3 stretching of intact frog skeletal muscle fibres 1Department of Physiological Sciences, University of Florence, M. Nocella, B. Colombini, G. Benelli, G. Cecchi and M. Bagni Florence, Italy, 2Department of Histology and Medical Embryology, Dipartimento di Scienze Fisiologiche, Università degli Studi di University of Rome “La Sapienza”, Rome, Italy and 3Interuniversity Firenze, Firenze, Italy Institute of Myology, Chieti, Italy The mechanical properties of the actomyosin bond were inves- Previous studies on activated frog muscle fibres (1-3) showed tigated by applying to activated frog muscle fibres, fast the presence of a Ca2+-dependent non-crossbridge stiffness, stretches (~25 nm hs-1 amplitude and ~350 μs duration) which called “static stiffness” (SS) which preceded tension genera- forcibly detached the crossbridge ensemble. Stretches were tion following stimulation. This effect was attributed to a Ca2+- applied before and up to 20 ms after a conditioning step release dependent stiffening of the titin filament. The experiments (~4 nm hs-1 amplitude, 120 μs duration) during the time course reported here were made to ascertain whether the SS was pres- ofthequickforcerecovery.Experimentsweremadeat5∞C. ent also in mammalian muscle fibres from either wild-type (WT) Frogs (Rana esculenta) were killed by decapitation followed or MLC/mIGF1 transgenic (TG) mice in which the local expres- by destruction of the spinal cord, according to the official reg- sion of the insulin like growth factor-1 (IGF1), under transcrip- ulation of the European Community Council (Directive tional control of Myosin Light Chain (MLC) promoter, induces 86/609/EEC). Force and sarcomere length were measured with muscle hypertrophy (4). Mice (3-6 month-old) were killed by a fast force transducer (~50 kHz natural frequency) and a stri- rapid cervical dislocation, according to the EEC (Directive ation follower device. To reduce fibre damaging by the 86/609) guidelines for animal care. Experiments were per- stretches,experimentsweremadeonthetetanusriseatten- formed at ~23∞C on single intact fibres from the flexor digi- sion (P ) of about 0.5 the plateau tension. All the force values torum brevis muscles. Fibres were mounted in an experimen- t0 reported here are normalized for P . The rupture force of the tal chamber between the lever arms of a force transducer and t0 crossbridge ensemble, the critical force P and the sarcomere of an electromagnetic motor to apply fast stretches. Sarcom- c elongation at P (critical length, L ), were measured. Before the ere length (l ) was measured by means of a videocamera. The c c 0 release P was 3.60 ± 0.15 (n=8) times P .CriticallengthL was results showed the presence of the SS in both WT and TG mouse c t0 c 12.02 ± 0.45 nm hs-1.Incontrastwithpreviousdataonthe fibres. SS value was about two times greater and developed tetanus rise (Bagni et al. 2005) during the quick force recov- faster than in frog, reaching the peak on average 4 ms after the ery P was almost constant independently of the tension P stimulus compared to 8 ms in frog. In WT fibres the peak of SS c t developed by the fibre at the time of the stretch. L increased was 2.75 ± 0.27 % (n = 7) of the maximum stiffness (S ) meas- c 0 immediately after the step release (0.2 ms delay) to 16.24 ± ured at tetanus plateau tension (P ) thus representing a negli- 0 0.75 nm hs-1 and remained elevated up to at the end of the gible fraction of the total. However 4 ms after the stimulation, phase 2 of the recovery (2 ms delay) when it started to decrease when the active tension is still very low (2.57 ± 0.32 % P ), SS 0 returning to the isometric value in about 20 ms. The ratio (P - represents about 50 % of the total sarcomere stiffness. SS was c P )/L , which represents the chord stiffness of the half-sar- about 10 times greater than passive stiffness. Thus due to the t c comere, started to decrease progressively after the release and increase of the intracellular Ca2+ the fibre stiffness rises before reached a maximum reduction of 27 ± 5%at2msdelay.The tension increasing significantly the mechanical stability of the return to the pre-release value occurred in about 20 ms. Data sarcomere. SS was significantly smaller (2.13 ± 0.15 % S ; n = 0 analysis suggested that: 1) crossbridge population remained 6, P<0.05, t test) in TG fibres. The finding that the static stiff- almost constant during the quick force recovery; 2) the forced ness is present in mouse muscle fibres strengthen our hypoth- rupture of the actomyosin bond produced by the stretches is esis that SS is due to a Ca2+ stiffening of titin. It is known in fact preceded by the reversal of the power stroke; 3) the average that mammalian skeletal muscle express the N2A titin isoform myosin lever arm movement during the power stroke can be whose stiffness is Ca2+-sensitive. The lower value of SS found measured by the changes of Lc; 4) after the execution of the

149P Poster Communications power stroke, myosin heads are progressively detached and substituted by new heads attached with the pre-power stroke PC135 configuration. The complete substitution occurs in a relatively short time of 20 ms. The effects of peroxynitrite on rat sternohyoid muscle force Bagni MA et al. (2005). J Physiol 565, 261-268. C. Shortt and K.D. O’Halloran Where applicable, the authors confirm that the experiments University College Dublin, Co.Wicklow, Ireland described here conform with The Physiological Society ethical Chronic intermittent hypoxia (CIH) is a central feature of the requirements. debilitating disorder- obstructive sleep apnoea. CIH has been shown to impair upper airway respiratory muscle function1 perhaps due to oxidative and/or nitrosative stress2. Peroxynitrite PC134 is both an oxidising and nitrating agent formed in vivo from the reaction of superoxide oxide and nitric oxide. Peroxynitrite may Effect of creatine and arginine alpha-ketoglutarate play an important role in modulating skeletal muscle function. supplementationduringshort-termheavyresistance-exercise Studies have suggested that peroxynitrite is capable of pro- 3 on lean tissue mass and muscle strength in young adults ducing diaphragmatic contractile dysfunction . We tested the hypothesis that peroxynitrite inhibits sternohyoid muscle con- D.G. Candow1, D. Gervais2 and S. Mailloux2 tractile force in vitro. Earlier observations have suggested that 1Kinesiology, University of Regina, Regina, SK, Canada and muscle contraction accelerates the effect of exogenous oxi- 2Laurentian University, Sudbury, ON, Canada dants due to the additive effect of oxidants produced from endogenous sources4 and therefore we also examined whether Creatine (Cr) supplementation increases muscle mass and increased muscle contraction would accelerate and amplify the strength. Arginine alpha-ketoglutarate (AAKG) acts as a pre- effect of peroxynitrite. cursor for nitric oxide production, and has the potential to Adult male Wistar rats were anaesthetized with 5% isoflurane improve blood flow and nutrient delivery (i.e., creatine) to exer- and killed by spinal transection. Longitudinal strips from the cising muscles. The purpose of this study was to compare sup- sternohyoid (upper airway dilator) muscle were mounted iso- plementation with Cr + AAKG to Cr alone or placebo during metrically in water-jacketed tissue baths at 27∞C and either heavy resistance-exercise (RE) on lean tissue mass and muscle bubbled with a hyperoxic (95% O2/5% CO2) or anoxic (95% strength. Young healthy adults (n=23, 14 male, 9 female, ~23 N2/5% CO2) gas mixture. Strips were set to optimal length and years) were randomized (double blind) to one of three isocaloric force-frequency relationship was assessed by stimulating the treatment conditions: (1) Cr + AAKG (0.1g.kg-1 body mass Cr muscle at a frequency of 10, 50 and 100 Hz at 0, 30, 60 and 90 + 0.75g.kg-1 AAKG, n=8, 5 male, 3 female), (2) Cr (0.1g.kg-1, minutes. In a separate series of experiments, the force-fre- n=8; 5 male, 3 female), or (3) placebo (1g.kg-1 sucrose, n=7, 4 quency relationship was assessed every 10 minutes for two male, 3 female) for 6 weeks. Prior to and following supple- hours. Studies were conducted under paired conditions i.e. con- mentation and RE, measurements were taken for lean tissue trol vs. peroxynitrite (500mM)-treated muscle strips. mass (under-water weighing) and muscle strength (1 repeti- Peroxynitrite had no effect on muscle function under hyper- tion maximum leg press and bench press). Repeated measures oxic (7.6 ±1.1 vs. 6.5±1.8 N/cm2, mean±SEM, control vs. per- ANOVA showed there were no differences (mean ± SD) between oxynitrite at 90 min, P>0.05, Student’s paired t test, n=9) or groups for changes in body mass (Cr + AAKG: hypoxic conditions (6.6±0.9 vs. 6.8±0.8 N/cm2, control vs. per- 83.8±9Ç84.1±11kg, 0.4%; Cr: 77.9±11Ç77.5±9kg, -0.2%; oxynitrite at 90 min, P>0.05, n=9). Increased muscle activation placebo: 80.1±13Ç80.8±10kg, 0.9%), lean tissue mass (Cr + i.e. tetanic contractions every 10 minutes did not unmask a per- AAKG: 68.1±10Ç70.1±12kg, 2.9%; Cr: 61.2±11Ç62.1±9.1kg, oxynitrite effect under hyperoxic (9.2±1.4 vs. 7.6± 1.5 N/cm2, 1.5%; placebo: 66.2±11Ç66.7±10kg, 0.8%) or leg press control vs. peroxynitrite at 120 min, P>0.05, n=6) or hypoxic strength (Cr + AAKG: 267±68Ç371±91kg, 39%; Cr: conditions (1.5±0.3 vs. 1.4±0.2 N/cm2, control vs. peroxyni- 271±90Ç330.1±73kg, 22%%; placebo: 261±125Ç350±120kg, trite at 120 min, P>0.05, n=6). Preliminary studies using ebse- 34%). The Cr + AAKG group experienced a greater increase in len, a peroxynitrite scavenger, showed no improvement in bench press strength (p<0.05) compared to Cr and placebo (Cr hypoxia-induced muscle dysfunction. This suggests that + AAKG: 105±14Ç125±16kg, 19%; Cr: 79±11Ç87.2±19kg, endogenous peroxynitrite is not a major contributor to hypoxia- 10.3%%; placebo: 84±8Ç94±11kg, 11%). These results suggest induced muscle dysfunction. that Cr + AAKG supplementation during short-term heavy In conclusion, peroxynitrite had no effect on sternohyoid mus- resistance-exercise may result in small improvements in upper cle contractile force under hyperoxic or hypoxic conditions. A body muscle strength in young adults. peroxynitrite-induced effect was not uncovered with increased Where applicable, the authors confirm that the experiments muscle activation. This study suggests that upper airway mus- described here conform with The Physiological Society ethical requirements.

150P Poster Communications cle dysfunction associated with chronic intermittent hypoxia, preparation kit and directly sequenced using the Illumna which is ameliorated by antioxidant treatment,2 is independ- Genome Analyser. This resulted in an average of 6.9 million tags ent of peroxynitrite production. per sample. Initial processing and alignment of tags to Equ- Bradford, A. et al. (2005) Respir. Physiol. Neurobiol. 147(2-3): 223-34 Cab2.0 was carried out using the ELAND global alignment strat- Dunleavy, M. et al. (2008) Adv. Exp. Med. Biol. 605:458-62 egy (Illumna). Subsequent annotation of tags and statistical Supinski, G., A. et al. J. Appl. Physiol. (1999) 87(2): 783-791 analyses were carried out using a custom written programme Plant, D.R., et al. (2001) J. Appl. Physiol. 90: 832-838 in the R-language (R Development Core Team, 2004). The online tool DAVID (Hosack et al. 2003) will be use for functional Where applicable, the authors confirm that the experiments clustering and overrepresentation analyses of differentially described here conform with The Physiological Society ethical expressed genes to identify gene pathways relevant to exer- requirements. cise. Results will be confirmed using real-time quantitative RT- PCR. This study will be the first to characterize global mRNA expression profiles in equine skeletal muscle following a period PC136 of exercise conditioning. R Development Core Team, 2004: A Language and Environment for Sta- Digital gene expression profiling of the Thoroughbred horse tistical Computing, R Foundation for Statistical Computing, Vienna, Austria skeletal muscle transcriptome following exercise conditioning Hosack DA et al. (2003) Genome Biol.: R70.1-R70.8 B.A. McGivney1, P. McGettigan1, J.A. Browne1, S.S. Eivers1, Where applicable, the authors confirm that the experiments A. Evans1,2, R.G. Fonseca1, L.M. Katz1, A. Lohan2, B. Loftus2, described here conform with The Physiological Society ethical D.E. MacHugh1,2 and E.W. Hill1 requirements. 1School of Agriculture, Food Science and Veterinary Medicine, University college Dublin, Dublin, Dublin, Ireland and 2Conway Institute of Biomolecular and Biomedical Research, University PC137 college Dublin, Dublin, Dublin, Ireland The effect of vitamin E on skeletal muscle mass in intact and Following the sequencing of the equine genome (EquCab2.0) ovariectomized female rats it is important to utilize this information in the laboratory in 1 2 1 conjunction with in silico studies. Digital Gene Expression (DGE, A. Abdullah , K. Abdul Kadir and I. Soelaiman Illumina) profiling was used to characterize the assembly of 1Department of Pharmacology, Universiti Kebangsaan Malaysia, genes expressed in equine skeletal muscle and to identify the Kuala Lumpur, Malaysia and 2School of Medicine and Health subset of genes which were differentially expressed following Sciences, Monash University Malaysia, Bandar Sunway, Selangor, a ten month period of exercise conditioning. DGE is a recently Malaysia developed alternative to microarray gene expression profiling, The aim of this study was to determine the effects of vitamin currently the main platform used for global transcriptomic E deficiency and supplementation on skeletal muscle mass in investigations. In contrast to microarray technology which is intact and ovariectomized female rats. One hundred and limited to the hybridisation of cDNA to probes printed on the twenty 3-month-old female Wistar rats were used in this study. array platform, DGE is not dependent on currently available Half of the rats were ovariectomized (OVX) while the other half sequence and thus provides a global, hypothesis free quanti- were left intact (sham operated). For ovariectomy, the rats were tative analysis of the transcriptome. The use of this technique anaesthesized with Ketapex (100mg/ml) and Xylazil (20mg/ml) will lead to a greater understanding of the molecular networks [combined in a mixture of 1:1 (v;v)] through intraperitoneal that control cellular function relating to muscle physiology in administration (0.25ml/100g body weight). The OVX rats were the horse. rested for two weeks for the wounds to recover. Intact and OVX The study cohort comprised seven Thoroughbred racehorses rats were further divided into 6 groups and given different from a single training yard with similar dietary and training dietary treatments i.e. vitamin E deficient diets (VED, 75%VED, management. The subjects undertook a regular exercise regime 50%VED, 25%VED), conventional rat chow diet (RC) and confor ten months which consisted of 15-30 minute walk, followed ventional rat chow diet with oral supplementation of 30mg/kg by 1,000 m trot and 2,000 m canter once a day six times a week body weight of alpha-tocopherol vitamin E (RC+ATF). After 15 on an all-weather gallop as well as intermittent periods of higher weeks, skeletal muscle mass, which is represented by lean soft intensity exercise (“work”) no more than once a week which tissue mass (fat-free mass), was measured using the DEXA (Dual consisted of 15-30 minute walk followed by 400 m trot, 500 Energy X-ray Absortiometry) technique. We found that there m canter and 1,000 m gallop (sprint). Exercise physiological was significant increase in skeletal muscle mass within all the data (heart rate, velocity, plasma lactate concentrations etc.) individual groups of intact rats after 15 weeks of dietary manip- were recorded throughout the conditioning period. Skeletal ulation [paired t-test: VED (P<0.05); 75%VED (P<0.001); muscle biopsies were collected at rest from the gluteus medius 50%VED (P<0.001); 25%VED (P<0.01); RC (P<0.01); RC+ATF following desensitisation of the site with 10 ml lidocaine (P<0.001)]. However, when the after-treatment values of skele- (20mg/ml) in the same individuals at two time points: T1 - tal muscle mass of the various intact groups were compared, unconditioned, (9±0.5 months old) and T2 - conditioned no significant difference was found. For OVX rats, there was (20±0.7 months old). RNA was isolated using the Trizol method, also significant increase in skeletal muscle mass within all the cDNA libraries were produced using the Illumna DGE sample individual groups after 15 weeks of dietary treatment [paired

151P Poster Communications t-test: VED (P<0.01); 75%VED (P<0.001); 50%VED (P<0.001); sion of those genes was not modified. Exogenous GH adminis- 25%VED (P<0.001); RC (P<0.001); RC+ATF (P<0.00001)]. How- tration increased liver IGF-I mRNA and serum IGF-I levels ever, when the after-treatment values of skeletal muscle mass (P<0.05). In control, but not in arthritic rats, GH and IGF-I admin- of the various OVX groups were compared, no significant dif- istration decreased atrogin-1 (P<0.05) and MuRF-1 gene expres- ference was noted. After-treatment skeletal muscle mass val- sion (P<0.05). However, neither GH nor IGF-I administration ues between the various groups of intact and OVX rats were were able to prevent arthritis-induced increase in atrogin-1 and not significantly different as well. These results indicate that MuRF-1 gene expression in the gastrocnemius muscle. vitamin E exerted only minimal effects on the growth and devel- Although the GH-IGF-I system has an inhibitory effect on ubiq- opment of skeletal muscle mass in young, growing female rats. uitin ligase enzymes, by decreasing atrogin-1 and MuRF-1 gene Supplementation of alpha-tocopherol vitamin E did not confer expression, in arthritis the GH or IGF administration was not much added benefits compared to giving conventional rat chow able to reduce the expression of these enzymes in the skeletal alone, indicating that the vitamin E content in conventional rat muscle. chow is adequate for the purpose of normal skeletal muscle This work was supported by CYCYT (BFU2006-11899), a grant development. These results also suggest that ovariectomy (and to M. Lopez-Menduiña (Ministerio de Educacion y Ciencia, BES- therefore lower circulating oestrogen levels) did not hinder the 2007-16001) and a grant to E.Castillero (Gobierno progress of skeletal muscle development in young, growing Vasco,BFI06.31). female rats. Where applicable, the authors confirm that the experiments TheauthorswouldliketothankMs.KiftiahAhmedandMs.Nor- described here conform with The Physiological Society ethical lindaDautfortheirtechnicalassistance.Thisstudyhasbeensup- requirements. ported by Universiti Kebangsaan Malaysia Grant UKM F28/99. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC139

The role of corticotrophin-releasing factor in the timing of puberty in the female rat PC138 J. Kinsey-Jones1, X. Li1, A.M. Knox1, S.R. Milligan1, S.L. Lightman2 and K.T. O’Byrne1 Effect of growth hormone and insulin-like growth factor-I administration on atrogin-1 and MuRF-1 gene expression in 1Department of Reproduction and Endocinology, Kings College control and arthritic rats London, London, UK and 2Henry Wellcome Laboratory for Integrative Neuroscience & Endocrinology, University of Bristol, M. López-Menduiña, E. Castillero, A. Martín, A. López-Calderón Bristol, UK and M. Villanúa Neonatal exposure to lipopolysacharride (LPS) programs long- Physiology, Universidad Complutense, Madrid, Spain term changes in hypothalamo-pituitary-adrenocotical (HPA) Adjuvant-induced arthritis is an animal model of chronic inflam- axis activity, resulting in an increased sensitivity to stress (1). mation and rheumatoid arthritis. Chronic arthritis induces Neonatal LPS (nLPS) exposure also causes long-term sensiti- cachexia associated with an inhibition of the growth hormone sation of the hypothalamo-pituitary-gonadal (HPG) axis to the (GH)-insulin-like growth factor-I (IGF-I) system together with inhibitory influences of stress in adulthood (2). Further, we an activation of the E3 ubiquitin ligase enzymes muscle atro- have shown that nLPS results in delayed puberty and a con- phy F-box (atrogin-1) and muscle Ring finger 1 (MuRF-1) in comitant decrease in kisspeptin (Kiss1) mRNA expression the skeletal muscle. Muscle wasting is believed to accelerate within the medial preoptic area (mPOA) in the female rat. morbidity and mortality in rheumatoid arthritis.The aim of this Whilst the mechanisms underlying the actions of nLPS on study was to analyzed the effect of GH and IGF-I administration pubertal development remain unknown, it is possible that the on E3 ubiquitin ligase system in the gastrocnemius muscle of stress neuropeptide, corticotrophin-releasing factor (CRF), may arthritic rats. Arthritis was induced in adult male Wistar rats by play a role. The aim of the present study is to examine the role an intradermal injection in the sole of the right paw of 1 mg of of CRF in both nLPS delayed puberty and normal pubertal complete Freund’s adjuvant. Fifteen days after adjuvant’s injec- development. tion, 300 μg /kg of GH or 200 μg /kg of IGF were administrated All surgical procedures were undertaken using ketamine (100 by subcutaneous injection 18 and 3 hours before being humanly mg/kg i.p) and Rompun (10 mg/kg i.p.) anaesthesia. Sprague- killed. Arthritis induces anorexia, for that reason we included Dawley pups received either LPS (50μg/kg, i.p.) or saline a pair-fed group to discard a possible effect of decreased food (0.05ml) on post natal day 3 and 5 (2), or had no neonatal treat- intake. Gene expression of IGF-I and GH receptor in liver and ments. At postnatal day 28, rats were implanted with intrac- ubiquitin ligase enzymes atrogin-1 and MuRF-1 in skeletal mus- erebroventricular (icv) cannulae connected to an osmotic mini- cle were quantified using RT-PCR. Serum IGF-I was measured pump. The neonatal saline (nSAL) or nLPS treated rats received by radioimmunoassay (RIA). Arthritis decreased IGF-I and GH- either a CRF receptor antagonist astressin (AST, 4nmol/day) receptor gene expression in liver (P<0.05), as well as circulat- or artificial cerebrospinal fluid (aCSF) (n=4-7 per group). The ing IGF-I levels (P<0.05). In skeletal muscle, arthritis increased neonatal none-treated rats were distributed between four atrogin-1 and MuRF-1 gene expression (P<0.01). These effects experimental groups; no surgery control, aCSF (0.5μl/day), are not the result of anorexia, since in pair-fed group the expres- 0.2nmol/day or 0.4 nmol/day CRF (n=3-7 per group). Com-

152P Poster Communications pounds were infused for 2 weeks; rats were then monitored for Nitroso-N-acetylpenicillamine, SNAP 5μg) and PVN and SON vaginal opening and first oestrus as markers of puberty. An were collected 60 min after ANGII stimulation. The results additional group of neonatal none-treated rats were infused are reported as means±SEM and the data were analyzed by t with either aCSF (0.5μl/day), CRF (0.4nmol/day) or AST student test (signif. P<0.05). Values obtained from control ani- (4nmol/day) (n=6-8) and seven days following icv implantation mals were 1.0±0.2 arbitrary units (au). ANGII induced an (post natal day 35) were decapitated and brains removed and increase in VP mRNA expression in both PVN and SON at 30, stored at −80 C. QPCR was used to determine Kiss1 and its 60, 90 and 120 min after stimulation (peak at 60min: PVN receptor (Kiss1r) mRNA levels in brain punches of the mPOA 2.7±0.3, SON 2.2±0.1 au). An increase in OT mRNA expression and arcuate nucleus (ARC). was observed only at 30 and 60 min after ANGII (peak at 60min: nLPS exposure resulted in a delay in both the day of vaginal PVN 2.2±0.3, SON 1.9±0.2 au). NOS expression after ANGII opening (nLPS-aCSF; 38±0.18 vs nSAL-aCSF; 36.71±0.2; stimulation was increased at 30 and 60 min in the PVN and at Mean±SEM; P<0.05) and first oestrus (nLPS-aCSF; 38.71±0.26: all experimental periods in the SON (peak at 60min: PVN nSAL-aCSF; 36.71±0.21; Mean±SEM; P<0.05). The CRF antag- 1.6±0.1, SON 2.1±0.2 au). Therefore, the highest VP, OT and onist was unable to reverse the nLPS-induced delay in puberty, NOS mRNA expression induced by central ANGII occurred at but advanced puberty in nSAL treated rats (nSAL-AST; 60 min after the stimulation. Animals submitted to injection 34.75±0.23; P<0.05). CRF resulted in a delay in puberty onset of LNAME alone showed an increase in VP (PVN 2.4±0.2, SON (P<0.05) and decreased Kiss1 mRNA expression in the mPOA, 1.9±0.2 au) and OT (PVN 2.4±0.1, SON 2.1±0.3 au) mRNA but not in the ARC. Kiss1r expression remained unchanged. expression, but a slightly reduction in NOS expression (PVN These data suggests that nLPS-induced pubertal delay is not 0.8±0.1, SON 0.8±0.1 au). Pretreatment with LNAME did not mediated by CRF. However, under normal conditions an change ANGII induced VP and OT expression, but it reduced inhibitory CRF tone is evident since a CRF antagonist advanced NOS mRNA expression (PVN 1.1±0.1, SON 1.1±0.1 au). In ANGII puberty and CRF per se delayed puberty and decreased mPOA stimulated rats, pretreatment with SNAP decreased the VP Kiss1 expression. (PVN 1.7±0.1, SON 1.4±0.1 au) and OT (PVN 1.5±0.1, SON ± (1) Shanks N et al. (2000). Proc Natl Acad Sci 97:5645-50. 1.5 0.1 au) mRNA expression, without effects on NOS expression. In conclusion, collectively with our previous results, the (2) Li XF et al. (2007).Endocrinology 148:5984-90. present data indicate that central angiotensinergic system facil- itates VP, OT and NOS expression and NO reduces VP and OT Supported by Wellcome Trust and BBSRC expression responses to ANGII, suggesting an inhibitory mod- ulationofNOinthecontrolofneurohypophysialhormones Where applicable, the authors confirm that the experiments synthesis and release. described here conform with The Physiological Society ethical requirements. Technical Assistance: Maria Valci Ados Santos,Marina Holanda andMileneMLopes.FinancialSupport:FAPESP,CNPqandCapes.

PC140 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. Nitric oxide reduces angiotensin-II-induced vasopressin and oxytocin mRNA expression in hypothalamic paraventricular and supra-optic nuclei PC141 W.L. Reis, A.L. Silva, S.G. Ruginsk, L.E. Silva, M. Castro, L.L. Elias and J. Antunes-Rodrigues Effect of growth hormone (GH) on the inflammatory process in pancreas obtained from old male senescence prone mice Physiology, School of Medicine of Ribeirao Preto - University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil S. Cuesta1, E. Vara2, C. García2, R. Kireev1, K. Forman1, C. Ariznavarreta1 and J. Fernández-Tresguerres1 We have shown that inhibition of nitric oxide syntase (NOS) 1Dep. Fisiology, University Complutense of Madrid, Madrid, Spain increases hormonal secretion of vasopressin (VP) and oxytocin 2 (OT) and that nitric oxide (NO) donor reduces plasma con- and Dep. Biochemistry and Molecular Biology, University centrations of VP and OT in response to central stimulation of Complutense of Madrid, Madrid, Spain angiotensin-II (ANGII). In the present study we analyzed in To investigate the effect of aging on various physiological hypothalamic paraventricular (PVN) and supra-optic (SON) parameters related to inflammation in the pancreas obtained nuclei the time course effect of central ANGII stimulation on from male, old and young senescence prone (SAMP8) mice and mRNA expression of VP, OT and NOS by Real Time PCR. We the influence of growth hormone (GH) treatment on the same also evaluated the influence of NO donor or inhibitor in these parameters in the old group. mRNA expressions. Wistar male rats (n=5-6), anaesthetized Nineteen animals were used, divided into three groups: old (10 with 2.5% tribromoethanol (1ml/100g bw, ip) with a stain- months), young (2 months), old GH-treated sc with 2mg/kg GH. less cannula placed into the right lateral ventricle were injected All animals were treated for one month with GH or vehicle. The with ANGII (50ng) and 30, 60, 90, 120 and 240 min later, the group of young animals was used as control. The expression of animals were decapitated and the PVN and SON nuclei interleukin4(IL4),interleukin10(IL10),Interleukin1β(IL1β),Inter- microdissected. Another group of rats received, 20 min before leukin2(IL2),Interleukin6(IL6),TumorNecrosisFactorα(TNFα) ANGII, a central pretreatment with NOS inhibitor (Nw-Nitro- and Monocyte chemoattractant protein 1 (MCP1) was deter- L-arginine methyl ester, LNAME 250μg) or NO donor (S- mined by specific ELISA methods in homogenates of pancreas.

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Anti-inflammatory cytokines like IL10 showed a significant gen receptor (ER)α agonist and weak ERβ antagonist, signifi- decrease in older individuals whereas IL4 did not showed dif- cantly reduced TST by 1.12 ± 0.12oC (paired Students t-test, ferences with young mice. Aging significantly increased the lev- P<0.0002; n=6). In contrast, 8-n-heptylnaringenin (20mg/kg), els of pro-inflammatory cytokines like IL1β, IL2, IL6, TNFα. But a weak ERα agonist and strong ERβ antagonist, did not signifi- no differences in MCP1 were observed. cantly reduce the elevated TST when administered alone and Administration of GH to old male mice significantly increased failed to block the E2 induced decrease in TST when co-admin- the levels of IL4 and IL10. GH treatment reduced also signifi- istered with E2. These results indicate that analogues of 8PN cantly IL1β, IL2, IL6, TNFα and MCP1. are effective in the rat model of hot flushes and deserve further Aging leads to an increase of inflammatory processes in the pan- investigation. creas. The administrations of sustitutive hormonal therapy with This work was supported by the BBSRC. GH inhibited the production of pro-inflammatory cytokines and increased anti-inflammatory cytokine production. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical This work was supported by grants: RETICEF RD 06/0013 (FIS) requirements. and SAF 2007, 66878-C02-01. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC143 Developing a Mathematical Model of Insulin release from Pancreatic b-cells: The Importance of Glutamate PC142 M. Salvucci1, Z. Neufeld2 and P. Newsholme1 The effect of synthetic analogues of the phyto-oestrogen 1School of Biomolecular and Biomedical Sciences, Health Science 8-prenylnaringenin on tail skin temperature in a rat hot Complex and Conway Institute, University College Dublin (UCD), flush model Dublin, Ireland and 2Complex & Adaptive Systems Laboratory L. Breen1, D. Sugden1, A. Heyerick2, K. O’Byrne1 and S. Milligan1 (CASL), University College Dublin (UCD), Dublin, Ireland 1 Division of Reproduction and Endocrinology, King’s College Pancreatic β-cells play a key role in the glucose homeostasis, 2 London, London, UK and Laboratory of Pharmacognosy and secreting insulin in response to blood nutrient fluctuations. This Phytochemistry, Ghent University, Ghent, Belgium is due to a complex relationship between metabolism and Hot flushes are a distressing symptom of the menopausal syn- insulin secretion which comprises both triggering (ATP and Ca2+ drome affecting over 75% of women, many of whom seek med- dependent [1]) and amplifying (mitochondrial metabolite ical treatment because of the severity of the symptoms. There dependent [2]) pathways of insulin secretion. has been growing interest in the use of phyto-oestrogens as This study has attempted to develop a mathematical model alternative therapies for hot flushes due to recent reports high- of insulin release from pancreatic β-cells with particular regard lighting adverse effects of oestrogen therapy. A potent oestro- to the role of glutamate. The model’s input is made up of genic compound in hops (Humulus lupulus) has been identi- three nutrient components: glucose, alanine [3] and gluta- fied as 8-prenylnaringenin (8-PN) and hop extracts containing mine [4] while the ouput of the model is represented by 8-PN have been used for treating menopausal symptoms, insulin secretion. including hot flushes. Synthetic analogues of 8-PN, 8- A systems biology approach was employed to integrate bio- neopentylnaringenin and 8-n-heptylnaringenin, have been logical experimental work with mathematical tools to gain demonstrated to be selective oestrogen receptor modulators novel insights about the role played by glutamate in insulin in vitro. We have investigated the in vivo oestrogenic activity of secretion. these compounds using a rat model of the hot flush phenom- A simplified kinetic model of the glucose-stimulated insulin enon in which the tail skin temperature (TST) is increased after secretion in pancreatic β-cells which takes into account gly- oestrogen deficiency induced by ovariectomy. colysis, Krebs cycle, alanine uptake and glutamine-glutamate Female Wistar rats were ovariectomized and implanted with metabolism was built [5]. chronic telemetry devices (TA10TA-F40 W/TP; Data Sciences Experimental work was carried out on a functional clonal International) under general anaesthesia induced by ketamine insulin-secreting cell line (BRIN-BD11) to validate the model. hydrochloride (100mg/kg) co-administered with medetomi- Cell viability and death assays were performed following incu- dine (0.5mg/kg) ip. On completetion of surgery, anaesthesia bation in various concentrations of nutrients. was reversed by atipamezole (1mg/kg) sc. After a two week The utilization and production of the major components of the recovery period TST was monitored for 7 sec every 5 min mathematical model include: glucose, alanine, glutamine, pyru- throughouttheexperimentalperiod.Followingathreedaymon- vate, lactate, glutamate, ATP/ADP ratio and insulin. All the itoring period to establish baseline temperatures, 17β-oestra- determinations described above were performed following diol (E2), vehicle, 8-neopentylnaringenin or 8-n-heptylnarin- BRIN-BD11 incubation in the presence of 10 mM of alanine and genin were administered subcutaneously daily for five days. various concentrations of glucose (5, 16.7, 30 mM) and gluta- μ Administration of E2 (4 g/kg) significantly reduced the ele- mine (0, 1, 2 mM). A quantitatively accurate description of the vated TST on the fifth and final day of injection by 1.89 ± 0.14oC oscillating behaviour of pyruvate and others metabolites of the from the baseline TST (paired Students t-test, P<0.0001; n=8). Krebs cycle and ATP was obtained. ATP, ADP, pyruvate and glu- In addition, 8-neopentylnaringenin (20mg/kg), a strong oestro-

154P Poster Communications cose-6-phosphate concentrations were associated with coor- Male Wistar rats (250-320g) were anaesthetised with 1ml i.p. dinated oscillations. chloralose/urethane (16.5/250 mg/ml) and cannulae were Differences in the oscillating features were found when values inserted into the right femoral artery, to measure mean arte- obtained by experimental procedures were used since desen- rial pressure (MAP), and vein to infuse saline (9g/lNaCl) at 3ml/h. sitization to glucose induced by 24hr incubation in high glu- The left kidney was exposed via the flank, its ureter cannulated cose (30mM) resulted in reduced oscillations of the metabo- and an ultrasonic flowprobe fitted to the renal artery. A small lites and lower insulin secretion. cannula was inserted into the cortex for 4.5 ml to lie at the cor- Cell viability was not altered by incubation in various nutrient tico-medullary boarder for the infusion of drugs (1.0 ml/h intra- combinations (maximum difference 12.1%). renally, i.r.). At the end of surgery, the i.v. saline was replaced Insulin secretion was biphasic in the presence of glucose and with saline plus inulin (2g/l) for measurement of glomerular fil- alanine (AUC value: 126 ± 12 and 117 ± 14 μg/mg protein dur- tration rate (GFR) and the animals were allowed 1-2 h to recover. ing the 1st and 2nd phaserespectively)butwasnotaffectedby A 30 min basal urine collection was taken and thereafter an acute (1–20 min) glutamine stimulation, indicating that only i.v. infusion of either tempol (a SOD mimetic) at 30 a small amount of glutamine-derived glutamate is converted μmmol/kg/min, or diethyl – dithio-carbamate (DETC) at into α-ketoglutarate in glucose stimulatory conditions. It is likely 2mg/kg/min was begun. After 30 min, a further 30 min urine that changes in glutamate and α-ketoglutarate production rep- collection was taken, the i.r. infusion was stopped and a 30 min resent a more important coupling pathway when low concen- recovery clearance was taken. Data, mean±S.E.M. were taken trations (below 5mM) of extracellular glucose are present. as significant when P<0.05 (ANOVA). ± Corless M, Kiely A, McClenaghan NH, Flatt PR, Newsholme P. Glutamine Infusion of tempol i.r. (n=5) had no effect on MAP, at 949 regulates expression of key transcription factor, signal transduction, mmHg and was unchanged in the recovery period. Under these metabolic gene, and protein expression in a clonal pancreatic β-cell conditions there was a rise (P<0.05) in GFR, from 7.6±2.8 line. Journal of Endocrinology. 2006;190: 719-27. ml/min/kg, of 100% which was accompanied by increase in both ± ± Maechler P, Wolheim CB. Mitochondrial glutamate acts as a messen- urine flow, from 37.61 1.2 to 79.4 24.5 ml/min/kg, and ger in glucose-induced insulin exocytosis. Nature. 1999 Dec absolute sodium excretion, from 2.6±0.9 to 8.22±.3 9;402(6762):685-9. μmol/kg/min (both P<0.05). Administration of DETC (n=6) was without effect on MAP but caused decreases in GFR, of some Brennan L, Shine A, Hewage C, Malthouse JP, Brindle KM, McClenaghan N, et al. A nuclear magnetic resonance-based demonstration of substan- 40%, urine flow of some 30% and sodium excretion of 50% (all tialoxidativeL-alaninemetabolismandL-alanine-enhancedglucosemetab- P<0.05). olisminaclonalpancreaticbeta-cellline:metabolismofL-alanineisimpor- These findings show that scavenging of superoxide anions tanttotheregulationofinsulinsecretion.Diabetes.2002;51(6):1714-21. within the kidney results in a marked increase in glomerular filtration and excretion of fluid and conversely blockade of SOD Brennan L, Corless M, Hewage C, Malthouse JP, McClenaghan NH, Flatt PR, et al. 13C NMR analysis reveals a link between L-glutamine metab- reduces filtration and fluid excretion. The changes in glomeru- olism, D-glucose metabolism and gamma-glutamyl cycle activity in a lar filtered load is most likely responsible for the changes in fluid clonal pancreatic beta-cell line. Diabetologia. 2003;46(11):1512-21. excretion but this remains to be resolved. Jiang N, Cox RD, Hancock JM. A kinetic core model of the glucose-stim- Guillermo BS et al (2006). Hypertension, 48, 467-472 ulated insulin secretion network of pancreatic β cells. Mammalian Genome. 2007;18:508-20. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

PC145

PC144 Effect of cystine dimethylester lysosomal loading on the viability of LLC-PK cells Impact of reactive oxygen species on renal function in 1 anaesthetised rats R.E. Sumayao and P. Newsholme Y. Margham and E.J. Johns School of Biomolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin 4, Ireland University college cork, Cork, Ireland Background. Cystinosis is an autosomal recessive disorder char- Reactive oxygen species (ROS) are a product of mutochondral acterized by excessive accumulation of cystine in the lysosomes respiration undertaken by a number of enzymes such as xan- of the cell. The disease is caused by a defective cystine trans- thine oxidase and NADPH oxidase. Although the level of one porter, cystinosin, in the lysosomal membrane which mediates ROS, superoxide anions, is limited by the enzyme superoxide cystine efflux from the lysosome to the cytosol (1). Cystinosis dismutase (SOD), it can affect physiological processes.There is affects almost all cells and tissues but the kidney involvement in vitro evidence (Gavin et al) that superoxide ions act directly remains the foremost clinical characteristic of the disorder to suppress epithelial transport along the nephron. The aim of which is manifested clinically by a generalized dysfunction of this study was to examine in vivo the impact of either increas- proximal tubular transport of glucose, amino acids, phosphate ing the level of SOD, or following inhibition of its action on renal haemodynamic and excretory function.

155P Poster Communications and essential ions resulting to Fanconi’s syndrome (2). Initial are also hypertensive but volume contracted[2]. This uncou- investigations on the pathogenesis of cystinosis were hampered pling of sodium and water reabsorption suggests a urine con- by inability to achieve elevated intracellular concentrations of centrating defect. We therefore assessed water balance and the cystine. This hurdle was eventually solved when it was demon- effect on urine concentration of a water deprivation challenge. strated that the methyl ester derivative of cystine leads to the Male C57/Bl and 11βHSD2-/- mice (n=5 per group) were indi- accumulation of cystine in the lysosomes in vitro and in vivo vidually housed in metabolic cages. After acclimatization, (3,4). cumulative water balance was taken over 4 consecutive days, Aim. In this study, we investigated the effect of cystine dimethyl after which drinking water was removed for 24h. Following 1 ester (CDME) on the viability of LLC-PK1 cells, an epithelial cell week recovery, mice were injected with desmopressin (ddAVP; line originally derived from porcine kidneys and expresses many 1μg/kg;S.C.) and water removed for 24h. At the end of the functions and characteristics of the proximal tubular cells. experiment mice were culled by decapitation. Data are pre- Methods. A colorimetric assay based on the reduction of tetra- sented as mean±S.E.M. Statistical analysis was performed using zolium salt WST-1 into a colored formazan dye by succinate- ANOVA, Mann-Whitney or t-tests as appropriate. tetrazolium reductase in viable cells was used as a measure of Water turnover during the baseline period was higher in the the net metabolic activity of cells following incubations at dif- 11βHSD2-/- mice than in controls, with the knockouts being ferent concentrations of CDME (0.1 to 1 mM). both polyuric (12.2±0.6 vs 2.1±0.1 ml/24hr P<0.001) and poly- ± ± Results. Incubation of LLC-PK1 cells with low CDME concentra- dipsic (15.9 0.6 vs 5.2 0.2 ml/24hr P<0.001). Despite this tions (0.1 and 0.2mM) for 60 and 120 minutes resulted in an cumulative water balance was not significantly different increase in WST-1 absorbance associated with enhanced mito- between the 2 groups. Urine osmolarity (Uosm) was signifi- chondrial metabolism, which may be related to higher demand cantly lower in the 11βHSD2-/- than controls (446±17 vs for ATP (139% of control at 0.1 mM CDME for 60 minutes to 1658±69 mOsm P<0.001). Urine flow rate fell in both groups 372% of control at 0.2 mM CDME for 120 minutes, p<0.05). following water deprivation, but 11βHSD2-/- mice continued However, the viability of the cells subsequently dropped to less to produce significantly more urine (1.7±0.1 vs 0.8±0.2 ml/24hr than 15% of control (p<0.05) upon incubation with higher con- P<0.01). Water deprivation caused an increase in Uosm in both centration of CDME (1mM) for 30, 60 and 120 minutes, indi- groups, however the increase was significantly blunted in the cating the acute lethal effect of CDME at high concentration. 11βHSD2-/- mice (1892±109 vs 4888±509 P<0.001). In both It can be speculated that exposure of non-cystinotic cells to groups of mice, ddAVP had no further effect on urine concen- high levels of CDME alters the metabolism and redox status of tration following water deprivation. This suggested the maxi- the cells sufficiently to trigger cell death. mal response to endogenous AVP had been achieved: notably -/- Taranta A, Palma A and Emma F. Pathogenesis of cell dysfunction in the response was significantly lower in 11βHSD2 mice nephropathic cystinosis. Pediatrics and Child Health 2007;17:S51-53. (2246±243 vs 4318±202 mOsm P<0.001). Weight loss during Gahl WA, Thoene JG and Schneider JA. Cystinosis. N Engl J Med 2002; water deprivation, used as an index of body water, was signifi- -/- 347:111-121. cantly greater in 11βHSD2 than the controls (3.0±0.1 vs 1.8±0.2 g P<0.001). Goldman R and Kaplan A. Rupture of rat liver lysosomes mediated by L-amino acid esters. Biochem Biophys Acta 1973;318:205-216. In conclusion we have identified a urine concentrating defect in 11βHSD2-/- mice, which may contribute to the contraction Foreman JW, Bowring MA, States JLB, and Segal S. Effect of cystine of plasma volume. Although 11βHSD2-/- mice retain a residual dimethyl ester on renal solute handling and isolated renal tubular transport in the rat: a new model of the Fanconi syndrome. Metabolism concentrating capacity, the maximum value is blunted relative 1987;36:1185-1191. to controls. We cannot exclude a defect in AVP release but the failure of ddAVP to restore urine concentrating ability suggests Where applicable, the authors confirm that the experiments a nephrogenic component to the diabetes insipidus. described here conform with The Physiological Society ethical Ferrari, P. and Z. Krozowski, Role of the 11beta-hydroxysteroid dehy- requirements. drogenase type 2 in blood pressure regulation. Kidney Int, 2000. 57(4): p. 1374-81. Bailey, M.A., et al., A switch in the mechanism of hypertension in the syndrome of apparent mineralocorticoid excess. J Am Soc Nephrol, PC146 2008. 19(1): p. 47-58.

A urine concentrating defect in 11b-Hydroxysteroid I would like to acknowledge Gillian Brooker for her technical Dehydrogenase Type 2 knockout mice assistance L.C. Evans, J.J. Mullins and M.A. Bailey Where applicable, the authors confirm that the experiments Molecular Physiology, Centre for Cardiovascular Science, University described here conform with The Physiological Society ethical of Edinburgh, Edinburgh, UK requirements. 11β-Hydoxysteroid Dehydrogenase Type 2 (11βHSD2), which inactivates cortisol, is co-expressed with mineralocorticoid receptors (MR) in aldosterone target tissues where it protects the receptor from activation by glucocorticoids. Mutations in 11βHSD2 cause the syndrome of apparent mineralocorticoid excess in which hypertension is thought to be driven by volume expansion secondary to sodium retention[1]. 11βHSD2-/- mice

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC147 requirements. Impact of Angiotensin 1-7 (Ang1-7) on renal haemodynamic and excretory function anaesthetised normotensive and hypertensive rats PC148 E.J. Johns and A. Albulushi Department of Physiology, University College Cork, Cork, Ireland ATP-mediated regulation of vasa recta diameter by pericytes in the renal medulla Increasingly, it is perceived that Ang1-7 has significant physio- 1 1 1 1 logical actions at the vascular and tubular levels of the kidney C. Sprott , R.L. Simmons , C. Crawford , T. Kennedy-Lydon , 2 1 1 which are generally opposite to those of angiotensin II (AngII). R.J. Unwin , S.S. Wildman and C.M. Peppiatt-Wildman Recent reports have shown Ang1-7 to cause a renal vasodila- 1Urinary Systems Physiology Unit, Royal Veterinary College, tion and a diuresis and natriuresis and that these actions were London, UK and 2Centre for Nephrology, University College London, enhanced in transgenic and renovascular models of hyperten- London, UK sion (Burgelova et al, 2005) and concluded that the response to the peptide was related to the degree of activation of the ATP-activated P2 receptors are expressed throughout the kid- renin-angiotensin system. This study tested the hypothesis ney in vascular smooth muscle, endothelial and tubular epithe- that the renal haemodynamic and excretory responses to Ang1- lial cells. Functional studies demonstrate that the renal vascu- 7 would be greater in the spontaneously hypertensive rat (SHR) lature is highly responsive to P2 receptor activation, and in vivo than normotensive rats. studies in rabbits have shown that the ATP analogue α, β-meth- Male Wistar and SHR (300-350 g) were anaesthetised with 1 ylene ATP reduces both cortical and medullary blood flow [1]. ml i.p. chloralose/urethane (16.5/250mg/ml) and cannulae In the renal medulla, blood flows via the vasa recta which are placed in a femoral artery to measure blood pressure and and devoid of contractile smooth muscle. In vitro studies looking at vein for the infusion of saline (9g/l NaCl) at 3 ml/h. The left kid- isolated descending vasa recta have however shown that ney was exposed, its ureter cannulated, an ultrasonic flow probe smooth muscle-like pericyte cells occur at regular intervals. placed on the renal artery and a small cannula inserted 4.5 mm Furthermore, it has been shown that these pericytes can con- into the kidney for infusion of saline/vehicle or Ang1-7 at strict isolated descending vasa recta in the presence of certain 1.0ml/h intra-renally (i.r.) at the cortico-medullary boarder. Fol- vasoactive substances [2]. In addition to studies on isolated lowing a 1-2 h recovery, four 20 min clearances were taken, 2 capillaries, pericytes have been shown to control capillary diam- before and 2 during the intrarenal infusion of saline or Ang1-7 eter in situ, in whole retina and in the CNS [3]. In the retina per- (3x10-7 M). Data, means ± S.E.M, were deemed significant icytes have been shown to respond to ATP [3, 4]. Here we aim when P<0.05 (Wilcoxon Signed Rank Test). to (i) investigate whether extracellular ATP and UTP constrict Infusion of saline i.r. in Wistar rats (n=5) had no effect on blood in situ vasa recta pericytes and (ii) identify expression of P2 pressure (BP), renal blood flow (RBF), glomerular filtration rate receptor subtypes in the medulla. (GFR), urine flow (UV), absolute (UNaV) or fractional sodium Kidney slices (200 μm) were obtained from Sprague Dawley excretion (FENa). In the Wistar group in which Ang1-7 was rats (killed by cervical dislocation) and maintained in a physi- infused i.r. (n=5), BP, at 92±5 mmHg, RBF, at 1.66±0.16 ml/min ological saline solution bubbled with 95% O2/ 5% CO2. Video and GFR, at 2.71±0.21 ml/min/kg, were unchanged but UV, at imaging techniques were utilized to study whether ATP and 19.7±3.8 μl/min/kg, UNa, at 0.29±0.11μmol/min/kg, and FENa, UTP regulate vasa recta pericytes in live kidney slices; pericytes at 0.69±0.27%, were increased (both P<0.05) by 2 to 2.5 fold. were identified by their ‘bump on a log’ morphology [3]. Infusion of Ang1-7 i.r. in the SHR (n=7) had no effect on either Immunohistochemistry techniques and confocal microscopy BP or RBF but increased UV, UNaV and FENa by between 2 and were employed to label and identify specific P2 receptor sub- 3 fold (all P<0.05), respectively, which were responses very sim- types in fixed kidney slices. ilar to those obtained in the Wistar rats. Superfusion of ATP (10 μM) evoked significantly greater vaso- These findings demonstrated that infusion of Ang1-7 locally constriction (p<0.05, Student’s t test) of vasa recta at peri- into the kidney at this dose, had minimal effects on renal cyte sites (24.37±4.94%, mean±s.e.m, n=5.) than at non-per- haemodynamics but caused a marked increase in both water icyte sites (2.15±2.11%, mean±s.e.m.). 100 μM UTP also and sodium excretion. This would suggest that the peptide was evoked significantly greater vasoconstriction (p<0.05, Stu- having a direct action on tubular reabsorptive processes. Sur- dent’s t test) of vasa recta at pericyte sites (8.63±1.38%, prisingly, the magnitude of the excretory responses in the SHR mean±s.e.m., n=12) than at non-pericyte sites (4.93±0.81%, were similar to those of the Wistar and did not support the pro- mean±s.e.m.). Higher concentrations of UTP (1 mM) evoked posed hypothesis. One possible reason may be due to the rel- a greater vasoconstriction of vasa recta at pericyte sites atively normal levels of plasma renin activity reported to be (15.48±1.84%, mean±s.e.m., n=5) but did not significantly present in the SHR (Bivol et al, 2007). constrict non-pericyte sites (5.17±0.84%, mean±s.e.m., Burgelova M et al. (2005). Kidney Int 67,1453-1461 p<0.05, Student’s t test). The P2 receptor antagonist suramin (100 μM) attenuated the ATP-evoked vasoconstriction by Bivol LM et al. (2007). Am J Physiol 293, F839–F845 56.12% (n=3). Immunohistochemistry experiments identified

expression of P2X3,P2X4 and P2X5 receptorsubtypesinthe renal medulla.

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drenaline (10nM) also evoked a significantly greater vasocon- Data presented here demonstrate that P2 receptors are striction of vasa recta at pericyte sites (20.77+5.05%, expressed throughout the medulla in rat kidney slices and that mean+s.e.m,n=7) than at non-pericyte sites (7.63+2.04%, both ATP and UTP can act specifically at pericytes to regulate mean+s.e.m, p<0.05, Student’s t test). The nitric oxide donor vasa recta diameter. We propose that pericytes play a key role S-nitroso-N-acetyl-l,l-penicillamine (SNAP 100μM) evoked a in regulating medullary blood flow at the capillary level in the significantly greater vasodilation of vasa recta at pericyte sites kidney. (14.05+3.52%, mean+s.e.m,n=12) than that at non-pericyte [1] Eppel GA, Ventura S & Evans RG (2006). Br. J. Pharmacol. 149 sites (4.23+1.62%, mean+s.e.m, p<0.05, Student’s t test). 523–531. Superimposing SNAP on Ang II significantly decreased (p<0.05, [2] Zhang Z, Rhinehart K, Kwon W, Weinman E & Pallone TL (2004) Am Student’s t test) the Ang II-evoked constriction (i.e. it dilated J Physiol Heart Circ Physiol. the vessel) by 84% (n=4) of the preconstricted diameter at the 287(2):H773-81. pericyte but had no effect elsewhere. [3] Peppiatt CM, Howarth C, Mobbs P & Attwell D (2006) Nature The techniques described here establish kidney slices as a viable 443(7112):700-4. model which can be used to investigate the role of in situ peri- [4] Kawamura H, Sugiyama T, Wu DM, Kobayashi M, Yamanishi S, Kat- cytes in the regulation of vasa recta. We have identified peri- sumura K & Puro DG (2003) J Physiol. 551(Pt 3):787-99. cytes along vasa recta capillaries in the renal medulla and have presented data that shows vasa recta pericytes, like CNS peri- Funded by the Medical Research Council. cytes, constrict vasa recta capillaries and may play a role in reg- Where applicable, the authors confirm that the experiments ulation of blood flow in the renal medulla. described here conform with The Physiological Society ethical Peppiatt CM et al., 2006 Nature 443(7112):700-704 requirements. Zhang Z et al., 2004 Am J Physiol Heart Circ Physiol. 287(2):H773-H781.

Funded by the Medical Research Council.

PC149 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. Pericyte-mediated regulation of vasa recta capillaries in situ C. Crawford1, C. Sprott1, T. Desai1, R.J. Unwin2, S.S. Wildman1 and C.M. Peppiatt-Wildman1 PC150 1Urinary System Physiology Unit, The Royal Veterinary College, 2 London, UK and Centre for Nephrology, University College London, Regulation of renal haemodynamics by reactive oxygen London, UK species in anaesthetised rats: a direct or indirect action via Pericytes have been shown to control capillary diameter in situ, nitric oxide? in whole retina and in the CNS (1). Pericytes occur at regular A.F. Ahmeda, E.E. O’Reilly and E.J. Johns intervals on renal medullary vasa recta capillaries and are known to constrict isolated perfused descending vasa recta (2). Here Department of Physiology, University College Cork, Cork, Ireland we aim to (i) identify renal pericytes on vasa recta capillaries The regulation of the blood flow through the cortex and in live kidney slices, (ii) establish kidney slices as a viable model medulla of the kidney is dependent on a range of factors one and (iii) investigate the role of pericytes in the regulation of of which may be the level of oxidative stress that can modu- vasa recta capillaries in situ. late vascular tone. Previously (Ahmeda and Johns, 2004), we Freshly isolated live kidney slices were labeled with calcein, pro- demonstrated that blockade of superoxide dismutase (SOD) pidium iodide and Hoechst and imaged using confocal imag- within the kidney decreased perfusion of blood through both ing techniques to confirm viability of kidney slices (n=3). Peri- the cortex and medulla. This study aimed to investigate cytes and vasa recta capillaries were labeled with fluorescently whether the vasoconstriction developed when superoxide conjugated NG2 (neural-glial 2) and IB (isolectin B ), respec- 4 4 anion production was increased was a consequence of a direct tively and pericyte location along vasa recta capillaries was con- action on the blood vessels or an indirect one due to scaveng- firmed in the medulla of kidney slices by confocal imaging tech- ing of nitric oxide (NO). niques (n=10). Video imaging techniques were utilized to Groups (n=6) of male Wistar rats (250-300 g) fed a normal (0.3% identify pericytes along vasa recta capillaries in live kidney slices Na+) or high salt (3% Na+) diet, were anaesthetised with 1 ml and to investigate whether pericytes regulate the diameter of i.p. chloralose/urethane (16.5/250 mg/ml, respectively) and vasa recta capillaries in response to vasoactive agents in adult supplemented as required. The right femoral vein and artery rat kidney slices. Kidney slices (200μm) were obtained from were cannulated for infusion of saline (154 mM NaCl) at 3 ml/h, adult Sprague Dawley rats (killed by cervical dislocation). and measurement of arterial blood pressure (BP). The left kid- Here we show for the first time, pericyte-mediated constric- ney was exposed via a flank incision, placed in a holder and a tion of vasa recta in situ, in live kidney slices. Superfusion of small cannula inserted 4.5 mm into the kidney for Angiotensin-II (Ang-II 100nM) resulted in an average constric- intramedullary (i.m.) infusion of drugs at 0.6-1.0 ml/h. Two tion of vasa recta capillaries at pericyte sites of 29.26+4.67% Laser-Doppler microprobes were inserted 1.5 and 4.0 mm into (mean+s.e.m,n=14). This was significantly greater than at non- pericyte sites (p<0.001, Student’s t test) where capillary diameter changes were 7.56+1.98%, (mean+s.e.m,n=14.). Nora-

158P Poster Communications the kidney to measure cortical (CP) and medullary (MP) blood lation and measurement of RSNA with a bipolar stainless steel perfusion, respectively After 90min, baseline values were taken electrode. GFR was quantified using FITC–Inulin clearance. Kid- then either diethyl-dithio-carbamate (DETC), a SOD inhibitor, neys were removed post infusion and homogenates were blot- 1, 2, and 4 mg/kg/min or a combination of DETC plus L-NAME ted for ENaCα, AQP2 and MC3R. Cultured inner medulla col- (Nitric oxide synthase, NOS, inhibitor) 10 μg/kg/min were lectingductcells,isolatedfrommaleWistarkidneys(1)were infused i.m. for 30 min. At the end of the experiments the ani- treated with 100nM NDP-γMSH for 3 hours in the presence/ mals were killed with an anaesthetic overdose. Data ± SEM were absence of Cytochalsin D & Brefeldin A. Total cell lysates and subjected to the Student′s t-test and significance taken at plasma membrane fractions prepared by differential centrifu- P<0.05. gation were blotted for ENaCα, AQP2 and MC3R. Baseline BP was similar in both normal and high salt rats, 110±5 Infusion of NDP-γMSH increased FeNa more in HNa (165±14% and 120±6 mmHg, as was CP,230±30 PU versus 208±28 PU but P<0.01) than NNa (10±4%); raised UV more in HNa, MP was lower in the high salt compared to the normal salt rats, 45±12.7μl/min/kg (P<0.03) than NNa, 6.23.1± 2.9μl/min/kg; 47±5PUversus62±6 PU (P<0.05). Infusion of DETC in the nor- increased RSNA more in HNa, 46±10% (P<0.001) than NNa, mal salt rats decreased CP and MP by 23±8% and 23±7% (both 0.4±0.1%; raised GFR only in HNa, 3.20±0.17ml/min/kg P<0.05), whereas in the high salt group CP fell by 15±4% and MP (P<0.05), NNa, 1.86±0.15 ml/min/kg. MAP (NNa, 96±3mmHg; by 21±4% (both P<0.05) and there were no differences in the HNa 96±1mmHg) and heart rate (NNa, 369±16bpm; HNa, degree of reduction in either group. The magnitudes of reduc- 415±15bpm) did not change during infusion. Results, presented tion in CP and MP in both groups after DETC plus L-NAME were as means+/-SEM, were compared using paired t-tests and post not different from those obtained when DETC was infused alone. hoc bonferroni correction. The lower level of MP in the high salt rats suggested that oxidative Infusion of NDP-γMSH significantly decreased ENACα expres- stresswasenhancedinthisregionasaconsequenceoftheelevated sion and increased AQP2 and MC3R expression in the infused sodium intake. Following the blockade of SOD, the raised level of kidney (n=4). In vitro treatment of collecting duct cells with superoxideanionsinthekidneymostprobablymediatedthisvaso- NDP-γMSH resulted in a significant decrease in expression of constrictoractioninthecortexandmedulla.Theobservationthat ENaCα and significant increases in expression of AQP2 and themagnitudesofthereductionsinMPwereunchangedwhenNO MC3R and was accompanied by a decrease in the plasma mem- production was blocked suggested that NO was not involved and brane abundance of ENaCα which was prevented by Cytochalsin thatthesuperoxideanionswereactingdirectlyonthevasculature. D & Brefeldin A (n=3). Ahmeda & Johns (2004). J Physiol 560P, C3l Data suggests that NDP-γMSH possesses same natri- Where applicable, the authors confirm that the experiments uretic/diuretic, but not cardiac pressor, effects as native γMSH. described here conform with The Physiological Society ethical Natriuretic effects may be achieved by redistribution from api- requirements. cal membranes and a down regulation in expression of ENaC in collecting duct cells mediated by MC3R. Ni XP, Bhargava A, Pearce D, Humphreys MH. Modulation by dietary PC151 sodium intake of melanocortin 3 receptor mRNA and protein abundance in the rat kidney. Am J Physiol Regul Integr Comp Physiol. (2006) 290:R560-7. Intra-renal infusion of NDP-gMSH evokes a potent natriuresis and diuresis and is associated with collecting duct changes Ni XP, Humphreys MH. Prevention of salt-induced hypertension by an in ENaCa expression analog of gamma-melanocyte-stimulating hormone in the rat. Am J Hypertens. (2007) 20:862-5. G. Cope1, H. Belinda L.1,2, V. Healy1 and J. Edward J.1 1Physiology,UniversityCollegeCork,Cork,Irelandand 2Physiologyand Science Foundation Ireland. Pharmacology,OregonHealth&ScienceUniversity,Portland,OR,USA Where applicable, the authors confirm that the experiments Gamma melanocyte stimulating hormone (γMSH) possesses described here conform with The Physiological Society ethical cardiac pressor effects and promotes natriuresis by binding requirements. to melanocortin 3 receptor (MC3R) in the inner medulla (1). In this study we examined the in vivo and in vitro effects of a stable analogue NDP-γMSH (2) on mean arterial pressure PC152 (MAP), heart rate, fractional sodium excretion (FeNa), urinary flow rate, (UV) renal sympathetic nerve activity (RSNA) and Acute and chronic effects of nerve growth factor on expression of epithelial Na+ channel (ENaC), aquaporin-2 cystometric parameters in female Wistar rats (AQP2) and MC3R. Male Wistar rats were fed 0.3% (NNa) or 3% (HNa) sodium for S. Cummings, J. Gardiner, G. McMurray and K. Conlon 6 weeks from 3 weeks of age. They were anaesthetised with 1 ml (I.P.) of a chloralose–urethrane (16.5 and 250 mg ml–1, Genitourinary Biology, Pfizer Global Research and Development, respectively) and maintained with supplemental doses of 0.05 Sandwich, UK ml every 30 min. Indwelling cannulae were inserted for measurement of blood pressure, i.v. fluid infusion (0.15M saline) and Nerve growth factor (NGF) elicits bladder hyperactivity following acute intravesical administration and is involved in collection of urine. Both kidneys were exposed, the left was can- (1) γ experimentally induced cystitis , whilst 2 week intrathecal nulated for direct infusion of 100nM NDP- MSH (300fmol/min) (2) into cortico-medullary border and the right for renal nerve iso- delivery of NGF or administration of NGF into the bladder wall reduces bladder capacity and increases voiding frequency in

159P Poster Communications rats(3). We investigated the duration of action of acute intravesical NGF on voiding parameters in anaesthetised cystome- PC153 try, and also the effects of chronic I.P. dosing of NGF in conscious rats. Radiotelemetry allows measurement of both urodynamic Female Wistar rats (225-260g) were anaesthetised with and haemodynamic parameters in conscious unrestrained isoflurane (2-5% in O2) and urethane (1.25g/kg I.V.). A jugu- female beagle dogs lar vein (anaesthetic infusion), carotid artery (BP measure- 1 2 2 2 2 ment) and trachea were cannulated. The bladder was can- G. Peacock , H. West , J. Wickens , H. Gunton , W. Attwell and 1 nulated via the urethra and tied at the external meatus, K. Conlon creating a closed system for isovolumetric cystometry. Fol- 1Genitourinary Biology, Pfizer Global Research and Development, lowing baseline cystometrograms with saline (75μl/min) Sandwich, UK and 2Experimental Biological Sciences, Pfizer Global until reflex bladder contractions occurred (threshold vol- Research and Development, Sandwich, UK ume), the bladder was filled to capacity with 20μg/ml NGF or vehicle (10% DMSO in saline) which was held in situ for 1 Urodynamics have been traditionally recorded in anaesthetised hour (h) before draining. Cystometrograms were then or tethered animals. We have evaluated a model for simulta- repeated at 1, 2, 3, and 4h post treatment. In a separate study neous measurement of urodynamic and haemodynamic rats were anaesthetised with isoflurane, and the bladder can- parameters in conscious, freely moving female dogs via nulated aseptically via the urethra. The bladder was filled radiotelemetry. All experiments were conducted in accordance with 1ml of either 20μg/ml NGF or vehicle which was held with UK legislation and local ethical guidelines. Four female in situ for 1h before draining. The catheter was removed and beagle dogs (9-15kg) were surgically anaesthetised with propo- animals allowed to recover for 24h before undergoing anaes- fol (70-80mg) and maintained with isofluorane anaesthetic (2% thetised cystometry as described above. In conscious stud- in 1:2 O2/NO2 mix) for implanting telemetry transmitters (D70- ies rats (225-260g) were dosed daily for 10 days with PCT) to measure bladder pressure, blood pressure, ECG and 0.1mg/kg NGF or saline 10ml/kg I.P. and voiding function temperature. An additional catheter was sutured into the blad- assessed the following morning via metaboles over 3h. Mean der and attached to an access port located under the skin to ± S.E.M.fromeachstudywerecomparedbyANOVAforNGF allow filling and emptying of the bladder. Animals were given vs. vehicle treated rats, P<0.05 was considered statistically appropriate antibiotic and analgesic treatment prior to and fol- significant. lowing surgery. After recovery, animals were habituated to Intravesical NGF (n=5) significantly decreased % baseline saline bladder filling via an infusion line connected to the blad- threshold volume vs. vehicle (n=4) at 2 (63.5±4.6 vs. der access port. Animals were singly housed in metabolism 106.2±10.7), 3 (78.9±4.5 vs. 102.2±7.7) and 4h post treat- cages during studies to allow urine collection, the size of the ment (75.7±7.5 vs. 108±7.4). Contraction frequency was signi- cages allowed free movement. The optimum infusion rate for ficantly increased in the NGF treated group vs. vehicle at 1 each animal was determined using a theoretical natural diure- (4±0.6 vs. 2±0.9), 2 (12±1.4 vs. 2±0.9), 3 (9±1.1 vs. 3±0.9) and sis rate of 360ml/hr [Hamaide et al, 2003] as a starting point, 4h post-dose (10±2.1 vs. 2±0.9). At 24h post NGF there were and for each animal was identified based on consistency of blad- no significant effects on either threshold volume or contrac- der fill volumes and pressure. After each study animals were tion frequency. In conscious rats an increase in voiding fre- returned to their home pens. All 4 animals’ bladder fill data was quency and reduction in volume per void developed over time consistent over subsequent fills in the same day and subsequent in the NGF treated group (n=4), reaching significance at day days. Table 1 shows maximum threshold and micturition pres- 5 for voiding frequency, and at day 6 for volume/void vs. vehi- sure, bladder fill volume, MAP prior to first bladder fill, MAP cle (n=4). No significant difference between NGF and vehicle prior to final fill and optimal infusion rate for each animal [mean treatment was observed more than 24h post the last treat- ± SEM (n)]. ment day. Simultaneous measurement of haemodynamic parameters These results indicate that depending on the mode of admin- showed that cystometry did not cause stress to the animals. istration, NGF can produce both an acute and more chronic In conclusion this model allows simultaneous measurement bladder hyperactivity that persists for up to 24h post dose. of urological and cardiovascular parameters in the absence Dmitrieva et al. Neuroscience Vol. 78, No.2, pp. 449-459, 1997 of anaesthetic or restraint providing a useful means to eval- Yoshimuraetal.ThejournalofNeuroscience26(42):10847-10855,2006 uate the effects of drugs on urological and cardiovascular function. Zvara & Vizzard. BMC Physiology 2007, 7:9 Table 1 Urological and haemodynamic parameters at the optimal infusion rate for each animal Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

Hamaide AJ et al (2003). Am J Vet Res: 64(5); 574-579

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC155 requirements. Thyroxine-induced heart hypertrophy: impact on cardiac function F. Lainis Department of Physiology, University College Cork, Cork, Ireland PC154 The impact of experimentally induced cardiac hypertrophy on cardiac haemodynamic parameters and inotropic responses is Vascular responses mediated by GPR30/GPER not fully understood. E. Brailoiu1, A.A. Tica1, C.G. Brailoiu1, J.B. Arterburn2, This study aimed to quantify the durational effect of thyrox- M. Barton3, E.R. Prossnitz4, T.I. Oprea5 and N.J. Dun1 ine(T4)-induced cardiac hypertrophy on the haemodynamic and cardiac function parameters and to investigate how they 1Pharmacology, Temple University School of Medicine, responded when challenged with the β-adrenoceptor agonist, Philadelphia, PA, USA, 2Department of Chemistry and isoprenaline, before and following blockade of nitric oxide (NO) Biochemistry, New Mexico State University, Las Cruces, NM, production with the NO synthase inhibitor NG-nitro-L-arginine USA, 3Departement für Innere Medizin, Universitätsspital (L-NAME). Zürich, Zürich, Switzerland, 4Department of Cell Biology and Heart hypertrophy was induced by 7- and 14-day regimes of Physiology, University of New Mexico School of Medicine, intra-peritoneal T4 treatment. Eight controls, nine T4-7 and Albuquerque, NM, USA and 5Department of Biochemistry and nine T4-14 cardiac hypertrophied male Wistar rats (250-290g), Molecular Biology, University of New Mexico School of Medicine, respectively, were anaesthetized i.p. with 1.0 ml chlo- Albuquerque, NM, USA ralose/urethane mixture (16.5/250 mg/ml, respectively) and prepared for in vivo measurement of haemodynamic and GPR30/GPER is a novel G protein-coupled estrogen recep- cardiac function parameters. tor. Using a rabbit polyclonal antiserum against the human Heart weights were higher in the group given T4 for both 7 days GPR30 C-terminus, we identified GPR30 immunoreactivity in and 14 days than control group 14% and 19% (P<0.05), respec- the cytoplasm of human aortic endothelial cells (HAEC). tively. This was associated with an increase in heart rate, max- Administration of G-1, a selective GPR30 agonist or 17b-estra- imum dP/dt and contractility index by 16%, 34% and 14% diol (E2) to HAEC produced a fast and sustained increase in (P<0.05) respectively in the T4-14 rats. The L-NAME increased μ intracellular Ca2+ concentrations [Ca2+]i. G-1 (0.01 M, 0.1 the basal levels of blood pressure, heart rate, contractility index μ μ M and 1 M) increased [Ca2+]i by 93 + 3.2, 176 + 4.2, and and maximum dP/dt by 39%, 23%, 32% and 27% (P<0.05) μ 558 + 6.9 nM, respectively. E-2 (15 M) increased [Ca2+]i by respectively in T4-14 rats. The L-NAME also augmented the iso- 127 + 2.1 nM. Activation of GPR30 caused NO production in prenaline’s inotropic effect in both T4-7 and T4-14 hypertro- μ HAEC and NO-dependent aortic ring relaxation. G-1 (1 M) phied hearts but had no effect on basal myocyte contractility. μ Δ or E2 (15 M) increased DAF (F/F0) fluorescence by 0.09 + The T4-14 heart hypertrophy rats showed reduced stress 0.009 and 0.07 + 0.009, respectively, while acetylcholine (15 endurance capacity compared to both control and 7 days heart μ Δ M) increased DAF by 0.31 + 0.01. Administration of G-1 hypertrophy rats. μ μ μ (0.01 M, 0.1 M and 1 M) produced HAEC hyperpolariza- The haemodynamic and cardiac function parameters were tion with an amplitude of 4 + 1.9 mV, 18 + 3.6 mV, and 28 + significantly enhanced after 14 days thyroxine-induced cardiac μ 3.8 mV, respectively. E2 (15 M) hyperpolarized HAEC by 16 hypertrophy indicating increased cardiac performance1. Fur- + 2.6 mV. Intravenous administration of G-1 (41.2 ng/kg to thermore, the L-NAME caused significant augmentation of the μ 41.2 g/kg) to urethane-anesthetized Sprague Dawley rats isoprenaline inotropic effect, in addition to the enhanced effect produced a dose-dependent decrease (by 2.6 + 1.6 % to 14.7 on the basal cardiac performance in the 14 days hypertrophy + 1.1%) in mean arterial pressure. Taken together, our results hearts. A more severe degree of cardiac hypertrophy was asso- indicate that GPR30 is involved in the vascular response to ciated with a reduction in stress endurance capacity. estrogen. Flanagan ET, Buckley MM, Aherne CM, Lainis F, Sattar M, Johns EJ. Impact of cardiac hypertrophy on arterial and cardiopulmonary baroreflex con- Where applicable, the authors confirm that the experiments trol of renal sympathetic nerve activity in anesthetized rats. Exp Phys- described here conform with The Physiological Society ethical iol 2008; 93(9): 1058-1064. requirements. Morgan HE, Baker KM. Cardiac hypertrophy. Mechanical, neural, and endocrine dependence. Circulation 1991; 83: 13-25. Kahaly GJ, Dillmann WF. Thyroid hormone action in the heart. Endocrine Reviews 2005; 26(5): 704-728. Polikar R, Burger AG, Scherrer U, Nicod P. The thyroid and the heart. Circulation 1993; 87: 1435-1441. Balligand JL, Kelly RA, Marsden PA, Smith TW, Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signalling system. Proc Natl. Acad Sci USA 1993; 90: 347-351. Supervisor: Professor Edward J. Johns: Department of Physiol- ogy, University College Cork, Republic of Ireland.

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Where applicable, the authors confirm that the experiments Milutinovi ´c S et al. (2006). Am J Physiol Regulatory Integrative Comp described here conform with The Physiological Society ethical Physiol 291:1579-1591. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC156

Cardiovascular short-term variability modulation by central PC157 V1a and V2 receptor blockade involve thermoregulation: transfer function study Time and Frequency Domain Analysis of the Cardiovascular Response to Shaker Stress in Borderline Hypertensive Rats S. Milutinovic-Smiljanic1 and N. Japundzic-Zigon2 O.D. Sarenac1, M. Lozic1, S. Drakulic1, D. Bajic3, J.F.R. Paton2, 1General and oral histology and embryology, School of Dentistry, D. Murphy2 and N. Japundzic Zigon1 Belgrade, Serbia and 2Institute of Pharmacology, School of Medicine, Belgrade, Serbia 1Institute of Pharmacology, School of Medicine University of Belgrade, Belgrade, Serbia, 2University of Bristol, Bristol, UK and We have previously shown that central vasopressin receptors 3University of Novi Sad, Novi Sad, Serbia contribute to cardiovascular (CV) short-term variability in normotensive freely moving rats(1). In this study we investigate We investigated the effects of shaker stress on blood pressure the effect of central vasopressin receptor blockade on blood (BP) and heart rate (HR) short-term variability and spontaneous pressure (BP) and heart rate (HR) short-term variability of con- baroreceptor reflex (BRR) sensitivity in adult normotensive Wis- scious rats and its relationship to changes in body temperature tar rats (WR) and borderline hypertensive rats with family his- (Tb) using transfer function. All procedures are in accordance tory of hypertension (BHR). Experiments were performed in with ECC Directive 86/609. Adult male Wistar outbred rats were accordance with Directive 86/609/ECC: under anesthetized with xylazine/ketamine anesthesia (0.4ml 10% xylazine/ketamine anesthesia (0.4 ml 10% ketamine IP,0.1 ml ketamine IP, 0.1ml 2%xylazine IP per animal) for insertion of 2% xylazine IP) TA11PA-C40 DSI implants were inserted in TL11M2-PA-C50-PXT DSI implant for concomitant measure- abdominal aorta and rats were treated with metamizol (200mg ment of BP in aorta and Tb subcutaneously. After full recovery IP) for pain refief. At the end of experiments rats were eutha- under the same anesthesia, rats were chronically instrumented nized with bolus overdose of thiopentone sodium. Only after with intracerebroventricular (icv) cannula (1). The role of cen- full recovery (10 days) rats were included in experimentation. tral vasopressin receptors was assessed using selective non- BP was recorded 20 minutes before, during acute exposure to peptide V1a, V1b and V2 antagonists injected icv, as described shaking platform at 200 cycles/min/10 minutes, and 30 min- previously (1). At the end of experimentation rats were eutha- utes after. This was followed by random exposure to 5 min- nized with bolus overdose of thiopentone sodium. Arterial BP utes long shaking period 18 times/day/3 days (1). BP was and Tb were digitalized at 1000Hz. Systolic BP (SBP), diastolic recorded during the last exposure to stress, as well as 30 min- BP (DBP) and HR were derived from the arterial BP as maximum, utes after. Arterial BP was digitalized at 1000Hz. Systolic BP minimum and inverse of interbeat interval, respectively. Time (SBP), diastolic BP (DBP) and HR were derived from the arterial spectra and transfer function (gain, phase and coherence) were BP as maximum, minimum and inverse of interbeat interval, calculated on 15 overlapping 2048 point time series involving respectively. Evaluation of the spontaneous BRR was performed 410-s registration period. Spectra were analyzed in very-low- by using the method of sequences and calculation of barore- frequency (VLF: 0.0195 - 0.195Hz), low-frequency (LF: 0.195 - ceptor reflex sensitivity-BRS and effectiveness index-BEI 0.8Hz) and high-frequency (HF: 0.8 - 3Hz) range. At neutral (2).Time spectra were calculated on 15 overlapping 2048 point ambient temperature (22∞C ± 2) 100ng of V1a antagonist time series involving 410-s registration period (3). Spectra were increased coherence between Tb and SBP in LF spectral band analyzed in very-low-frequency (VLF:0.0195-0.195Hz), low- (0.56 ± 0.03, p < 0.05). Moreover SBP and HR coherence in LF frequency (LF:0.195-0.8Hz) and high-frequency (HF:0.8-3Hz) spectral band was increased by 100ng (0.76 ± 0.02, p < 0.05) band. Statistical significance was assessed with one-way and 500ng of V1a antagonist (0.8 ± 0.03, p < 0.05). 100ng of ANOVA. Basal values of SBP, DBP and HR were comparable in V2 antagonist increased coherence between SBP and HR (0.83 both strains. Exposure of WR and BHR to acute shaker stress ± 0.04, p < 0.05), Tb and SBP (0.74 ± 0.1, p < 0.05) and Tb and increased SBP and DBP (WR:124.5 ± 4.5 mmHg, p<0.05 and HR (0.58 ± 0.03, p < 0.05). 100ng and 500ng of V2 antagonist 95.3 ± 2.0 mmHg, p<0.05; BHR:149.3 ± 3.4 mmHg, p<0.01 and increased coherence between Tb and SBP in VLF band (0.66 ± 109.9 ± 3.9 mmHg, p<0.05), and reduced VLF SBP and VLF DBP 0.15, p < 0.05; 0.59 ± 0.1, p < 0.05). Phase analysis in LF band (WR:2.8 ± 0.7 mmHg2/Hz, p<0.05 and 1.5 ± 0.1 mmHg2/Hz, under V2 receptor blockade indicate that changes in temper- p<0.05; BHR:2.0 ± 0.3 mmHg2/Hz, p<0.05 and 2.3 ± 0.4 ature precede those of SBP and HR. In rats exposed to 34∞C ± mmHg2/Hz, p<0.05). In HR spectra of WR, but not of BHR, LF 2 ambient temperature we noted increase of coherence HR (13.2 ± 0.4 bpm2/Hz, p<0.05) and HF HR (5.6 ± 0.6 bpm2/Hz, between SBP and HR in LF spectral band under V2 receptor p<0.05) increased. Chronic stress increased HF SBP and HF DBP blockade (0.83 ± 0.02, p < 0.05). Results suggest that central in both strains (WR:0.5 ± 0.1 mmHg2/Hz, p<0.05 and 0.4 ± 0.1 V1a and V2 receptors contribute to CV short-term variability mmHg2/Hz, p<0.05; BHR: 0.5 ± 0.1 mmHg2/Hz, p<0.05 and 0.4 in LF and VLF spectral band involving baroreceptor reflex - ± 0.1 mmHg2/Hz, p<0.05), and enhanced LF HR (5.9 ± 1.3 dependent and - independent thermoregulation, respectively. bpm2/Hz, p<0.05) and HF HR (2.5 ± 0.6 bpm2/Hz, p<0.05) only

162P Poster Communications in WR. BRS and BEI did not change during acute and chronic discharge of rat vagal paraganglia during normoxia, it can exposure to stress in both strains. Spectral analysis results show modulate hypoxic responses. Therefore IL-6 may not play a that BHR exhibit reduced HR variability response to acute and role in modulating discharge during exercise when active chronic shaker stress in comparison to normotensive control, muscles release it, but rather modulate discharge when para- suggesting remodeling of cardiac regulation in absence of ganglia become inflamed, e.g. during chronic hypoxia (Liu hypertension. et al., 2009). Farah V et al.(2004).Physiology&Behavior 83:135-142 O’Leary DM, Murphy A, Pickering M, Jones JFX. Arterial chemoreceptors in the superior laryngeal nerve of the rat. Respiratory Physiology Di Rienzo M et al.(2001).Am J Physiol Regulatory Integulatory Comp and Neurobiology. 2004; 141(2): 137-44. Physiol 280:744-751 Liu X, He L, Stensaas L, Dinger B, Fidone S. Adaptation to chronic hypoxia Stojicic et al.(2008).Neuropharmacology 54(5):824-36 involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. American Journal of Physiology - Lung Where applicable, the authors confirm that the experiments Cellular and Molecular Physiology. 2009;296: L158-L166. described here conform with The Physiological Society ethical requirements. This research was funded by the School of Medicine and Med- ical Science, University College Dublin and the Wellcome Trust (UK).

Where applicable, the authors confirm that the experiments PC160 described here conform with The Physiological Society ethical requirements. The Effect of IL-6 on the Hypoxic Sensitivity of Vagal Nerve Paraganglia in the Rat

E.T. O’Connor, K.D. O’Halloran and J. Jones PC161 School of Medicine and Medical Science, University College Dublin, Dublin, Ireland Correlation of plasma B-type natriuretic peptide level with ejection fraction in primary hypertensive patients Paraganglia of the vagus (Xth cranial) nerve are minute clus- 1 2 3 ters of glomus cells resembling the carotid body – an organ that N.J. Nordin , L. Muhamed and R. Harun senses changes in blood chemistry. They are chemosensitive 1Physiology, Science Islam University of Malaysia, Kuala Lumpur, to cyanide and reductions in the partial pressure of oxygen Wilayah Persekutuan, Malaysia, 2Cardiology, Cyberjaya University (PO2) (O’Leary et al., 2004). We proposed that they may also College of Medical Science, Bangi, Selangor, Malaysia and 3National be sensitive to circulating cytokines released from exercising University of Malaysia, Kuala Lumpur, Wilayah Persekutuan, muscles. Specifically, we wished to test our hypothesis that the Malaysia myokine- interleukin-6 (IL-6) can cause an acute change in superior laryngeal nerve (SLN) activity. Currently we are in the midst of a chronic disease epidemic of We exposed isolated superfused rat glomus cells - located heart failure, one of the worst complications for primary hyper- at the bifurcation of the SLN – to hypoxia (~20mmHg) to tensive patients. Ejection fraction is an important measure measure chemosensitivity to changes in PO2. Next, we forleftventricularfunction,aswellasanindicatorforthepres- exposed the cells to IL-6 at concentrations of 0.1ng/ml, enceofheartfailure(1&2).EarlierdatasuggeststhatB-type 0.3ng/ml and 1ng/ml during both normoxia (~100mmHg) natriuretic peptide level partially reflects the ventricular pres- and hypoxia while monitoring single axonal action potential sure (3). The aim of this work is to investigate whether plasma frequency. This was followed by hypoxia in the absence of IL- concentration of BNP reflects the heart’s capability with the 6 to check that the preparation was still responsive. One sin- ejection fraction as the indicator. This study was approved by gle fibre per animal was tested and values are expressed as the ethical committee and consents from all the subjects were mean ± S.E.M. collected prior taking samples. It was conducted on 50 hyper- The results confirm previous findings that these cells respond tensive patients referred for echocardiography to evaluate the strongly to changes in PO2, significantly increasing their dis- ventricular function. Patients with diabetes mellitus, previous charge rate in response to hypoxia (from 0.71 ± 0.36 to 6.0 ± history of myocardial infarction were excluded from the study. 2.4Hz). IL-6 did not affect the baseline discharge rate of the SLN The cardiologist making the assessment of left ventricular func- units at any concentration; however, it did modulate the tion was blinded to BNP levels. The BNP levels were assessed response to hypoxia. In 5/6 animals the smallest IL-6 concen- using the Triage Meter from Biosite Diagnostics. Results tration (0.1ng/ml) caused a small increase in peak frequency showed that BNP levels display a negative correlation with ejec- of the SLN (mean peak frequency 6.6 ± 2.6 Hz). Also, in 5/6 ani- tion fraction (Pearson correlation test) and it is clearly shown mals the largest IL-6 concentration (1ng/ml) caused a decrease in the BNP versus ejection fraction scatter plot graph. The signi- in peak frequency of the SLN ficant result (paired t test, p < 0.05) proves that both predic- (4.7 ± 2.1 Hz). There was a statistically significant difference tors are very important and relates to each other. The study in peak discharge from the lowest to the highest concentra- which is the first to be conducted in Malaysia may be helpful tion of IL-6 (Wilcoxon signed-rank test, p=0.03). This data in ruling out the diagnosis of heart failure or as the best screen- shows that although the myokine IL-6 has no effect on basal ing method.

163P Poster Communications

ogy at different temperatures, temperature can become an important parameter to control in electrophysiological experiments. By heating the bath solution and the solution entering the bath of a planar patch clamp system, the temperature of the external solution can be constantly maintained at a given temperature. Data will be presented showing hERG recordings at room temperature and at physiological temperature, including a full concentration response curve to quinidine at physiological temperature. Not only can the amplitude and kinetics of currents be changed by temperature, indeed, some ion channels can actually be activated by elevated temperature. The TRPV1 channel, for example, can be activated by ligands such as capsaicin, but also has been reported to be activated by temperatures above ~42∞C. Using a heated pipette, the temperature of the added solution can be increased and then rapidly applied to the cell. Very rapid changes in temperature can be achieved at the cell (within ms), from room temperature up to 60∞C, whilst continuously Dao Q, Krishnaswamy P, Kazanegra R, Harrison A, Amirnovin R, Lenert recording. Exemplar traces will be presented showing heat acti- L. 2001. Utility of B-type natriuretic peptide in the diagnosis of con- vation of TRPV1 expressed in CHO cells using a range of tem- gestive heart failure in an urgent care setting. J Am Coll Cardiol.37(2): 379-385. peratures. TRPV1 channels could prove to be important targets for the treatment of pain and, therefore, the temperature acti- Magliano D, Liew D, Ashton E, Sundararajan V, McNeil J. 2003. Novel biomedical risk markers for cardiovascular disease. JCardiovasc Risk. vation of TRPV1 could have important implications for drug 10(1):41-55. design as well as investigations into physiological modes of action. Remme WJ & Swedberg K. 2001. Task force for the diagnosis and treatment for chronic heart failure, European Society of Cardiology. Guide- Where applicable, the authors confirm that the experiments lines for the diagnosis and treatment of chronic heart failure. Eur Heart described here conform with The Physiological Society ethical J. 22(17):1527-1560. requirements. This study is funded by UNISEL and IMU. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical PC165 requirements. Phagocytic activity is increased in dissociated CD11b- positive cells prepared from brains of aged rats but this is PC163 not affected by treatment with amyloid-beta in vitro 1 2 2 1 Expanding applications of automated planar patch clamp: J. O’Reilly , W. Chen , Y.K. Gun’ko and M.A. Lynch A focus on internal exchange and heat activation 1Trinity College Institute of Neuroscience, Trinity College Dublin, 2 A.R. Haythornthwaite, S. Stoelzle, C. Haarmann, C. Farre, Dublin, Ireland and CRANN Institute, Trinity College Dublin, Dublin, A. Brueggemann, M. Kreir, M. Beckler, M. George and N. Fertig Ireland Nanion Technologies, Munich, Germany Microglia are the resident macrophage-like cells of the CNS and are partly responsible for the clearance of amyloid-beta (Aβ) in Planar patch clamp devices are finding their place in academic the healthy brain. They display similar properties to peripheral and industrial labs because of their ease of use and higher macrophages and therefore they are phagocytic and capable throughput as compared with conventional patch clamp. Pla- of cytokine production. Alzheimer’s Disease (AD) is a common nar borosilicate glass chips are used for performing whole cell, age-related neurodegenerative disease characterised by the perforated patch, or cell attached electrophysiological record- formation of insoluble amyloid plaques that are associated with ings. High quality data is acquired with a high success rate for activated microglia, but it is unknown whether these microglia obtaining giga-seals (typically 60-80%). Given the design of the are involved in phagocytosis. Here we set out to assess whether borosilicate glass chip, the internal recording solution is much phagocytic activity was altered in cells prepared from aged, more accessible to exchange. This means that experiments compared with young, rats and to evaluate whether Aβ mod- involving exchange of the internal solution can be performed ulated phagocytic activity in these cells. with relative ease. Exemplar traces will be shown for block of We investigated phagocytic activity in rat primary CD11b-pos- KV1.3 channels by the internal perfusion of ions and small mol- itive cells using flow cytometry to assess uptake of quantum ecules. In addition, data will be presented showing planar lipid dots and found pre-treatment with Aβ1-42 (2μM, 24h) exerted bilayers which were used to estimate the time required to fully no significant effect on uptake. Phagocytosis of quantum dots exchange the internal solution. (QD) was significantly decreased by pre-treatment of cells with Since many ion channels are sensitive to temperature and some H2O2 (**p<0.01; ANOVA; n=18) or NaF (***p<0.001; ANOVA; compounds have been shown to exhibit different pharmacol- n=18). Phagocytosis was significantly increased in CD11b-pos-

164P Poster Communications itive cells prepared from aged, compared with young, rats tions between the auditory and other sensory circuits in this (*p<0.05; Student’s t-test; n=26). We investigated microglial structure. We recently developed a method that allows deliv- activation in brain tissue prepared from the same rats and found ery of fluorescent dyes within precisely localised tissue areas that there was a significant age-related increase in CD11b (Barker et al., 2008). Using this system, in excised brain tissue mRNA expression in both the hippocampus (*p<0.05; Student’s from decapitated rats, we were able to deliver dextran amine t-test; n=7) and the cortex (*p<0.05; Student’s t-test; n=7), rhodamine conjugates to the Lister Hooded rat DCN and trace while CD40 mRNA expression was significantly increased in hip- retrograde projections coming from the lateral vestibular pocampus (***p<0.001; Student’s t-test; n=7). Expression of nucleus. Dextran amine conjugates were also delivered to the a lysosomal membrane protein, CD68, which is upregulated lateral vestibular nucleus and synaptic terminals could be during phagocytic activity was also significantly increased in localised within the DCN confirming anterograde projections the hippocampus (**p<0.01; Student’s t-test; n=7) and the cor- from the lateral vestibular nucleus to the DCN. Vestibulo- tex of aged, compared with young, rats (**p<0.01; Student’s cochlear projections were characterised within the sagittal and t-test; n=7). the coronal plane. The projections were mainly within the deep We investigated the effect of Aβ1-42 treatment on phagocytic and fusiform cell layers of the DCN and fibre tracts could only activity of CD11b-positive cells prepared from brain tissue of be discriminated in the sagittal plane. Therefore to assess func- aged, compared with young, rats and found differential age- tional synaptic projections from the lateral vestibular nucleus relatedeffects. Treatmentofcellspreparedfromyoungratswith to the DCN, whole cell recordings were performed in sagittal Aβ1-42significantlyincreasedphagocyticactivity(*p<0.05;Stu- slices. Stimulating the lateral vestibular nucleus elicited gluta- dent’st-test;n=27)butAβ1-42inducednofurtherincreaseinthe matergic synaptic currents in fusiform and granule cells that phagocytic activity of cells isolated from the brains of aged rats. were blocked by glutamate receptor antagonists NBQX and D- The data suggest that that the age-related increase in microglial AP5. Vesicular glutamate transporters (VGLUT’s) selectively activation is associated with an increase in phagocytic activity package glutamate into synaptic vesicles. By combining the and that microglial cells prepared from aged rats cannot be fur- dextran amine neuronal tracing method with immunocyto- ther stimulated to increase their phagocytic activity by Aβ1-42. chemistry, we were able to show projections from the lateral vestibular nucleus predominantly colabeled with VGLUT2 in The authors acknowledge grant support from the Health the DCN, whereas very few colabeled with VGLUT1. In conclu- Research Board and Science Foundation Ireland. sion, we have shown that the lateral vestibular nucleus sends Where applicable, the authors confirm that the experiments functional glutamatergic projections into the DCN, mainly described here conform with The Physiological Society ethical mediated by the VGLU2 vesicular transporter. This suggests requirements. functional links between the auditory and the vestibular system and ultimately that balance and locomotion could interact with the sound localization in the vertical plane. PC167 Focal macromolecule delivery in neuronal tissue using simultaneous pressure ejection and local electroporation. Barker M, Billups B, Hamann Temporal change in occipito-frontal propagation of repeated M. J Neurosci Methods. 2009 Mar 15;177(2):273-84. cortical spreading depressions in rats This work was supported by the Wellcome Trust. V.B. Bogdanov1,2, S. Multon1, V. Chauvel1, M. Makarchuk2 and Where applicable, the authors confirm that the experiments J. Schoenen1 described here conform with The Physiological Society ethical 1 GIGA Neuroscience, University of Liege, Liege, Belgium and requirements. 2Human and animal physiology department, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine TITLE ONLY PC169 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Assessment of the intracellular activities of ROS and NO requirements. induced by passive stretching of isolated skeletal muscle fibres from aged wild type and dystrophic mice J. Palomero, D. Pye, T. Kabayo and M.J. Jackson PC168 Pathophysiology Research Unit, School of Clinical Sciences, University of Liverpool, Liverpool, UK Vesicular glutamate transporter VGLUT2 is associated with synaptic projections from the lateral vestibular nucleus to Skeletal muscle constantly produces reactive oxygen species the dorsal cochlear nucleus (ROS) and nitric oxide (NO) which may play a role in signalling and regulatory pathways. Isolated muscles in vitro release NO M. Barker, N. Pilati, H. Hashimoto and M. Hamann to the extracellular space [1] and passive stretching of muscle CellPhysiologyandPharmacology,UniversityofLeicester,Leicester,UK increases the release of NO from rat skeletal muscle in vitro The dorsal cochlear nucleus (DCN) is the first relay of the cen- [2]. We have developed a model to study the generation of ROS tral auditory system and is involved in the localization of sound and NO in real time in isolated single muscle fibres [3, 4]. in the vertical plane. The DCN is also the site of integration of The aim of this study was to evaluate the effect of passive other sensory inputs. Therefore knowledge of synaptic inputs stretching on the intracellular generation of ROS and NO in sin- to the DCN is of importance to understand possible interac-

165P Poster Communications gle muscle fibres isolated from young and old wild type and young mdx mice. PC170 We used young (2-4 month-old) and old (26-28 month-old) C57BL/6 mice, and young mdx mice, a mouse model of GTPase-activating protein Rap1GAP2 and synaptotagmin- Duchenne muscular dystrophy. Muscle fibres were isolated from like protein 1 are involved in the control of aggregation and the Flexor Digitorus Brevis and attached to a flexible silicone dense granule secretion in platelets membrane which had been previously coated with a collagen A. Smolenski1, O. Neumüller2 and M. Hoffmeister2 Matrigel™ matrix. Fibres were loaded with different fluorophore 1 probes: 2’, 7’-dichlorodihydrofluorescein-diacetate (DCFH-DA) UCD School of Medicine and Medical Science, University College 2 which is a general detector of ROS, 4-amino-5-methylamino- Dublin, Dublin, Ireland and Institute of Biochemistry II, University 2’,7’-difluorofluorescein diacetate (DAF-FM-DA) which is a of Frankfurt, Frankfurt, Germany detector of NO, and dihydroethidium (DHE) which can detect The small guanine-nucleotide-binding protein Rap1 plays a key superoxide. A passive stretching protocol was applied for eight role in platelet aggregation and hemostasis and we recently minutes to fibres using the FX-4000™ Flexercell® system. Using identified Rap1GAP2 as the only GTPase-activating protein of fluorescence microscopy, the fluorescence emission from fibres Rap1 in platelets. Yeast-two-hybrid screening revealed 14-3-3 at different time points was quantified by image analysis to and synaptotagmin-like protein 1 (Slp1) as binding partners monitor the intracellular ROS and NO. Experimental groups of Rap1GAP2. Using GST pull down assays in platelets and were consisted of 6-12 fibres. Statistical analysis: one-way immunoprecipitation studies in transfected HeLa cells we could ANOVA followed by post hoc LSD test for multiple comparisons show that ADP and thrombin induce 14-3-3 binding to and unpaired Student’s t test for single comparisons, statisti- Rap1GAP2 whereas prostacyclin and nitric oxide induced cal significance was set at P < 0.05. platelet inhibition involves the detachment of 14-3-3 from Results from positive control experiments indicated that the Rap1GAP2. The interaction of Rap1GAP2 and 14-3-3 is regu- technique is able to detect changes in intracellular ROS and NO. lated by two phosphorylation events at serines 7 and 9 of The rate of increase in DAF-FM fluorescence (indicating NO) in Rap1GAP2. In transfected HeLa cells 14-3-3 binding abolished fibres from young mice decreased significantly after the appli- Rap1GAP2 mediated inhibition of cell adhesion suggesting that cation of passive stretching. However, fibres from old mice the GTPase-activating function of Rap1GAP2 towards Rap1 showed a slight increase in DAF-FM fluorescence after the same might be regulated by 14-3-3. In contrast, binding of Slp1 stretching protocol, and fibres from young mdx showed no appears to link Rap1GAP2 to dense granule release. Slp1 con- change with the protocol. Fibres from old and young mice dis- tains a Rab27-binding domain and by co-immunoprecipitation played different patterns of ethidium fluorescence following we demonstrate that Rap1GAP2, Slp1 and Rab27 form a DHE loading, with the activity of superoxide appearing to trimeric complex in platelets. By mutagenesis studies we increase in fibres from young mice and decrease in fibres from mapped the Rap1GAP2/Slp1 interaction sites to a new TKXT old mice after the passive stretching protocol. motif within Rap1GAP2 and to the C2A domain of Slp1. Puri- We conclude that the application of passive stretching to iso- fied recombinant Slp1 dose-dependently decreased dense gran- lated single muscle fibres produces minor changes in the intra- ule secretion in streptolysin-O permeabilized platelets treated cellular activities of ROS and NO, and induces different patterns with calcium or GTPgammaS. The isolated C2A domain of Slp1 of intracellular fluorescence depending on the age of the mice had a stimulatory effect on granule secretion and could reverse and muscular dystrophy. the inhibitory effect of full-length Slp1. Purified Rap1GAP2 aug- Balon TW & Nadler JL (1994). J Appl Physiol 77, 2519-2521. mented dense granule secretion of permeabilized platelets. Tidball JG et al. (1998). Am J Physiol 275, C260-C266. Deleting the Slp1-binding TKXT motif in Rap1GAP2 abolished the effect of Rap1GAP2 on secretion. We conclude that Slp1 Palomero J et al. (2008). Antioxid Redox Signal 10, 1463–1474. inhibits dense granule secretion in platelets whereas Rap1GAP2 Pye D et al. (2007). J Physiol 581, 309–318. has a modulatory function in secretion. Our data suggest pos- This work was funded by the Wellcome Trust, UK. sible connections between the regulation of granule secretion and aggregation in platelets. Where applicable, the authors confirm that the experiments Schultess J et al. (2005). Blood 105, 3185-3192 described here conform with The Physiological Society ethical Danielewski O et al. (2005). Thrombosis Haemostasis 93, 319-325 requirements. Hoffmeister M et al. (2008). J Biol Chem 283, 2297-2306 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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PC172 PC171

The influence of cannabinoid CB2 receptor in adult rat The role of the endocannabinoids system in the regulation mesenchymal stem cell viability of adult rat mesenchymal stem cell differentiation B. Maloor and V. Campbell A. Gowran1, K.K. McKayed1,2, S. Hussey1 and V.A. Campbell1,2 Trinity College Dublin, Dublin, Ireland 1Physiology, Trinity College Institute of Neuroscience, Dublin, Ireland Adult mesenchymal stem cells (MSCs) are a multipotent popu- and 2Trinity Centre for Bioengineering, Trinity College Dublin, lation of stem cells that can differentiate along the osteogenic, Dublin, Ireland chondrogenic and adipogenic lineages. They offer a potentially exciting source of cells for engineering skeletal tissues for the Adult mesenchymal stem cells (MSCs) are multipotent stem treatmentofosteochondraldefects.Cannabinoidsarelipophilic cells that can differentiate along the osteogenic, chondrogenic signallingmoleculesthatinteractwithG-proteincoupledcannabi- or adipogenic lineage (1). The exact physiological function of noid(CB)receptors.Recently,theskeletonhasbeenidentifiedas MSCs remains unclear at present, however, MSCs do play a role a major endocannabinoid target through both the CB1 and CB2 in tissue healing (2). Therefore, MSCs represent a source of cannabinoid receptors1, 2. The aim of this study was to examine adult stem cells which could have immense potential for use the role of the CB2 receptor in the regulation of MSC viability. in reparative medicine. The endocannabinoid system is com- MSCs were isolated from the bone marrow of adult male Wis- prised of the cannabinoid (CB) G-protein coupled receptors tar rats and expanded in culture. MSCs were treated with the (CB1 and CB2), their endogenous ligands and degradative CB2 antagonist, AM630 (0.001-100μM) alone or in the pres- enzymes. The endocannabinoid system has recently been ence of the CB2 agonist, JWH015 (10nM, 100nM and 1μM) for established as a major regulator of the musculoskeletal system 24h and colorimetric TUNEL was performed to detect the per- (3). The aim of this study was to examine the role of the endo- centage of apoptotic cells. Phospho-c Jun N-terminal kinase cannabinoid system in the differentiation of MSCs along the (JNK) and c-Jun expression were assessed by western osteogenic lineage. immunoblotting. Caspase-3 activity was measured using a col- MSCs were isolated from the bone marrow of adult Wistar rats orimetric assay to assess cleavage of the caspase-3 substrate, andexpandedinculture.MSCsweretreatedwithosteogenicfac- β Ac-DEVD-pNA. tors (OFs; 10mM -glycerolphosphate, 10nM dexamethasone, μ AM630 (10 and 100μM) significantly increased the percentage 50 Mascorbicacid)toinduceosteogenesis.Insomeexperiments of apoptosis; thus, in control conditions 9.08±0.38% (mean± MSCs were treated with the CB1 receptor antagonists, AM251 μ S.E.M)ofcellswereapoptoticandthiswassignificantlyincreased or SR141617A (both 1 M).MSCsweretreatedfor2weeks,RNA to 23.08±0.60% and 42.33±0.60% by AM630 at 10μM and extractedandosteocalcinmRNAandCB1mRNAweremeasured 100μM, respectively (p<0.001, ANOVA, n=3, Newman-Keuls by qPCR. Osteocalcin immunoreactivity was assessed by Multiple Comparison test) and this induction of apoptosis was immunocytochemistry. Hydroxyapaptite deposits in the extra- preventedbyJWH015(100nM).AM630(10μM,24h)significantly cellular matrix were measured using a commercial kit following increasedphospho-JNKimmunoreactivtyfrom1.58±0.20(mean treatmentofthecellswithOFs,AM251orSR141617Afor5weeks. ± arbitrary units ± S.E.M) to 3.00 ± 0.37 (p<0.05, n=5, Student’s t- OsteocalcinmRNAwassignificantlyincreasedfrom0.3 0.1(arbi- ± ± test) and also significantly increased caspase-3 activity from traryunits,mean SEM)to7.4 3.1(p<0.01,unpairedt-test,n=3- 647.5±101.2 pmol pNA/min/mg to 2571±89.4 and this was 6) by osteoinductive factors. Also, osteocalcin mRNA was signi- ± ± ± significantly reduced to 1878±67.9 in cells exposed to AM630 ficantly increased from 0.3 0.1 to 1.2 0.1 and 2.3 0.8 when μ μ inthepresenceofJWH015(100nM;p<0.001,ANOVA,n=5,New- MSCs were exposed to AM251 (1 M) and SR141617A (1 M), man-Keuls Multiple Comparison test). AM630 (10μM,24h) respectively(p<0.01vs.,controlcells,unpairedt-test,n=6),indica- induced a significant increase in phospho-c-Jun activity from tiveofosteogenicdifferentiation.IncubationwithSR141617Ain 0.62± 0.10 (mean arbitrary units ± S.E.M) to 1.20±0.08 and this the presence of OFs further increased the expression of osteo- ± wassignificantlyreducedto0.85±0.08incellsexposedtoAM630 calcin mRNA from 7.4 3.1 in cells treated with OFs alone to ± in the presence of JWH015 (100nM) (p<0.05, p<0.01, ANOVA, 15.7 5.1(p<0.05,Newman-KeulsMultipleComparisontest,n=3- n=5, Newman-Keuls Multiple Comparison test). 6). Hydroxyapaptite deposits in the extracellular matrix were ± ± This study demonstrates that inhibition of the CB2 receptor significantlyincreasedfrom1.9 0.1(arbitraryunits,mean SEM) ± ± leads to MSC apoptosis and suggests that activation of the CB2 to 5.9 0.6 by OFs and further significantly increased to 7.3 0.3 receptorisnecessarytomaintainthesurvivalofMSCs.Thispro- when MSCs were exposed to AM251 in the presence of OFs videsevidenceofanothercellulartargetforcannabinoidrecep- (p<0.05,unpairedt-test,8replicates).CB1mRNAexpressionwas ± ± torswhichmaybenecessaryforthemaintenanceofbonehealth. significantlyincreasedfrom1.6 0.8(arbitraryunits,mean SEM ) to 5.5±1.2 by OFs and further increased to 6.3±0.6 in MSCs Idris AI, van’t Hof RJ, Greig IR, Ridge SA, Baker D, Ross RA, Ralston SH (2005) Nat Med. 11(7):774-9. treated with SR141617A (p<0.01, unpaired t-test, n=3-7). These data show that the cannabinoid receptor system is upreg- Bab IA (2007) Ann N Y Acad Sci. 1116:414-22. ulated during MSC osteogenesis and that osteogenesis is Where applicable, the authors confirm that the experiments enhanced following pharmacological blockage of the CB1 described here conform with The Physiological Society ethical receptor. requirements. Pittenger MF et al., (1999) Science 284:1335-1347. Devine MJ et al., (2002) J Orthop Res 20:1232-1239. Bab IA et al., (2007) Ann N Y Acad Sci 1116:414-22.

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Funded by Science Foundation Ireland. Nakanishi M and Rosemberg DW (2006). Biochim Biophys Acta 1761(11),1335-43 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Thomas W et al. (2008). Front Biosci 13, 2604-13 requirements. The work presented was funded by the Higher Education Authority of Ireland and the RCSI Research Committee. Where applicable, the authors confirm that the experiments PC173 described here conform with The Physiological Society ethical requirements. Role of cPLA2 in Endocrine-sensitive and Endocrine-resistant breast cancer cell growth F. Caiazza, W. Thomas and B.J. Harvey PC174 MolecularMedicine,RoyalCollegeofSurgeonsinIreland,Dublin,Ireland Heme oxygenase (HO)-1 as a potential biomarker in the 17β-estradiol (E ) is a steroid hormone that regulates many bio- 2 pathogenesis of endometriosis logical activities - including cell proliferation and invasiveness - in breast cancer, contributing to tumorigenesis and progres- L. Noordin and E.M. Ellis α β sion. E2 acts through its intracellular receptors (ER and ER ) Strathclyde Institute of Pharmacy and Biomedical Sciences, to regulate gene transcription in the nucleus and activate rapid University of Strathclyde, Glasgow, Scotland, UK signaling pathways from the plasma membrane. Recent stud- The pathogenesis of endometriosis, an estrogen-dependent ies demonstrated a rapid, E2-induced increase in intracellular Ca2+ concentration through activation of the calcium-depend- disease, remains elusive. Early work proposed the initiation of α the disease is due to “retrograde menstruation” and this has ent cytosolic phospholipase A2 (cPLA2 ) in the breast cancer- α been widely accepted. However, to date, the mechanism of derived MCF-7 cell line (1). cPLA2 catalyzes the hydrolisis of membrane glycerophospholipides to release arachidonic acid endometriotic cell proliferation is still unexplained. Oxidative (AA), which is converted to biactive eicosanoid lipid mediators stress has been implicated as contributing to cell growth. The (including prostaglandins produced through cycloxigenases) expression of heme oxygenase 1 (HO-1) is upregulated by oxida- which can activate downstream proliferative signals (2). The tive stress and is capable of protecting cells against oxidant- rapid release of bioactive lipids may play a role in the signalling mediated injury. HO-1 has been suggested as being involved in the mechanism of cell proliferation, and estradiol has also responses stimulated by E2 in breast cancer cells and contribute α been implicated in proliferative events. to proliferation, however the role of cPLA2 in breast cancer is not established. We used specific pharmacological inhibitors In the present study, we determined the effects of oxidative on model breast cancer cell lines to study the molecular mech- stress and estradiol on endometriotic cell proliferation and on α the expression of HO-1 using immortalized human endometri- anism of cPLA2 activation by western blotting and confocal microscopy. otic epithelial cells (12-z). Cells were treated with the oxidants μ μ The E -induced rapid activation of cPLA α was dependent on H2O2 (10 M) or menadione (25 M), or the lipid peroxidation 2 2 μ EGFR/HER2 trans-activation, heterodimerisation and down- product acrolein (35 M) for 48 hours and the proliferation of stream signalling through ERK1/2 MAPK to phosphorylate cells was determined by MTT [(3-(4,5-Dimethylthiazol-2-yl)- α α 2,5-diphenyltetrazolium bromide] assay. cPLA2 on Ser505. E2 also promoted cPLA2 trafficking to per- inuclear membranes, and this effect was subsequent to, and The results show that H2O2, menadione and acrolein signifi- μ μ μ dependent on, MAPK-induced phosphorylation on Ser505. cantly induced cell proliferation at 1 M, 20 M and 25 M EGFR and/or HER2 over-expression correlates with a poor clin- respectively, showing that H2O2 is the most potent compound. -9 ical outcome and resistance to endocrine therapy in breast can- Cells were also pre-treated with estradiol at 10 M for 24 hours cer. The endocrine-resistant SKBR3 cell line over-expressed EGFR followed by the addition of compounds at similar concentra- and HER2 and also showed elevated cPLA expression (at both tions for 24 hours. Interestingly, in the presence of estradiol at 2 -8 the mRNA and protein level) compared to MCF-7, confirming 10 M, the cells that were treated with lower concentrations the correlation between the eicosanoid and the EGFR signalling of compounds showed a significant increase in proliferation, pathways, as suggested by other reports in the literature (3). α Pharmacological blockade of cPLA2 with a specific inhibitor impacted on cell growth in both cell lines, by reducing E2- induced proliferation and inducing apoptotis and necrosis, with- α out affecting the invasiveness of surviving cells. cPLA2 is likely to play a key role in regulating the already established growth- promoting effects of estrogen and COX-2 in breast cancer, balancing the cytotoxic effects of free arachidonic acid with the α proliferative effects of prostaglandins. Furthermore, cPLA2 could play a role in the transition of breast cancer from estrogen-dependence to estrogen-resistance through the over-activation of the EGFR signaling pathway. Thomas W et al. (2006). Steroids 71(3),256-265

168P Poster Communications but this was not the case in the presence of estradiol at 10-9 M. ever, ΔN117 mutant cells have a more acidic pH than KCC3- In contrast, at high concentrations, all compounds caused signi- i overexpressed cells in bicarbonate-buffered solution (BBS). A ficant growth inhibition. The expression of HO-1 mRNA in number of Na-dependent and acid extruding transporters were treated cells was determined by Quantitative Real Time-Poly- assessed by RT-PCR. Only the expression of NHE3 correlated merase Chain Reaction (Q-PCR). A significant up-regulation of with [Cl-] (Fig. 1C). The present results suggest that bicarbon- HO-1 mRNA was observed in all the treated cells and further i ate can replace Cl- as the intracellular anion during changes of increased in the presence of estradiol at 10-9 M. KCC transport activity. These results indicate that oxidative stress may contribute to the proliferation of endometriotic cells. In addition, there may be some interplay with estradiol to further increase cell proliferation. Expression results indicate that estradiol may be involved in the regulation of the HO-1. The up-regulation of HO-1 mRNA during proliferation indicates that this enzyme could be used as a reliable marker in the pathogenesis of endometriosis. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

PC175

Effects of altered KCl cotransporters expression on intracellular chloride levels and pH in cervical cancer cells Figure 1 (A) Intracellular chloride concentration measured by MEQ. (B) Intra- cellular pH measured by BCECF-AM. (C) Expression of membrane trans- W. Wei1, J. Davis1, M. Shen2, R.J. Wilkins1 and J.C. Ellory1 porters measured by RT-PCR. 1Department of Physiology, Anatomy & Genetics, University of Shen, M.R., Chou, C.Y., and Ellory, J.C. (2000) Pflugers Arch. 440, 751–760. Oxford, Oxford, UK and 2Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan Mercado, A., Song, L., Vazquez, N., Mount, D.B., and Gamba, G. (2000) J. Biol. Chem. 275, 30326-30334. KCl cotransporters (KCCs) play significant roles not only in ionic Shen, M.R., Chou, C.Y., Hsu, K.F., Liu, H.S., Dunham, P.B., Holtzman, and osmotic homeostasis but also tumor biology. Overex- E.J., and Ellory, J.C. (2001) Proc. Natl. Acad. Sci. USA. 98, 14714-14719. pression of KCC1, KCC3 and KCC4, with KCC3 the most abun- Shen, M.R., Chou, C.Y., Hsu, K.F., Hsu, Y.M., Chiu, W.T., Tang, M.J., Alper, dant isoform, has been shown to correlate with human cervi- S.L., and Ellory, J.C. (2003) J. Biol. Chem. 278, 39941-49950. cal carcinogenesis (1). KCC1 and KCC4 are osmotically-sensitive and involved in volume regulation (2) while KCC3 contributes Supported by the M.R.C. Grant G0700759. to cell proliferation (3). KCC3 overexpression enhances proliferation, migration and invasiveness of cervical cancer cells. Where applicable, the authors confirm that the experiments Removal of the N-terminal 117 amino acids from KCC1 (ΔN117) described here conform with The Physiological Society ethical produces a dominant-negative loss-of-function phenotype for requirements. KCl cotransport in human cervical cancer cells which exhibit inhibited cell proliferation, tumor growth and invasiveness (4). We have shown previously that expression levels of KCC3 cor- - - relate inversely with intracellular Cl levels ([Cl ]i) estimated by PC176 36Cl- equilibrium. There is no significant difference in cell volume between wild-type, KCC3-overexpressed, and ΔN117 Genetic dissection of potassium chloride cotransporter (KCC) mutant cervical cancer cells (4). To maintain electroneutrality, functions in epithelial development and carcinogenesis in - changes in [Cl ]i associated with altered KCC activity must result Drosophila in alterations to the levels of other ions, most likely HCO3-. In the present study, the properties of these KCC transfectants Y. Chen1,2, M. Shen1,2, R.J. Wilkins3, J.C. Ellory3 and C. Wilson3 in regulating their intracellular pH (pH ) with changes in [Cl-] i i 1InstituteofBasicMedicalSciences,CollegeofMedicine,NationalCheng have been investigated. 6-Methoxy-N-ethylquinolinium chlo- Kung University, Tainan, Taiwan, 2Department of Pharmacology, ride (MEQ) was used to determine [Cl-] in the three cell lines. i College of Medicine, National Cheng Kung University, Tainan, Taiwan [Cl-] estimated by MEQ was 73.80±15.82 mM (n=5) in wild- i and 3Physiology,Anatomy&Genetics,UniversityofOxford,Oxford,UK type cervical cancer cells, 45.63±1.65 mM (n=3) in KCC3-overexpressed cervical cancer cells and 104.01±6.66mM (n=3) in Electroneutral potassium chloride cotransporters (KCC) belong Δ N117 mutant cells (Fig. 1A). The resting pHi was determined to the superfamily of electroneutral cation-chloride cotrans- by cuvette fluorimetry using BCECF-AM (10 μM for 15 min at porters and are encoded by four genes in vertebrates (KCC1 37∞C). As shown in figure 1B, there are no significant differ- through KCC4) (1). The activity of KCC plays an important role ences in resting pHi between KCC3-overexpressed cells and in variety of physiological and pathological cellular functions, ΔN117 mutant cells in HEPES-buffered solution (HBS). How- 169P Poster Communications including cell volume regulation, epithelial ion transport, and Shen MR, Chou CY & Ellory JC (2000). Volume-sensitive KCl cotrans- osmotic homeostasis. Our previous studies have highlighted port associated with human cervical carcinogenesis. Pflugers Archiv important roles of KCC in tumor progression. Malignant trans- 440, 751-760. formation of cervical epithelial cells is associated with the dif- Shen MR, Chou CY, Hsu KF, Hsu YM, Chiu WT, Tang MJ, Alper SL & Ellory ferential expression of volume-sensitive KCC activities result- JC (2003). KCl cotransport is an important modulator of human cervi- ing from the up-regulation of mRNA transcripts for KCC1, KCC3 cal cancer growth and invasion. J Biol Chem 278, 39941-39950. and KCC4 isoforms (2). Loss-of-function KCC mutant cervical Hekmat-Scafe DS, Lundy MY, Ranga R & Tanouye MA (2006). Mutations cancer cells exhibit inhibited cell growth accompanied by in the K+/Cl- cotransporter gene kazachoc (kcc) increase seizure sus- decreased activity of the cell cycle proteins retinoblastoma and ceptibility in Drosophila. J Neurosci 26, 8943-8954. cdc2 kinase (3). In this study, we aim to investigate the in vivo Brand A H & Perrimon N (1993). Targeted gene expression as a means functions of KCC in epithelial development and carcinogene- of altering cell fates and generating dominant phenotypes. Develop- sis using Drosophila melanogaster as the model. There is a sin- ment 118, 401-415. gle KCC homologue in Drosophila, but its functional properties Work supported by the Medical Research Council, UK. remain largely unknown. Hypomorphic mutations in the Drosophila KCC homologue, CG5594, render flies susceptible to Where applicable, the authors confirm that the experiments epileptic-like seizures associated with excitatory GABAergic sig- described here conform with The Physiological Society ethical naling, consistent with the function of mammalian KCC2 (4). requirements. However, strong loss-of-function alleles are lethal, suggesting other critical developmental roles. Using the GAL4/UAS (Upstream Activation Sequence) expression, we have overexpressed CG5594 in multiple tissues (5). Crosses were performed PC177 at 25∞C, and 1-2 day old adult progeny were anesthetized with CO2 for eye and wing analyses. Overexpression of CG5594 in Multiple purinergic receptors regulate anion secretion in differentiating cells of the eye using the GMR-GAL4 driver had the bovine oviduct epithelium no significant effect on the size of fly eye but produced a mild disarrangement of the ommatidial array (Fig. 1A). Additionally, N. Keating and L. Quinlan CG5594 overexpression in the posterior compartment of the Physiology, NUI Galway, Galway, Ireland developing wing, under the control of engrailed-GAL4, resulted in drastic size reduction in the region of overexpression with Introduction: Fluid within the oviduct provides the appropri- associated reduction in cell size (Fig. 1B). We conclude that KCC ate environment essential for gamete transport, fertilization can regulate cell growth and cell size of fly eyes and wings. and early embryo development. However, little is known about Analyses of mutant clones in the fly eye and wing will provide the potential mechanisms underlying the formation of oviduct an excellent assay system in which to investigate how modu- fluid. Our previous studies [1] have demonstrated a role for lation of KCC function influences epithelial development and basolateral purinoceptor activation in promoting chloride secre- how overexpression might lead to metastasis in an appropri- tion across the bovine oviduct epithelium. The present study ate genetic background. explored the regulation of anion secretion by luminal purinergic receptors. Methods: Ion secretion was measured as changes in short-cir- μ 2 cuit current (ISC( A/cm ) across voltage-clamped polarized oviduct epithelial cell monolayers. Changes in intracellular calcium were measured by fluorescence microscopy using fura-2. mRNA expression was confirmed by reverse transcriptase PCR (RT-PCR). Results are expressed as mean ± SEM and statistical analyses were made by one way ANOVA and the Bonnfer- roni/Dunn post hoc test Results and Discussion: ATP (100μM) application to the luminal surface of polarized bovine oviduct epithelial cells induced a rapid and transient increase in current of 28.82 ±1.7 μA/cm2 followed by a sustained current of 1.91±0.1μA/cm2 (n=18). Removal of extracellular chloride or bicarbonate ions or both reduced the ATP-induced ISC response by 30%, 40% and 70% respectively (n=5). Furthermore, the ATP response was pre-

Adragna NC, Di Fulvio M & Lauf PK (2004). Regulation of K-Cl cotransport: from function to genes. J Membr Biol 201, 109-137.

170P Poster Communications served in the presence of amiloride but was reduced by 60% in HbS and HbA cells were collected from volunteers with full eth- the presence of the CFTR inhibitor, glybenclamide ical consent. Lysis experiments were performed at 37oC (Ellory (p<0.001,n=4) and almost completely abolished in the pres- et al., 2008). Briefly, streptomycin (0.1 to 10 mM) was added ence of thapsigargin (p<0.001, n=4).Ca2+ measurements to erythrocytes (haematocrit 4%) suspended in buffered revealed that ATP also induced a rapid transient increase in intra- sucrose solution (300 mM) and the suspension maintained cellular Ca2+ which was sensitive to thapsigargin, U73122 and deoxygenated for 60 min. Lysis was determined by measuring BAPTA-AM (p<0.001, n=4). Other P2Y agonists also rapidly acti- haemoglobin release optically. Erythrocyte membrane currents vated anion secretion with a potency profile of ATP = UTP > ADP were recorded at room temperature (Browning et al., 2007), suggesting the involvement of the P2Y2 subtype of purinocep- usingconventionalwhole-cellpatchclamptechniques.I-Vrela- tor. In addition a range of P2X agonists rapidly activated anion tionships were obtained by applying a series of 300 ms test secretion with a potency profile of ATP = 2MeSATP >α,β meATP potentials from -80 to +80 mV in 10 mV increments from a > BzATP, suggesting the presence of multiple functional P2X holding potential of 0 mV. Currents were analysed by averag- receptors on the luminal membrane. P2X1, P2X4 and P2X7 ing the current values of the final 50 ms evoked by each test receptor expression was confirmed by RT-PCR. Finally the P1 potential. receptor agonist, adenosine, also induced a rapid transient In control experiments, about 25% of HbS cells lysed after 60 ± μ 2 increase in ISC by 7.46 0.86 A/cm which was associated with min in sucrose. About 60% of lysis was inhibited by strepto- a subsequent sustained current of 4.31 ± 0.38 μA/cm2 (n=4). mycin, IC50 0.13 ± 0.06 mM (n = 3) (Figure 1). Whole-cell cur- In conclusion, we have demonstrated that luminal ATP rapidly rent measurements were compared between HbA and HbS cells - - + activates Cl and HCO3 secretion across the bovine oviduct, in using Na -containing bath and pipette solutions. A represen- a Ca2+ dependent fashion most likely through P2Y2 receptor tative I-V curve is shown for HbS cells before and after the addi- activation. Furthermore activation of P2X and P1 receptor sub- tion of 50 μM streptomycin (Figure 2). HbS cells demonstrated types on the luminal membrane are also capable of promoting considerably larger whole-cell currents (p < 0.05) than HbA cells, anion secretion. The purinergic regulation of anion secretion although both had a reversal potential of approximately 0 mV. in the oviduct provides an important method of regulating Application of streptomycin (50 μM) to the bath inhibited 74% oviduct fluid formation and optimizing conditions for fertil- of membrane current at +80 mV in HbS cells, compared to only ization and early embryo development. 24% in HbA cells (not shown). Keating N and Quinlan L.R. (2008) Biol Reprod 78, 1119-1126 Results show that streptomycin can inhibit an electrophysiological conductance, thought to be Psickle, in addition to a Where applicable, the authors confirm that the experiments selective permeability pathway in HbS cells. We hypothesise described here conform with The Physiological Society ethical that this transport pathway may represent Psickle. Further stud- requirements. ies can help elucidate the molecular identity of Psickle, which can have important therapeutic implications for SCD patients.

PC178

Evidence for a streptomycin-sensitive transport pathway in human sickle erythrocytes S. Dalibalta1, J.A. Browning1, J.S. Gibson2, D. Rees3 and C. Ellory1 1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK, 2Veterinary Medicine, University of Cambridge, Cambridge, UK and 3Molecular Haematology, King’s College London School of Medicine, London, UK HbS erythrocytes from individuals with sickle cell disease (SCD) have altered membrane transport compared to HbA erythrocytes from healthy individuals (Brugnara, 2004). In particular, HbS cells show increased permeability to cations upon deoxygenation. This pathway, termed Psickle, contributes to erythrocyte shrinkage (Tosteson et al.,1952), accel- erating sickling. The identity of Psickle is elusive. We have demonstrated electrophysiologically that whole-cell conductance of HbS cells was higher than in HbA cells, and potentiated by deoxygenation. This pathway shares properties with Psickle (Browning et al., 2007). Deoxygenated HbS cells, unlike HbA cells, are permeable to certain organic osmolytes, including sucrose, and this pathway is inhibited by dipyrimadole and DIDS (Ellory et Figure 1. Representative data showing percentage of lysed deoxygenated al., 2008). Here we show that streptomycin, an inhibitor of HbS cells in 60 min with inhibition by streptomycin stretch-activated channels (Shen et al., 2003), inhibits both these pathways.

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Occlusion of small blood vessels is implicated and involves exposure of phosphatidylserine (PS) on the outer leaflet of the RBC membrane (Kuypers, 2008). This phospholipid is normally confined to the inner leaflet through the action of an aminophos- pholipid translocase. On exposure, it makes RBCs stickier. We have investigated the extent to which calcium entry through the deoxygenation-induced pathway, Psickle (Lew and Bookchin, 2005), contributes to PS exposure. Blood samples were obtained with ethical approval from con- senting volunteers homozygous for HbSS. RBCs were washed into low (LK) or high potassium (HK) saline, comprising (in mM) NaCl 140, KCl 4, glucose 5, HEPES 10 for LK saline, and NaCl 55, KCl 90, glucose 5 and HEPES 10 for HK saline, all pH 7.4 at 37oC. They were incubated at different extracellular [calcium]s for up to 18h. RBCs were subsequently treated with vanadate (1mM), harvested, and labelled with FITC-annexin (Becton Dick- inson, BD). Percentage of RBCs with PS exposed on their external membrane was then measured by flow cytometry. Data are given as means±SEM for blood samples from n patients. After 18h, the percentage of RBCs expressing PS when incubated under oxygenated LK conditions was 5.3±1.4, 5.7±1.0, 5.7±1.1 and 6.2±0.9 (n = 4) at 0.5, 1.1, 2 and 5mM [calcium], respectively. At the same [calcium]s, when deoxygenated, values increased to 11.1±2.3, 11.1±1.4, 13.3±0.9 and 18.2±1.7. When deoxygenated at 5mM Ca2+ in LK saline, 10.3±4.4% (n = 3) RBCs exposed PS, falling to 5.4±1.7% in HK saline (compared with 5.3±0.8% in oxygenated conditions). The deoxygenation- induced increase in PS exposure was abolished by loading cells Figure 2. Representative I-V current measurement in HbS cells, before and with the calcium chelator, MAPT-AM. In deoxygenated LK saline after addition of streptomycin with 5mM calcium, the number of RBCs with PS exposure fell Brugnara, C. (2004). Sickle cell disease. In: Bernhardt I, Ellory JC, by about 15% when incubated with either dipyridamole eds. Red Cell Membrane in Health and Disease. Berlin: Springer; (100μM) or clotrimazole (10μM). 139-152. These results agree with previous findings that PS exposure Browning JA, Staines HM, Robinson HC, Powell T, Ellory JC, Gibson JS. increases upon deoxygenation. They also show that the effect (2007). Blood. 109, 2622-2629. is dependent on extracellular calcium and is prevented by its Ellory JC, Sequeira R, Constantine A, Wilkins RJ, Gibson JS. (2008). Blood chelationintracellularly.PSexposureislargelypreventedby Cells Mol Dis. 41, 44-49. removing the gradient for loss of potassium efflux by suspension in HK saline. There is modest inhibition by dipyridamole Shen MR, Chou CY, Chiu WT. (2003). FEBS Lett. 554, 494-500. (a partial Psickle inhibitor) and clotrimazole (which inhibits the Tosteson DC, Shea E, Darling RC. (1952). J. Clin. Invest. 48, 406-411 Gardos channel). We conclude that PS exposure is induced by calcium entry following deoxygenation-induced P activa- The authors would like to acknowledge the Wellcome Trust and sickle the British Heart Foundation. tion, and that intracellular calcium has its main effect via cell shrinkage due to solute loss following activation of the Gar- Where applicable, the authors confirm that the experiments dos channel. described here conform with The Physiological Society ethical Lew, V. L. and Bookchin, R. M. (2005). Physiol. Rev. 85, 179-200. requirements. Kuypers, F. A. (2008). Curr. Mol. Med. 8, 633-638.

We thank the British Heart Foundation for financial support.

PC179 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Ca2+ and phosphatidylserine exposure in red blood cells from requirements. patients with sickle cell disease E. Weiss1, D. Rees2 and J.S. Gibson1 1Veterinary Medicine, University of Cambridge, Cambridge, UK and 2Molecular Haematology, King’s College London School of Medicine, London, UK Patients with sickle cell disease (SCD) have HbS (rather than normal HbA) in their red blood cells (RBCs). How HbS results in the symptoms of SCD remains incompletely understood.

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PC180

Voltage-dependent gating currents from the CLC-5 Cl-/H+ exchanger J.D. Lippiat and A.J. Smith Institute of Membrane & Systems Biology, University of Leeds, Leeds, UK Much of our understanding of the CLC family has come from functional studies of the Torpedo CLC-0, a voltage-gated Cl- channel, and from structural studies of prokaryotic CLC-ec1, a voltage-independent Cl-/H+ exchanger. Mammalian CLC-5, however, exhibits voltage-dependent activation and Cl-/H+ exchange. We carried out high resolution whole-cell patch clamp recordings of wild-type and mutant CLC-5 expressed in HEK293 cells, as described in Smith et al. (2009). Outward currents, corresponding to Cl- into and H+ out from the cytosol, were activated by positive potentials (Fig. 1A). Upon repolarisation, brief (< 1ms) inward tail currents were observed, which according to the apparent reversal potential did not relate to the movement of permeating ions. As seen previously (Zdebik et al., 2008), neutralisation of an internal proton acceptor (E268A) resulted in ablated ionic flux across the membrane. With this mutant we observed non-linear capacity transient currents lasting approximately 1 ms (Fig. 1B), which had charge- ± ± movement kinetics (V1⁄2 = 94.0 0.8 mV, ze = 1.3 0.03; mean ± s.e.m. of recordings from 5 cells) that correlated with the volt- ± Figure 1: Whole-cell currents recorded from HEK293 cells expressing wild- age-dependent activation of wild-type CLC-5 (V1⁄2 = 108.6 ± type (A) and E268A (B) CLC-5. 2.0 mV, ze = 1.4 0.05, n = 5). The brief tail current that were observed with wild-type CLC-5 appeared to relate to gating Smith et al. (2009). Am J Physiol Renal Physiol 296, F390-397. rather than permeation and had similar charge-movement Zdebik et al. (2008). J Biol Chem 283, 4219-4227. kinetics. We found that the properties of the E268A transient currents - - Supported by the Wellcome Trust. were similar when extracellular Cl was replaced by Br (ze = ± 1.3 0.10, n = 3), an anion that also permeates wild-type CLC- Where applicable, the authors confirm that the experiments 5. In contrast, the voltage-dependence was reduced in the pres- described here conform with The Physiological Society ethical ± ence of the impermeant anions aspartate (ze = 0.9 0.05, n = requirements. ± 3) and methanesulphonate (ze = 0.9 0.08, n = 3). Furthermore, similar gating transients were recorded from cells expressing wild-type CLC-5 in the presence of the impermeant anions. The PC181 higher gating charge (ze) observed with permeant anions suggest that these interact with or modify an intrinsic voltage-sen- Identification of tryptophan analogues as non-transported sor with a ze of 0.9 and may provide the additional charge which inhibitors of the proton-coupled amino acid transporter moves through a fraction of the voltage field (δ≈0.4). The PAT2 (slc36a2) impermeant anions are unlikely to contribute to the gating 2- N. Edwards, C. Anderson and D.T. Thwaites charge since it was similar with extracellular divalent SO4 ions ± (ze = 0.9 0.04, n = 3) and persisted in near-complete removal Epithelial Research Group, Institute for Cell and Molecular of ions. Biosciences, Newcastle University, Newcastle upon Tyne, UK Our data suggest that the voltage-sensor in CLC-5 may be PAT2 (slc36a2) is a pH-dependent, Na+-independent transporter formed by an intrinsic protein domain in combination with per- of small, dipolar amino and imino acids (1,2). PAT2 is defec- meating anions. As demonstrated by the lack of sustained Cl- tive in the renal reabsorptive disorder iminoglycinuria (3). Tryp- /H+ current with the E268A mutant, “gating” of the pore also tophan analogues act as non-transported inhibitors of the requires H+ likely to be delivered from the intracellular E268 amino acid transporters PAT1 (4) and ATB0,+ (5). The purpose proton acceptor. of this study was to identify the nature of the interaction of tryptophan analogues with PAT2. Rat PAT2 was expressed in X. laevis oocytes following standard procedures (2). Data are mean ± SEM (n) with statistical significance determined by ANOVA and Tukey’s multiple comparison post-test. Under optimal uptake conditions (pH 5.5, Na+-free), 5-hydroxy-L-tryptophan

173P Poster Communications and α-methyl-DL-tryptophan both significantly reduce is established that extracellular ATP signalling, originating from (p<0.001) [3H]proline uptake (10μM, 5μCi.ml-1, 40min, 22∞C) stretch-evoked ATP release and necessarily involving activation in PAT2-expressing oocytes in a concentration-dependent man- of a variety of P2 receptors, is involved in bladder sensation(2). ± ± ner with IC50 values of 4.2 0.8 (20) and 3.5 0.6 (30) mM, Furthermore, inflammation is associated with increased ATP respectively. Rheogenic H+/proline symport via PAT2 was meas- release from epithelial cells(3). Taken together, we hypothesise ured using two-electrode voltage-clamp (-60mV, pH 5.5, Na+- that in pyuric OAB patients, there is increased ATP release and/or free). In contrast, 5-hydroxy-L-tryptophan and α-methyl-DL- increased P2 receptor expression, caused by inflammation, tryptophan (both 20mM) failed to induce inward current in which ultimately results in increased sensory nerve excitation PAT2-expressing oocytes. However, both 5-hydroxy-L-trypto- and the exacerbation of OAB symptoms. Here we begin to inves- phan and α-methyl-DL-tryptophan (20mM) significantly tigate our hypothesis. reduced (p<0.001) the proline- (0.2mM) induced current by Bladder urothelium biopsies were obtained from i) asympto- 81.0 ± 2.3 (6) and 76.7 ± 3.6 (5) %, respectively. To confirm that matic patients, ii) pyuric OAB patients, and iii) non-pyuric OAB both tryptophan analogues are non-transported inhibitors of patients, using flexible cystoscopy. Basal and hypotonicity- PAT2, trans-stimulation of [3H]proline efflux (oocytes were pre- evoked (i.e. stretch-evoked) ATP release from urothelium was loaded with 5mM proline, 0.1μCi.ml-1) was measured under quantified using a luciferin/luciferase assay, P2 receptor mRNA optimal uptake conditions. The PAT2 substrate glycine (10mM) levels were investigated using quantitative real time-PCR, and significantly trans-stimulated (p<0.001) [3H]proline efflux via P2 receptor expression was investigated in snap-frozen sliced PAT2 [266.5 ± 17.2 (10) pmol.oocyte-1.(10min)-1] compared tissue using immunohistochemistry. to water-injected oocytes [13.6 ± 2.5 (10) pmol.oocyte- Basal ATP release was 50-fold greater from the urothelium of 1.(10min)-1]. In contrast, both 5-hydroxy-L-tryptophan and α- pyuric OAB patients (P<0.01; n=10) than from non-pyuric OAB methyl-DL-tryptophan (both 10mM) failed (p>0.05 versus (n=9) or asymptomatic patients (n=9). In contrast, in all three water) to trans-stimulate [3H]proline efflux via PAT2 [9.3 ± 1.0 patient groups, the concentration of ATP released following (10) and 11.9 ± 1.4 (10) pmol.oocyte-1.(10min)-1, respectively]. stretch was similar. Basal and stretch-evoked ATP release was In conclusion, the combination of measurements demonstrate significantly abolished (P<0.01; n=3) by addition of the P2 that 5-hydroxy-L-tryptophan and α-methyl-DL-tryptophan are receptor antagonist suramin (1 mM). In asymptomatic patient non-transported inhibitors of PAT2. These compounds should urothelium, we detected significant levels of P2X1-3,5-7 subunit prove useful experimental tools to help elucidate the physio- and P2Y1,2,6,11-14 receptor mRNA (n=6). Urothelium from pyuric logical role of PAT2. OAB patients showed a significant increase in abundance of Boll M et al. (2002) J Biol Chem 277, 22966-22973. P2X5 subunit and P2Y2,11,12 receptor mRNA, and a significant Chen Z et al. (2003) Biochem Biophys Res Commun 304, 747-754. decrease in abundance of P2X1 subunit mRNA (P<0.01; n=3). Urothelium from non-pyuric OAB patients showed a significant Bröer S et al. (2008) J Clin Invest 118, 3881-3892. increase in P2Y11 mRNA (P<0.01, n=3). Immunohistochemisty Metzner L et al. (2005) FASEB J 19, 1468-1473. confirmed our real time-PCR data (n=3). Karunakaran S et al. (2008) Biochem J 414, 343-355. In summary, this data demonstrates that in a subset of OAB patients (those with pyuria) there is increased basal ATP release Supported by the Wellcome Trust (grant 078640/Z/05/Z). NE from the urothelium, which is abolished by the P2 receptor holds a BBSRC postgraduate studentship. antagonist suramin, and, altered P2 receptor expression. This Where applicable, the authors confirm that the experiments data suggests that increased ATP release from the urothelium, described here conform with The Physiological Society ethical involving activation of P2 receptors, may play a role in the requirements. heightened symptoms associated with pyuric OAB patients. Malone-Lee et al, Urodyn. (International Continence Society Proceed- ings), Ab. 42, 2007. PC183 Burnstock, Pharmacol. Rev. 58(1):58-86, 2006. Davis et al, Am. J. Physiol. Lung Cell Mol. Physiol. 286:L112-L120, 2004. Altered urothelial ATP signaling in human Overactive Bladder (OAB) patients We are grateful to the University of London Central Research Fund and Bloomsbury Consortium. W. Tan2, A. Contreras-Sanz1, T. Kennedy-Lydon1, S.L. Alexander1, S. Janska1, S. Bishara2, R. Lunawat2, R. Khasriya2, Where applicable, the authors confirm that the experiments K.M. Taylor3, C. Crawford1, J. Malone-Lee2 and S.S. Wildman1 described here conform with The Physiological Society ethical requirements. 1Urinary System Physiology Unit, Royal Veterinary College, London, UK, 2Department of Medicine, University College London, Whittington Campus, London, UK and 3Department of Pharmaceutics, The School of Pharmacy, University of London, London, UK Overactive bladder (OAB) is characterized by frequency, urgency and urge-incontinence, in the absence of urinary infection. We have identified a previously unrecognized pyuria (≥10 white blood cell/μl urine) in a majority of patients diagnosed as having OAB; associated with more severe symptoms(1). It

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PC184 PC185

Ursodeoxycholic acid exerts antisecretory actions on colonic Epidermal growth factor enhances colonic epithelial epitheial cells secretory capacity through activation of O. Kelly1,2, F.E. Murray2 and S.J. Keely1 phosphatidylinositol 3-kinase, mitogen-activated protein 1Molecular Medicine, Royal College of Surgeons in Ireland, Dublin, kinase and upregulation of multiple transport proteins 2 Irelandand Gastroenterology,RCSI/BeaumontHospital,Dublin,Ireland M.S. Mroz, F. Toumi, F. O’Mahony and S. Keely Background:Theprimarybileacid,chenodeoxycholicacid(CDCA) Molecular Medicine, RCSI, Dublin, Ireland stimulates colonic fluid and electrolyte secretion. Increased colonic delivery of CDCA contributes to diarrhea in conditions of Background: There is increasing evidence that growth factors, bileacidmalabsorption.However,CDCAismetabolisedbycolonic such as epidermal growth factor (EGF), are important regula- bacteriatoursodeoxycholicacid(UDCA)andlithocholicacid(LCA) tors of epithelial transport function. Previous work from our and little is known of their effects on colonic secretory function. laboratory has shown that EGF chronically enhances colonic Aims: To investigate the effects of UDCA on colonic epithelial epithelial secretory function through a mechanism involving secretory function. Methods: Cl- secretion was measured as increases in NKCC1 expression. Aim: Here we set out to further changes in short circuit current (Isc) across voltage-clamped investigate molecular mechanisms underlying EGF potentia- monolayers of T84 colonic epithelial cells.Results were express- tion of colonic epithelial secretory capacity. Methods: Mono- esdasmean+/-SEMforaseriesofnexperiments.Statisticalanaly- layers of T84 cells, grown on permeable supports, were mounted sesweremadebyonewatANOVAusingNewmanKeulsmultiple in Ussing chambers and Cl- secretion measured as changes in comparisontest.Pvalues<0.05wereconsideredtobesignificant short-circuit current (Isc). Transport protein mRNA expression Results: At high concentrations (500 μM– 1mM) CDCA rapidly was measured by RT-PCR. Results are expressed as mean ± stan- stimulated Cl- secretion across T84 cells. In contrast, UDCA (50 dard error of the mean for a series of n experiments. Statistical μM – 1 mM) was devoid of prosecretory activity. However, pre- analyses were made by one way ANOVA with the Newman-Keuls treatment of T84 cells with UDCA (500 μM) significantly attenu- post test. P values < 0.05 were considered to be significant. atedsubsequentsecretoryresponsestotheCa2+-dependentago- Results: In addition to NKCC1, acute treatment with EGF (100 nist, carbachol (CCh; 100 μM) and the cAMP-dependent agonist, ng/ml for 15 min) induced increases in the mRNA levels of CFTR forskolin (10 μM) to 11.9 ±4.2% (n=9; p<0.001) and 43.0 ±13.0% (270 ± 20 % of controls; n = 3; p < 0.01), KCNN4 (150 ± 20 of β ± (n=6;p<0.05)ofcontrols,respectively.TheeffectsofUDCAwere controls; n = 5; p < 0.05) and Na/K-ATPase 1 subunit (370 100 concentration-dependentwithantisecretoryactionsbeingappar- % of controls; n = 4; p < 0.01) but did not alter expression of the μ α ± entatconcentrationsaslowas50 M.However,UDCA(1mM)did 1 subunit of Na/K ATPase (130 30 % of controls; n=4). To not alter transepithelial resistance implying it did not exert toxic determine signaling pathways involved in mediating the effects effects. In further experiments we measured Na/K-ATPase pump of EGF, we examined the effects of PI3-K (LY294002), ERK activityinapicallypermeabilisedmonolayersandfoundthatUDCA (PD98059) and p38 MAPK (SB203580;) inhibitors on the abil- ± 2+ inhibitedNa/K-ATPasepumpactivityto16.2 3.9%ofthatincon- ityofEGFtoenhanceIsc responses to either the Ca or cAMP- trolcells(n=4;p<0.001).Inexperimentsdesignedtoisolatebaso- dependent secretagogues, carbachol (CCh) and forskolin (FSK) lateral K+ conductance, UDCA inhibited activity to 13.7 ±2.4% of (Table1). Conclusions: EGF chronically increases colonic epithe- controls (n=5, p<0.001). Similar to UDCA, LCA was also without lial secretory function through upregulation of key transport effect on basal Cl- secretion in T84 cells. However, pretreatment proteins that comprise the Cl- secretory pathway. PI3-K and ERK ofcellswithLCA(50–200μM)significantlypotentiatedresponses MAPK are important in mediating EGF potentiation of both Ca2+ toCCh.LCA(100μM)increasedCCh-inducedresponsesto146.67 and cAMP-dependent responses, while p38 MAPK is involved ±8.7% of those in controls (n = 8; p < 0.001). However, at concen- only in enhancing cAMP-dependent responses. The precise roles trations > 500μM, LCA exerted antisecretory actions similar to of these effector pathways in mediating EGF-induced increases UDCA. Conclusion: Bacterial metabolism of CDCA alters its abil- in transport protein expression are currently under study. Our ity to regulate colonic epithelial secretion. While UDCA exerts data suggest that agents targeting EGFR-dependent signaling mainlyantisecretoryeffects,LCAenhancessecretionatrelatively pathways may be useful in therapy of intestinal disorders asso- low concentrations but is antisecretory at high concentrations. ciated with dysregulated epithelial transport. Our data also suggest that altering bile acid metabolism by phar- macologicalmeansorthroughantibiotic/probioticmanipulation of the enteric flora may prove useful in treating intestinal transport disorders associated with dysregulated epithelial transport. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Table1. Isc responses to CCh and FSK were measured 24 hrs after pretreat- requirements. ment with EGF (100 ng/ml for 15 min). Data are expressed as % control response to CCh (n = 6) or FSK (n = 5) alone (**p<0.01; ***p<0.001).

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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lates the STC-1 enteroendocrine cell line. This preliminary study therefore examines the effect of colestyramine on gastric emp- PC186 tying and appetite in vivo in healthy humans in order to assess its potential use as a non-nutrient tool in gastric emptying stud- Differential expression of UT-B urea transporters in the ies. Nine healthy volunteers were given liquid test meals human gastrointestinal tract (500ml) containing 4g colestyramine, 12g colestyramine or G. Stewart water alone, on three occasions, in a randomised order. The School of Biology and Environmental Science, University College effect of colestyramine on gastric emptying was determined Dublin, Dublin, Ireland non-invasively using the 13C-acetate breath test and the effect on appetite was assessed by visual analogue scales. Colestyra- Facilitative UT-B urea transporters enable the passage of urea mine significantly delayed gastric emptying (overall treatment across cell membranes (1). Gastrointestinal urea transporters effect, p < 0.001). The cumulative 13CO2:12CO2 expired over are thought to play a significant role in the symbiotic urea nitro- the 45-minute period after vehicle was 427.7 ± 28.1 gen salvaging process that occurs between mammalian hosts mean/SEM), compared to 342 ± 26.7 after 4g, and 280.6 ± 14.3 and their gut bacteria (2). This study investigated the expres- after 12g colestyramine. Pairwise comparisons revealed that sion of UT-B urea transporters along the human gastrointesti- the significance of the emptying delay was greater in response nal tract, particularly in the colon. to 12g (p<0.001) than 4g colestyramine (p = 0.048) compared Immunoblot analysis showed that UT-B proteins (purchased to vehicle. The delay in gastric emptying in response to 4g was from AMS Biotechnology) were present throughout the human considerably variable between individuals, whereas the gastrointestinal tract and most strongly expressed in the cae- response to 12g was relatively consistent. Colestyramine also cum and colon. Within the colon, a 35 kDa signal was degly- significantly reduced hunger (p=0.007), the amount of food cosylated to a 30 kDa protein after one hour pre-incubation participants felt able to eat (p=0.001) and bloating (p=0.01). It with PNGaseF enzyme. Glycosylated UT-B protein expression did not evoke nausea. After vehicle, it took subjects ~20 min was greater in the proximal colon than in the distal colon. This to reach the average baseline hunger score, whereas the base- differential pattern of colonic expression was also observed for line score was not reached within the 45-min period with several other proteins involved in nutrient transport mecha- colestyramine. These data demonstrate that colestyramine nisms – such as NHE3 and NaKATPase. Finally, at the cellular significantly delays gastric emptying and reduces appetite in level UT-B transporters were located in the basolateral region humans. Therefore, it has potential as a novel non-nutrient tool of colonocytes situated in the upper portion of the colonic in gastric emptying studies in health and disease (eg diabetic crypts. gastroenteropathy). Colestyramine is particularly interesting These results illustrate a differential expression of glycosylated in that it is not absorbed from the lumen and therefore cannot UT-B transporters along the human gastrointestinal tract, cor- exert post-absorptive effects, which are problematic in inter- relating to regions that contain the largest populations of intes- preting the effects of absorbable nutrients. Furthermore, in tinal bacteria. These data therefore suggest an important role light of its effects on gastric emptying and appetite, under- for urea transporters in maintaining the symbiotic relationship standing the underlying mechanisms could lead to new ther- between humans and their gut bacteria. apeutic applications. C.P. Smith and G. Rousselet (2001). J.Memb.Biol 183, 1-14. Where applicable, the authors confirm that the experiments G.S.Stewart and C.P.Smith (2005). Nutr.Res.Rev. 18, 49-62. described here conform with The Physiological Society ethical ThisworkwasfundedbyTheRoyalSocietyandKidneyReseachUK. requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC190

Influence of insulin on membrane-bound Ca2+ content in permeabilized rat hepatocytes PC187 S. Bychkova Colestyramine: a Novel Non-nutrient Tool in Human Gastric Department of Human and Animal Physiology, Ivan Franko Emptying Studies National University of Lviv, Lviv, Ukraine A. Psichas1, T. Little2, M. Case1 and J. McLaughlin2 Insulin induces change of Ca2+ content in hepatocytes (Benze- 1Faculty of Life Sciences, Manchester University, UK and 2School of roual K. et al., 1997; Rodrigues M.A. et al., 2008). However, the Translational Medicine, Manchester University, UK role of IP3Rs and RyRs in insulin-induced change of Ca2+ con- Two established physiological actions of the enteroendocrine tent in hepatocytes is still unknown. Here we present results of peptide cholecystokinin (CCK) include inhibition of gastric emp- our investigation of insulin effect on on IP3Rs and RyRs in rat tying and mediation of satiation after a nutrient meal. There is permeabilized hepatocytes. evidence to suggest that the particulate resin colestyramine Experiments were carried out on humanly killed 4-5 months stimulates secretion of CCK in humans. It also directly stimu- old Wistar rats (n = 29, weight 0,18-0,2 kg). Isolated liver was perfused with insulin-content solution (external incubation solution contained (in mM): NaCl – 140,0; KCl – 4,7; CaCO3 – 1,3; MgCl2 – 1,0; HEPES – 10,0; glucose – 10,0; pH = 7,4 ; insu-

176P Poster Communications line - 0,04 u.) for 10 min at room temperature. Then hepato- compared to the left ventricle (LV), thus the aim of this study cytes were isolated by lidase digestion (16 u., 10 min, 37 ToC) was to investigate alterations in Ca2+ handling in RV hyper- and permeabilised by incubating with saponine (0.1 mg/ml, 10 trophy and HF in pulmonary hypertensive rats. min), which was added to internal incubation solution con- Male Wistar rats (200 g) were injected with monocrotaline at tained (in mM): NaCl – 20,0; KCl – 120,0; MgCl2 – 1,13; ATP 30 mg/kg to induce RV hypertrophy (HYP) or 60 mg/kg to («Sigma», USA) – 2,0, CaCl2 – 1,3, HEPES – 10,0; pH=7,0. Con- induce RV failure (FAIL) which was identified by clinical signs. centration of membrane-bound Ca2+ was measured using chlor- Control animals were injected with an equivalent volume of tetracycline. saline (CON). Animals were humanely killed 3-4 weeks after We showed that perfusion of liver with insulin-content solution injection and RV cardiomyocytes were enzymatically isolated. causes statistically significant increase of membrane-bound All experiments were conducted at 20-24∞C. Cell shortening Ca2+ concentration in permeabilized hepatocytes by 33,57 ± and Ca2+ transients were recorded in fura-4 AM loaded 7,93 % (n = 29, P < 0,001) in comparison to membrane-bound myocytes. Caffeine (20 mM) was used to assess the sarcoplas- Ca2+ concentration after liver perfusion with control solution mic reticulum (SR) calcium load. Calcium sparks were meas- (no insulin present). This liver perfusion with insulin also ured by confocal microscopy using the fluorescent indicator changed IP3 and ryanodine effect on membrane-bound Ca2+ fluo-4 AM. Statistical comparison between CON, HYP and FAIL in hepatocytes. We found that IP3 decreased the membrane- myocytes was performed by 1-way ANOVA. All procedures bound Ca2+ by 32,00 ± 4,08 % (n = 9, P < 0,05) after perfusion accorded with current UK legislation. liver by insulin-content solution, but caused an increase of mem- Compared to CON hearts; there was a statistically significant brane-bound Ca2+ content after perfusion with control solu- increase in the ratio of heart weight: body weight by 50% in HYP tion (without insulin). Also ryanodine (5 and 500 nM) decreased and by 78% in FAIL heart (P < 0.05, n = 5-6 hearts in each group). concentration of the membrane-bound Ca2+ by 16,24 ± 5,21 % Cell shortening was significantly decreased by 17% in HYP and by (n = 7, P < 0,05) and 26,83 ± 9,45 % (n = 4, P = 0,03), respec- 29% in FAIL myocytes but Ca2+ transient amplitude significantly tively. However, ryanodine (50 nM) did not elicit significant increased by 43% in HYP and 56% in FAIL myocytes, (P < 0.05 n = changes in the membrane-bound Ca2+ content in permeabi- 17-20 myocytes in each group). SR load was also increased in HYP lized hepatocytes after perfusion of liver with insulin-content by20%andFAILby61%myocytes(P<0.05,n=19-23myocytesin solution. In control experiments ryanodine (5, 50 and 500 nM) eachgroup).ConsistentwithanincreaseinSRload,thefrequency, caused statistically significant increase of membrane-bound duration and width of calcium sparks significantly increased in Ca2+ content in permeabilized hepatocytes by 22,26 ± 7,54 % (n = 7, P < 0,05), 22,95 ± 7,88 % (n = 9, P < 0,05), 18,85 ± 7,50% (n = 6, P < 0,05), respectively. Thus we conclude: 1) perfusion of liver by insulin-content solution prevented ryanodine’s (50 nM) action and changed on reverse action of ryanodine (5 and 500 nM) and IP3; 2) insulin changes concentration of membrane-bound Ca2+. We suppose that perfusion of liver by insulin-content solution increase content Ca2+ in intracellular store that is why action of ryanodine and IP3 is opposed to control. Benzeroual K, van de Werve G, Meloche S, Mathé L, Romanelli A, Had- dad P. Insulin induces Ca2+ influx into isolated rat hepatocyte couplets // Am J Physiol. – 1997. – V. 6 Pt 1/ – P. G1425-32. Rodrigues M.A., Gomes D. A.,. Andrade V. A,. Leite M. F,. Nathanson M. H. Insulin induces calcium signals in the nucleus of rat hepatocytes // Hepatology. – 2008. – Vol. 9999, Is. 999A. – P.NA.

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

PC192

Altered calcium handling in monocrotaline-induced, right ventricular hypertrophy and failure in rats D. Benoist, Z. Yang, D. Steele and E. White Institute of Membrane and Systems Biology, Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, UK The altered contractility that occurs in cardiac hypertrophy and heart failure (HF) is often attributed to changes in Ca2+ handling properties (Hasenfuss and Pieske, 2002). Such changes have been less extensively studied in the right ventricle (RV),

177P Poster Communications thedevelopmentofhypertrophyandHFbutsparkamplitudepro- cytoplasmic(Cacyt)calciumandiii)aSERCAformulationdepend- gressively reduced (P< 0.05, n= 24-32 myocytes in each group). ent on both Cacyt and CaSR (all but two). To investigate which of Ca2+-handling is altered in the RV hypertrophy and heart fail- thesemodelpropertiesareessentialfortheproductionofCaOSS ure associated with monocrotaline-induced pulmonary hyper- (and thus DADs) we perform in-silico experiments removing the tension.TheincreasedSRloadmayexplaintheobservedincrease dependence of RyR on Cass/Cacyt, or the dependence of SERCA in Ca2+ transient amplitude and the increased number of Ca2+ on CaSR. The change in RyR abolishes CaOSS, whereas SERCA sparks. However, these changes cannot be directly responsible modulation changes just their frequency and amplitude. Modu- for the reduced cell shortening, this is probably related to de- lation of RyR by CaSR is included in some models, but can be sensitizationofthemyofilamentstoCa2+(Lambertsetal.,2007). removedwithoutpreventingCaOSS–indicatingthatSRoverload Hasenfuss, G. et Pieske, B. (2002). J. Mol. Cell. Cardiol. 34, 951-969. modulates DADs, but is not the key mechanism. Lamberts, R.R. et al. (2007). J. Physiol. 582, 695-709. Our results confirm that the main trigger for CaOSS and DADs is an increased level of calcium within the cell, which is also Supported by The British Heart Foundation, D.B. is an Emma the common feature of the experimental results of calcium- and Leslie Reid Ph.D. student. induced DADs. The “spontaneous release” of calcium from the Where applicable, the authors confirm that the experiments SR occurs by the same mechanism as calcium induced calcium described here conform with The Physiological Society ethical release, i.e., the modulation of the RyR opening by calcium in requirements. the dyadic subspace. Future work will investigate the correlation between Markov formulations of RyR and DAD appearance.

PC193

Delayedafterdepolarisationsinducedbydyadiccalciumwithout sarcoplasmic reticulum calcium overflow. A simulation study P.J. Noble, D. Noble and M. Fink Physiology,AnatomyandGenetics,UniversityofOxford,Oxford,UK Delayed AfterDepolarisations (DAD) are depolarisations that occur after full repolarisation and have been related to sodium and/or calcium overload in the cell. They are triggered by “spontaneous release” of calcium from the sarcoplasmic reticulum (SR) leading to calcium oscillations and via the electrogenic sodium-calcium exchanger (NCX) to changes in membrane Figure 1. CaOSS and DADs in the Noble ’98 Model. Membrane potential potential (1). Due to the variety of experimental findings the appears in the top two panels and [Ca]i in the bottom two panels. On the trigger of the release is still under debate: SR overload (2) vs. left we see CaOSS (panel C) in the absence of membrane currents (V=-94 dyadic calcium (3). mV in panel A). On the right “spontaneous” SR calcium release (panel D) is We investigate the underlying mechanism of DAD induction triggering DADs leading to action potentials (panel B) in the full model. usingmathematicalatrialandventricularcellmodelsofhuman, Sipido KR et al. (2006). Handb Exp Pharmacol 171, 159-199. dog,rabbit,guinea-pig,ratandmouse(31models).Tocompare Pogwizd SM & Bers DM (2004). Trends Cardiovasc Med 14, 61-66. inducibility of calcium oscillations, each model is reduced to its Shannon TR et al. (2005). Biophys J 89, 4096-4110. “calcium subsystem”; trans-membrane potential, intracellular sodium ([Na]i) and potassium concentrations are fixed at their Noble D et al. (1998). Can J Cardiol 14, 123-134. end-diastolic values, all trans-membrane currents but the NCX The work was supported by BHF (PG/08/019/24600). are set to zero. Intracellular calcium handling is unchanged. We find that in all models showing Calcium Oscillations in the Where applicable, the authors confirm that the experiments SubSystem (CaOSS) the same parameter changes also produce described here conform with The Physiological Society ethical DADs in the full model, which in the case of the Noble’98 model requirements. (4) lead to action potentials (Fig 1). We do not find parameter combinations that initiate DADs but do not show CaOSS. Thus the initiation of DADs in the full model is concurrent with the appearance of CaOSS. ReducedmodelsaretestedforCaOSSbyincreasing[Na]i(increases [Ca]i via NCX) and the sarcoplasmic reticulum calcium ATPase (SERCA) current (reflecting increased sympathetic tone). All 19 reducedmodelsthatshowCaOSShavei)Markovformulationsfor RyR, ii) the RyR modulated by either dyadic subspace (Cass) or

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Where applicable, the authors confirm that the experiments PC194 described here conform with The Physiological Society ethical requirements. Simulation of the effects of BAPTA and EGTA on inactivation of ICa in a model of the rat ventricular myocyte M. Pásek1,3, M.R. Fowler2, J. ˇSimurda3, G.L. Smith2 and C.H. Orchard4 PC195 1Institute of Thermomechanics - branch Brno, Czech Academy of Science, Brno, Czech Republic, 2Faculty of Biomedical and Life Sciences, Glasgow, UK, 3Department of Physiology, Masaryk Revisiting the differential effects of left sided and right sided University Brno, Brno, Czech Republic and 4Department of sympathetic outflows on the whole heart: Studies in the Physiology and Pharmacology, University of Bristol, Bristol, UK isolated innervated rabbit heart 2+ In cardiac ventricular myocytes, the fast Ca buffer BAPTA has A. Tanko1, K.E. Brack1, J.H. Coote2 and G. Ng1 a greater effect than the slow Ca2+ buffer EGTA on inactivation 2+ 1 of the L-type Ca current, ICa (e.g. Brette at al. 2004, Sham, Cardiovascular Sciences, University of Leicester, Leicester, UK and 1997). The aim of the present study was to refine an existing 2Pharmacology, University of Birmingham, Birmingham, UK model of the rat ventricular myocyte (Pasek et al. 2006) to reproduce the differential effect of BAPTA and EGTA on inacti- Historical studies demonstrating differential effects between left and right sympathetic outflows to the heart have employed vation of ICa in the rat myocyte and to investigate its possible cause. The principal modifications to the model were: (i) refor- canine models (Ardell et al, 1988). The aim of this study was mulation of the description of sarcoplasmic reticulum (SR) Ca2+- to re-examine these differences in the rabbit using a novel in ± release according to Shannon at al. (2004); (ii) reformulation vitro preparation. Adult male New Zealand rabbits (3.0 0.1kg; of the description of voltage- and Ca2+-dependent inactivation n=4) were used. The isolated heart preparation with intact autonomic nerves was obtained under propofol anaesthesia (1 of ICa to simulate the experimental data of Brette at al. (2004); (iii) modification of the parameters of the SR Ca2+-pump to give mg/kg, i.v.) as previously described (Ng et al, 2001). Left ven- the relation between free Ca2+ in the cytosol and SR described tricular pressure (LVP) was measured using a fluid filled balloon. by Shannon and Bers (1997); (iv) incorporation of equations The left and right paravertebral sympathetic chains were iden- controlling exchange of free EGTA and Ca2+-EGTA and of free tified and isolated from adjacent tissues from the level of T4 up BAPTA and Ca2+-BAPTA between the pipette, cytosol and dyadic to T1/2 where fine custom-made silver bipolar electrodes were space. In the model, both buffers could inhibit the cytosolic positioned and electrically isolated with dental cement. The Ca2+-transient, and thus contraction. However, only BAPTA threshold voltage that caused an increase in both heart rate inhibited efficiently the rise of Ca2+ in the dyadic space, thus (HR) and LVP was determined and left (LS) and right (RS) sym- causing significant inhibition of Ca2+-dependent inactivation pathetic chains were stimulated separately at twice threshold voltage at 2.3±1.1V (LS) and 1.2±0.4V (RS) respectively at 2Hz of ICa. The principal reason for the different potencies of BAPTA and EGTA in reducing the rise of Ca2+ in the dyadic space was [Low], 5Hz [Med] and 10Hz [High] during 1) sinus rhythm and 2+ - 2) during right ventricular pacing at 250bpm to examine their different rates of Ca binding (BAPTA: kon= 500000 mM 1 -1 -1 -1 chronotropic and inotropic effects. Data are mean±SEM, sta- s ; EGTA: kon= 5000 mM s ). As a consequence, the concentration gradients of free BAPTA and Ca2+-BAPTA, and of free tistical analysis was performed using 2-factor ANOVA. Results: EGTA and Ca2+-EGTA, between the dyadic space and bulk LS and RS increased both HR and LVP, to differing degrees cytosol were substantially different after the onset of depolar- (Fig1A). The peak HR achieved during RS was significantly isation. In the case of BAPTA, the large gradients provided a greater than during LS (Table 1). Whilst there was a trend for large driving force, causing rapid transport of Ca2+-BAPTA mol- LS to have a greater effect in increasing LVP than RS (see Table ecules out of the dyadic space, and movement of free BAPTA 1 and Fig1B). 2-factor ANOVA revealed a significant difference into the dyadic space, so that BAPTA appeared to act as a fast in the percentage change in HR and LVP between LS and RS ‘shuttle’. The slower rate of Ca2+ binding to EGTA and devel- (Fig1B). In conclusion, stimulating the right sympathetic out- opment of substantially smaller free EGTA and Ca2+-EGTA gra- flow to the heart has a more prominent effect on the sino-atrial dients between the dyadic space and cytosol reduced the abil- node but a weaker effect on the left ventricle, whereas the 2+ reverse is true when stimulating the left sympathetic outflow. ity of EGTA to affect Ca -dependent inactivation of ICa. Brette F at al. (2004). Circ Res 95, e1-e7. This may reflect regional differences on sympathetic innervation to the heart. Sham JS (1997). J Physiol 500, 285-295. Pásek M at al. (2006). Phil Trans R Soc A 364, 1187-1206.

Shannon TR at al. (2004). Biophys J 87, 3351–3371.

Shannon TR & Bers DM (1997). Biophys J 73, 1524–1531.

Supported by the British Heart Foundation and project AV0Z 20760514.

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equilibration cardiomyocytes were perfused with 20mM caffeine for 1 minute followed by washing with normal buffer. Ca2+ transients were monitored using photometry throughout. Data is expressed as mean ± standard error. Exposure of adult cardiomyocytes to caffeine induced the characteristic marked increase in Ca2+ transient amplitude that was followed by complete abolition of the transients indicating emptying of the SR of Ca2+. This effect was also seen in cardiomyocytes from all other age groups. Upon reperfusion and washing out the caffeine, the SR gradually refills its Ca2+ stores and normal transients are restored. The rate of SR refilling was significantly slower in 14 day cardiomyocytes compared to those from adult (ANOVA, p= 0.027, n=10). The 14 day old cardiomyocytes on average took approximately 172 ± 12 seconds to refill the SR in comparison to adult cardiomyocytes which took 120 ± 10 seconds when exposed to the same concentration of caffeine for the same duration of time. This work shows an age-related difference in rate of SR refilling following caffeine induced emptying. This could be due to difference in SR development. Additionally, refilling of the SR with Ca2+ in early postnatal development depends on extracellular Figure 1: A) Heart rate (HR) and left ventricular pressure (LVP) during high Ca2+ coming via the Na+/Ca2+ exchanger rather than the Ca2+ frequency left (LS) and right sympathetic (RS) chain stimulation. B) The per- channel (Artman, M., 1992). The exchanger is likely to be slower centage change in HR and LVP during LS and RS. *P<0.05. in transporting Ca2+ across the sarcolemma which would explain the slower rate of SR refilling. Artman M. (1992) Sarcolemmal Na+-Ca2+ exchange activity and exchanger immunoreactivity in developing rabbit hearts. Am J Phys- iol Heart Circ Physiol 263: H1506–H1513. Table 1: Chronotropic and inotropic effect of left (LS) and right sympathetic O’Neill SC, Eisner DA. (1990) A mechanism for the effects of caffeine (RS) chain stimulation. on Ca2+ release during diastole and systole in isolated rat ventricular *P<0.05 vs. BL: LS vs. RS. myocytes. J Physiol 430:519–536. Ardell JL, Randall WC, Cannon WJ, Schmacht DC, Tasdemiroglu E 1988. This work is supported by the BHF. AJP;255:H1050-H1059. Ng GA, Brack KE, Coote JH 2001. Exp Physiol;86.3:319-329. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical We would like to acknowledge the Garfield Weston Trust requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC199

In middle aged and old individuals 20 weeks of resistance training restores the increases of leg blood flow after acute PC196 exercise and feeding to values seen in young subjects The effect of caffeine on cardiomyocytes isolated from B. Phillips1,2, W. Hildebrandt1, K. Smith1, M. Baker1, A. Gates1, different age groups P.L. Greenhaff2, M.A. Ian2 and M.J. Rennie1 S. Martin, N. Shukla, L. Hua, J. Jeremy and S. Suleiman 1School of Graduate Entry Medicine and Health, University of Nottingham, Derby, UK and 2Biomedical Sciences, University of Bristol Heart Institute, Bristol, UK Nottingham, Nottingham, UK The aim of this study was to determine whether the effect of Muscle blood flow can be increased by feeding and after exer- caffeine on calcium (Ca2+) transients in isolated perfused rat cise, and thus may be an important determinant of the size and cardiomyocytes is age-related. Caffeine is known to induce Ca2+ efficiency of the anabolic responses to these stimuli. We have release from the sarcoplasmic reticulum (SR) (O’Neill SC, Eis- tested the hypothesis that progressive resistance exercise train- ner DA., 1990). Whether this effect of caffeine is the same in ing would increase the response of leg blood flow to food and all age groups postnatally, is not presently known. to the combination of an acute resistance exercise bout and Cardiomyocytes were enzymatically isolated from 14, 21, 28 food. We studied three groups of subjects (young, n=7, 5 men, days old and adult rat hearts. Freshly isolated cardiomyocytes 2 women, 25.2 ±1.18 y, Body mass index (BMI) 24.1±0.54; mid- were loaded with the Ca2+ fluorescent dye Fura-2, then per- dle aged, n= 6, 3 men, 3 women, 50.9±1.5 y, BMI 27.5±1.3 and fused with HEPES buffer (1mM Ca2+) in a chamber under an old, n=6, 3 men, 3 women, 68.8±1.3 y, BMI 25.4±0.6) who inverted microscope at 34οC and stimulated at 0.2Hz. After underwent 20 weeks of progressive whole body resistance exer-

180P Poster Communications cise training (four weeks induction at 40-60% 1RM then three sessions/week at 70% 1RM, three each of upper and lower body and two torso-based exercises). Before and after training, femoral artery blood flow was measured (Doppler ultra-sound) in three conditions: (i) postabsorptive state at rest, (ii) in both legs 120 min after oral feeding (Fortisip: 16% protein, 49% carbohydrate, 35% fat) providing energy at 4.25× BMR for 2.5 h supplied as a 3×bolus and 4 further aliquots every 30 min with one leg at rest, and (iii) the other leg recovering from a previous exercise bout of 6 × 8 repetitions of leg extensions (75% 1RM). All procedures were approved by University of Notting- ham ethics committee. Leg strength (extension, curl and press) increased by 23.7±4.49, 39.6±2.86 and 32.3±4.13% respectively in the young, middle aged and old groups. Basal blood flow in the middle and old groups was identical (0.32±0.03 and 0.33±0.03 l.min-1 respectively) with a strong trend for the flows to be less than in the young (0.51±0.1 l.min-1). The young group showed no training effect in leg blood flow in response to feeding and exercise (66.2±12.3% pre-training vs. 74.6±14.0% post- training). In contrast, in both the middle aged and old groups, responses to feeding plus exercise (Figure 1) were significantly enhanced by training (middle 101±16% vs. 147±32% and old 59±15% vs. 115±47%). In middle aged subjects feeding alone did not increase leg blood flow in the untrained state, but induced responsiveness after training. The results show (i) that leg blood flow responses to exercise plus food are diminished in middle aged and old subjects and (ii) are restored to the pattern seen in young subjects by resistance training.

Figure 1. Effect of resistance training on increases in femoral artery blood flow after feeding or exercise plus feeding in young, middle-aged and old subjects. Values are means ±SEM. ** = P<0.01 vs. basal pre-training; =P<0.01 vs. basal post-training; = P<0.01 vs. feeding plus exercise pre-training, all via ANOVA with Tukey’s post analysis.

This work was supported by grants from UK BBSRC ((BB/XX510697/1 and BB/C516779/1) European Union EXE- GENESIS program and The Dunhill Trust.

Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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PC200

Molecular evidence for blood-brain barrier disruption in the absence of structural tissue damage during acute exposure to inspiratory hypoxia

K.A. Evans1, S. Taudorf2, R.M. Berg2, C. Lundby3, J. McEneny4, I.S. Young4, P.E. James5, J.M. McCord6, B.K. Pedersen2, K. Moller2,7 and D.M. Bailey1 ± Values are mean SD; NSE; a-vD, arterial minus venous concentration dif- 1Neurovascular Research Laboratory, University of Glamorgan, ference; main effect for condition indicates a pooled (arterial + venous) difference (P < 0.05) between normoxia vs. hypoxia; main effect for sample Pontypridd, UK, 2Centre of Inflammation & Metabolism, site indicates a pooled (normoxia + hypoxia) difference (P < 0.05) between Department of Infectious Diseases, Rigshospitalet, University of arterial vs. venous. Copenhagen, Copenhagen, Denmark, 3Copenhagen Muscle Bailey DM et al. (2009). J. Physiol. 15, 73-85 Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 4Centre for Public Health, Queen’s Kety SS & Schmidt CF (1945). Am J Physiol 143, 53-66. University, Belfast, UK, 5Wales Heart Research Institute, School of Where applicable, the authors confirm that the experiments Medicine, Cardiff University, Cardiff, UK, 6Division of Pulmonary described here conform with The Physiological Society ethical Sciences & Critical Care Medicine, University of Colorado, Denver, requirements. CO, USA and 7Departments of Cardiothoracic Anaesthesia & Intensive Care Unit 4131, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

It has been suggested that inspiratory hypoxia may cause PC201 molecular opening of the blood brain barrier (BBB) in the absence of structural neuronal-parenchymal damage subse- Influence of hyperoxia on the power-duration relationship quent to a systemic accumulation of free radicals (Bailey et al., during severe-intensity exercise in humans: a 31P-MRS study 2009). Therefore the current study aimed to establish changes 1 2 1 3 in regional metabolism, specifically the trans-cerebral exchange A. Vanhatalo , J. Fulford , F. DiMenna , D.C. Poole and 1 of S100β and neuron-specific enolase (NSE) to distinguish A.M. Jones potential “barrier disruption” from structural “brain tissue dam- 1School of Sport and Health Sciences, University of Exeter, Exeter, age” caused by hypoxia. Devon, UK, 2Peninsula NIHR Clinical Research Facility, University ± Ten healthy males aged 27 (mean) 4 (SD) years were exam- of Exeter, Exeter, Devon, UK and 3DepartmentsofKinesiologyand ined in normoxia and following 9h passive hypoxia (12.9%O2). Anatomy and Physiology, Kansas State University, Manhattan, Internal jugular venous and radial arterial bloods were col- KS, USA lected simultaneously for the measurement of serum S100β and NSE by ELISA. Global cerebral blood flow (CBF) was meas- The purpose of this investigation was to explore the mecha- ured using the Kety-Schmidt technique (Kety & Schmidt, nistic bases of the hyperbolic relationship between power and 1945) and global cerebral plasma flow (CPF) determined as the tolerable duration of severe-intensity exercise by assess- CBF x (1-haematocrit). Trans-cerebral net exchange was cal- ing the kinetics of intramuscular phosphocreatine concentra- culated as the arterio-jugular venous concentration difference tion ([PCr]) and pH during prediction trials performed both in x CPF. Data were analysed using a two-way repeated meas- normoxia and hyperoxia. It was hypothesised that the inspira- ures ANOVA and post-hoc Bonferroni-corrected paired sam- tion of hyperoxic gas would increase the asymptote of the ples t-tests. power-duration relationship, i.e. the critical power (CP), with- Hypoxia was associated with a general increase in S100β (arte- out altering the curvature constant (W′). It was also hypothe- rial inflow and venous output) whereas NSE decreased (Table sised that the tolerable duration of exercise, irrespective of the 1). The net cerebral output of S100β and uptake of NSE work-rate within the severe-intensity domain, would be asso- observed during normoxia was shown to persist without any ciated with the attainment of the same, critically low [PCr] and further changes being incurred during hypoxia. pH values as determined using 31P magnetic resonance spec- These findings provide the first regional molecular evidence to troscopy (31P-MRS). suggest that hypoxia causes subtle barrier disruption in the absence of neuronal-parenchymal brain injury. Table 1 Brain-specific proteins

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Following ethical approval, seven male subjects (mean ± SD, placed over the sternum approximately 2cm above the xiphoid age 30 ± 9 years) completed four constant work-rate, knee- process. Thereafter, a 30-second ballistocardiogram was extension exercise bouts to exhaustion (Tlim range: 3-10 min) recorded and stored for later analysis. A total of 15 dBG wave- both in normoxia (N) and hyperoxia (H; 70% O ) inside the bore 2 forms (heart beats) were analysed for each subject per day and of 1.5 T superconducting magnet. The inspirates and work- then averaged (Mean±SD). Results showed that average heart rates were administered in a single-blind, randomised order. rate on D2 (58.4±11.7 bpm) was significantly (ANOVA) lower Individual power-time relationships were established using the from D3 (60.2±7.7 bpm), but not D1 (59.1±8.2 bpm). When hyperbolic model [time = W′/(power-CP)] and the overall rate the dBG data was then corrected for heart rate there were no of decline in [PCr] was calculated as the mean response time significant differences between any of the cardiac cycle tim- (MRT). Paired samples t-tests were used to assess differences ing intervals, including Q-wave (EKG) to: atrial systole (AS) (D1=- between conditions in CP and W′. End-exercise [PCr] and pH 40.6±12.6msec; D2=-43.5±13.9msec; D3=-44.9±11.5msec); were compared across inspirates and work-rates using two- mitral valve close (MVC) (D1=42.6±9.8msec; way repeated measures ANOVAs. Significance was accepted at D2=41.3±11.5msec; D3=42.2±8.1msec); aortic valve open P<0.05. (AVO) (D1=75.5±8.0msec; D2=75.4±9.6msec; The CP was significantly higher in hyperoxia (N:16.1 ± 2.6 vs. D3=72.0±9.8msec); rapid ejection period (REP) H:18.0 ± 2.3 W; P<0.05), while the W′ parameter was not signi- (D1=145.1±10.9msec; D2=141.5±10.0msec; ficantly altered (N:1.92 ± 0.70 vs. H:1.48 ± 0.31 kJ; P>0.05). The D3=138.7±5.7msec); aortic valve close (AVC) [PCr] at the limit of tolerance (~8% of resting baseline) during (D1=329.7±30.3msec; D2=333.3±28.9msec; each of the four trials was not significantly different either in D3=331.0±27.2msec); mitral valve open (MVO) normoxia or hyperoxia (F6,18 = 1.43, P>0.05). The end-exer- (D1=433.7±31.3msec; D2=435.1±29.5msec; cise pH (~6.65) was likewise unaffected by work-rate and the D3=436.2±16.0msec); early diastole (ED) inspired oxygen fraction (F6,18 = 1.35, P>0.05). The overall rate (D1=518.9±33.2msec; D2=518.3±29.3msec; at which [PCr] fell was attenuated in hyperoxia (MRT 116 ± 46 D3=519.2±23.6msec); and late diastole (LD) s) compared to normoxia (59 ± 20 s) (P<0.05). (D1=995.2±160.2msec; D2=1025.6±236.2msec; This study is the first to demonstrate that hyperoxia resulted in a D3=996.1±139.6msec). The percent difference for these vari- significantincreaseinCPwithoutalteringtheW′,andthathyper- ables day-to-day was 9.7% (AS), 3.1% (MVC), 4.6% (AVO), 1.9% oxiaextendedthetimetoexhaustionduringsevere-intensityexer- (REP), 1.1% (AVC), 0.6% (MVO), 0.2% (ED), and 5.7% (LD). These cise by delaying the point at which [PCr] and pH reached values data suggest that: 1) day-to-day cardiac cycle mechanics in which might be considered to be limiting for continued muscle human subjects is reliable; and 2) digital ballistocardiography function.ThepresentfindingssupportthenotionthattheCPrep- can be used to reliably monitor differences in cardiac cycle tim- resentsthehighestsustainableoxidativemetabolicrateandindi- ing intervals from day-to-day. cate that the W′ (and thus the nature of fatigue >CP) is related to musclePCravailabilityand/ortheattainmentofalowmusclepH. Wewishtothankthesubjectsthatvolunteeredforthisstudy.Sup- port:CanadianInstitutesofHealthResearch(JPN),Saskatchewan Where applicable, the authors confirm that the experiments Health Research Foundation (JPN), Heart Force Medical. described here conform with The Physiological Society ethical requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC202

Cardiac Cycle Timing Events are Reliably Measured Day-to- PC203 Day Using Digital Ballistocardiography Characterization of in vitro differentiated aged human 1 1 2,3 J. Neary , D.S. MacQuarrie and E.F. Busse skeletal muscle satellite cells by transcriptional profile 1Kinesiology & Health Studies, University of Regina, Regina, SK, R. Mancinelli, C. Puglielli, R. La Rovere, T. Pietrangelo and S. Fulle Canada, 2Faculty of Graduate Studies, University of Regina, Regina, Università “G. d’Annunzio” Chieti-Pescara, Chieti, Italy SK, Canada and 3Cardiology, Regina General Hospital, Regina Qu’Appelle Health Region, Regina, SK, Canada Sarcopenia is an age-related non-pathological condition that includes a progressive loss of muscle mass, strength and func- Human myocardium has an amazing ability to contract reliably tion(Vandervoortetal.,2001).Severalfactorsbothintrinsic(meta- day-to-day as reflected by the timing events of the cardiac cycle. bolicpathwaymodifications,hormonalandcellularchanges)and We measured the mechanical activity of the heart to record extrinsic(lifestyleandcaloricintake)contributetosarcopenia.The cardiac cycle timing events using digital ballistocardiography aging process is associated with a consistent decrease in the abil- (dBG) in a group of healthy subjects (N=7; 3 females) with no ity of muscle tissue to regenerate following injury or overuse due known cardiovascular disease. We hypothesised that dBG would totheimpairmentofinterveningsatellitecells(HawkeandGarry, reliably record the timing events of the cardiac cycle from day- 2001). Satellite cells are muscle stem cells (Charge and Rudnicki, to-day. Subjects (mean ± SD, age= 31.7±10.5yrs) were assessed 2004). In response to stimuli such as myotrauma, satellite cells on three consecutive days (D1, D2, D3), at the same time of the becomeactivated,proliferate,andexpressmyogenicmarkersand day and under similar conditions in a quite laboratory setting. aretermedmyoblasts.Ultimately,thesecellsfusetoexistingmus- Each subject had the dBG-300 sensor attached to the skin using clefibersorfusetogethertoformnewmyofibers(myotubes)dur- solid gel electrodes (single lead EKG). The dBG-300 sensor was ing regeneration of damaged skeletal muscle. Previously we

183P Poster Communications demonstrated that in old subjects the decreased muscle regen- receptors (KARs) is less known. A number of studies have now erativecapabilityisnotduetoareducednumberofquiescent begun to reveal a role for KARs in mediating ischaemic damage satellite cells but, more probably, due to an impairment of their (1). Small Ubiquitin like Modifier (SUMO) is greatly upregulated differentiationprogram(Beccaficoetal.,2007;Fulleetal.,2005). during ischaemia and has been suggested that this may be a Although this cells population was identified 40 years ago, lit- neuroprotective mechanism (2,3). We discovered that the KAR tle is known regarding molecular basis of activated elderly satel- subunit GluR6a is SUMOylated by SUMO1 and that this controls lite cells in muscle repairing process. KAR trafficking causing removal of KARs from the postsynap- The aim of the present study was to characterize the transcrip- tic membrane (4). We hypothesised that neurones may be pro- tional profile of myoblasts and myotubes obtained from eld- tected from ischaemic damage by increasing SUMOylation of erly human skeletal muscle biopsies (after informed consent). KARs causing their removal from the cell surface and thereby We have isolated and cultured quiescent satellite cells on which reducing glutamate-mediated excitotoxicity. we determined myogenic percentage using immunocyto- We exposed hippocampal slices taken from P13-15 rats to chemistry and the fusion index percentage of myotubes stain- 15 minutes of oxygen glucose deprivation (OGD, oxygen ing fast and slow myosin heavy chains. To obtain the goal, we and glucose replaced with nitrogen and sucrose respec- performed microarray experiments on myoblasts and tively), an in vitro model of ischaemia, and measured synap- myotubes at early stages of differentiation (4, 24 and 72 hours) tic KAR-EPSCs using whole-cell electrophysiological record- comparing the transcriptional profile of older adults than young ings. We manipulated the degree of SUMOylation within individuals. the cell by including SUMO1, SUMO2 or the deSUMOylat- The present study suggest that the failure of the differen- ing enzyme SENP1 in the patch pipette. We replicated pre- tiative program is due to the disregulation of genes involved vious results demonstrating that inclusion of SUMO1 μ in: i) oxidative damage accumulation in molecular substrates, (4.2 M) reduced KAR-EPSCs and that SENP1 (100nM) probably due to impaired antioxidant activity and repair increased KAR-EPSCs (4). Inclusion of SUMO2 had no effect capability (upregulation of polymerase K and SHC1), ii) on the amplitude of KAR-EPSCs. This suggests that SUMO1 altered cytoskeleton turnover and extracellular matrix degra- and SUMO2 play different roles in the regulation of KAR traf- dation, and iii) activation of atrophy mechanism via a spe- ficking. cific FOXO-dependent program as well an impairment of the OGD decreased the amplitude of KAR-EPSCs (measured 20- ± protein balance. 25 minutes after OGD) by 60.5 10% (n=8). The reduction in KAR-EPSCamplitudesuggestsinternalisationofKARsfrom Beccafico, S., Puglielli, C., Pietrangelo, T., Bellomo, R., Fanò, G., Fulle, S., 2007. Age-dependent effects on functional aspects in human satel- the membrane since it was not due to changes in pre-synap- lite cells. Ann. N.Y. Acad. Sci. 1100, 345-352. tic fibre volley. SUMO1, SUMO2 and SENP1 and corresponding Charge, S.B., Rudnicki, M.A., 2004. Cellular and molecular regulation inactive control proteins were infused and KAR-EPSC ampli- of muscle regeneration. Physiol. Rev. 84, 209-238. tude stabilised before OGD application. OGD caused a 69.4±10% (n=12) decrease in KAR-EPSC after SUMO1 infusion Fulle,S.,DiDonna,S.,Puglielli,C.,Pietrangelo,T.,Beccafico,S.,Bellomo, R., Protasi, F., Fanò, G., 2005. Age-dependent imbalance of the antiox- that was significantly greater than the decrease induced in idative system in human satellite cells. Exp. Gerontol. 40, 189-197. interleaved controls using infusion of the inactive version of SUMO1, SUMO1ΔGG (47.1±10%, n=10, p=0.047, unpaired t- Hawke, T.J., Garry, D.J., 2001. Myogenic satellite cells: physiology to molecular biology. J. Appl. Physiol. 91:534-551. test). In contrast, OGD after infusion of SUMO2 caused significantly less reduction in the KAR-EPSC compared to Vandervoort, A.A., Symons, T.B., 2001. Functional and Metabolic Con- Δ ± ± sequences of Sarcopenia. Can. J. Appl. Physiol. 26, 90-101. SUMO2 GG (40.9 10% and 63.4 10% respectively, n=7, p=0.048, unpaired t-test). Infusion of SENP1 had no effect on We thank Dr Luigi d’Amelio (Orthopaedic Division of Ospedale the recovery of the KAR-EPSC after OGD compared to the inac- di Atri) for the surgical operations. This study was supported tive version of SENP1, SENP1-C603S (p=0.049, unpaired t- by a research grant from MIUR (Ministero Istruzione Università test). e Ricerca) and by University “G. d’Annunzio” of Chieti-Pescara The results presented suggest that SUMOylation can alter the local grants to Stefania Fulle. trafficking of KARs after OGD. However, SUMOylation does not Where applicable, the authors confirm that the experiments appear to play a major role in the loss of KARs during OGD; as described here conform with The Physiological Society ethical the KAR-EPSC never fully recovered after OGD. requirements. All errors given are standard errors of the mean. 1.Lee Y J et al. (2007), J Cereb Blood Flow Metab. 2(5):950-62. 2.Pei D S et al. (2005), J Neurosci Res. 1;82(5):642-9. PC204 3.Cimarosti H et al. (2008), Neuropharmacology. 54;2:280-289. 4.Martin S et al. (2007), Nature. 17;447(7142):321-5. The role of SUMOylation in Kainate Receptor trafficking during Oxygen Glucose Deprivation Where applicable, the authors confirm that the experiments S. Dennis and J. Mellor described here conform with The Physiological Society ethical requirements. University of Bristol, Bristol, UK Ischaemic brain damage is thought to be largely caused by excitotoxicity due to over-activation of glutamate receptors. The roles of NMDA and AMPA receptors in mediating this excito- toxic damage are well documented but the role of kainate

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Where applicable, the authors confirm that the experiments PC205 described here conform with The Physiological Society ethical requirements. Gut peptide hormones ghrelin and obestatin differentially effect structure and synaptic function of rat hippocampal neurones in vitro PC206 R.P. Murphy, K.J. Murphy and M. Pickering School of Biomolecular and Biomedical Sciences, University College Methamphetamine toxicity in PC12 cells is accompanied Dublin, Dublin 4, Ireland by an increase in BDNF: Possible neuroprotection Ghrelin and obestatin, both products of the ghrelin gene which A.M. Santos1,2, J. Kelly1 and K.M. Doyle2 have been implicated in regulating appetite, have also been 1Pharmacology and Therapeutics, NUI Galway, Galway, Ireland and shown to have pro-cognitive effects in vivo. However, it is not 2Physiology, NUI Galway, Galway, Ireland clear if cognitive effects result from direct effects on relevant brain structures (e.g. hippocampus), or what, if any, interac- Methamphetamine (MA) has been shown to induce toxicity in tion exists between both peptides. We examined the effect of vivo and in vitro [1] affecting in particular, but not only, both peptides, alone and in combination, on neuronal struc- dopaminergic neurons [2]. The molecular and cellular mecha- ture and synaptic function in-vitro, by exposing primary hip- nisms involved in this process remain to be clarified. Previously, pocampal neuronal cultures to a range of doses of ghrelin (0.05, in this laboratory, we found that repeated MA administration 0.5, 1.25, 5, 50 μM), obestatin or both in the medium for 48 increases BDNF levels in the frontal cortex and striatum of hours prior to testing. Results of all experiments are shown in Sprague Dawley rats [3], which may be an attempt to protect table 1. Neuronal structure was analysed by calculating the area the brain against MA toxicity. PC12 cells have dopaminergic under curve (AUC) of the Sholl plots (Sholl 1953). No dose of characteristics [4] and is a widely used model. The objective of ghrelin or obestatin had an effect on AUC, but a significant this study was to establish toxic doses of MA in PC12 cells and increase was seen at 5 μM dose when both peptides were co- to correlate apoptotic/necrotic morphology with cell viability applied. Additionally, while no ghrelin doses increased the as measured by MTT assay, and BDNF levels, measured by ELISA. length of the longest neurite, obestatin caused an increase at PC12 cells were seeded on poly-L-lysine coated well plates at a the 0.5 and 5 μM doses, and no increases were seen when both density of 5x 105/ml for ELISA and 2.5x 105/ml for morphol- peptides were co-applied. ogy and MTT analysis. Preliminary data demonstrated that MA Synaptic release rate was calculated from the half life of FM 1- did not affect morphology, cell viability or BDNF expression in 43 destaining with 65 mM KCl stimulation. Ghrelin at a dose PC12 cells at sub-millimolar concentrations. Cells were treated of 5μM decreased synaptic release rate, as did obestatin at 0.05, with MA (1mM, 3mM, 6mM or 15mM), staurosporine (STS) 0.5 and 50 μM doses. No effect on synaptic release was seen 2mM (positive control known to induce apoptosis) or medium when both peptides were co-administered. (negative control) for 24 hours. The results (see table) showed Whilebothpeptideswerefoundtohaveeffectsonhippocampal that methamphetamine reduced cell viability, induced apop- neurones, no simple dose response relationship or interaction totic and necrotic cell death and increased BDNF expression in wasfound,suggestinganunderlyingcomplexityinthesignalling PC12 cells. This study shows that MA induced toxicity is accom- ofthesepeptideswhichhasyettobeelucidated.Inparticular, panied by an increase in the expression of BDNF. Further eluci- the complex dose response to obestatin raises the possibility of dation of the mechanism by which MA reduces cell viability and more than one receptor for obestatin, which may have different induces cell death is needed, as is a better understanding of the affinitiesandwhichmayfunctionthroughinteractingsignalling role of BDNF in these events. pathways.Elucidatingthesepathwaysislikelytobekeytounder- standing the pro-cognitive effects of both peptides. Table 1.

Results are expressed as mean ± S.E.M. All data were analysed using one way ANOVA, followed by Student-Newman-Keuls test. *p<0.05 vs. Control, **p<0.001 vs. Control. Wallace, T.L., et al .(1999). J Neurosci. 19 (20): p. 9141-8. Jang, J., et al.(2007). Neural Plast. 2007: p. 29821. Santos, A.M. et al.(Inpress). J Med Sci All values expressed as mean ± SEM. * denotes p<0.05, ANOVA. Greene, L.A. and A.S. Tischler.(1976). Proc Natl Acad Sci U S A. 73(7): Sholl DA (1953) J Anat. 87, 387-406. p. 2424-8. This work was supported by Science Foundation Ireland. This study was supported by NUI Galway Scholarship.

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Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

PC207

In vivo behaviours of squid motor units during denervation of neighbouring units A. Packard stazione Zoologica Naples, La Garde-Freinet, France Figure 1. Detail of the mantle of a squid showing myogenic enhancement of motor unit end-organ activity resulting from DSS in the hours (18-40h) The chromatophore system of the squid Loligo vulgaris, run by following section of the nerve supply to surrounding skin. (Mature squid central motor neurones, generates images that can be recorded seen from left side, dorso-lateral view: LPN incompletely sectioned, right photographically (in the laboratory) at intervals while animals side of mantle (above) intact; dashed line = dorsal midline; yellow circle = are being maintained in their holding tanks. The poster reports ROI; below, selected spots in black: numbers = expanded size ratios relative to resting size (d); arrows indicate identical spots in a) to d). Temperature on image-analysis of two phenomena (supersensitivity, sprout- 20-22∞C) ing) visible in the output history of ‘residual’ chromatophore motor units in a partially denervated environment during the days following incomplete section of the pallial nerve on one side of the body [1,2]. Supersensitivity. The output amplitude of single units of multiply innervated dark chromatophores (spots) [3] is affected by their denervated neighbours through the intra- chromatophore coupling of a spot’s several muscle fibres (~25 on any one spot). The progress of this myogenic enhancement during the hours following section of the left pallial nerve (LPN) under light anaesthesia (<1% ethyl alco- hol in seawater) is seen in Figure 1. Whenever the animal changes colour, the ‘residual’ unit on the left side of the mantle (yellow ring) behaves synchronously with similar ones on the right side (upper in the figure); expanded amplitude of individual spots is low at 18h post-operation (a), Figure 2 Single ‘residual unit’ (see text) a) 5days, b) 16days post-operation while neuromuscular endings of neighbouring units are still Packard A (1995) J Physiol, 489: 134-135P intact, and increases as muscle denervation supersensitivity (DSS) develops to a maximum at 40h (b, c). Interestingly, Packard A (1995). In Abbott N.J., Williamson R. and L. Maddock, eds. Cephalopod Neurobiology. University Press, Oxford: pp.331-368 when the squid turns pale (d), DSS is suppressed through unknown mechanisms. Florey E (1966). Comp. Biochem. Physiol. 18, 305-324 Sprouting. Figure 2 shows a second residual unit that had been Euan Brown and staff of Stazione Zoologica “Anton Dohrn” and spared by LPN section, a) at 5 days post-operation, b) at 16 professional fishermen of the Bay of Naples. days. Over the intervening 11 days, the motor field of this unit nearly doubled in area through the capture of neighbouring Where applicable, the authors confirm that the experiments (denervated) spots of several size classes. Further enlarge- described here conform with The Physiological Society ethical ment of this unit did not occur in the remaining weeks before requirements. sacrifice.

PC208

Optimized voxel-based morphometry of the rat brain reveals cortical and hippocampal volume decline with age R.J. Kelly, C. Blau, E. O’ Hanlon, C.G. Connolly, C. Kerskens and M. Lynch Physiology, Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland Voxel-based morphometry (VBM) is commonly used for analysing differences in human brain volumes [1], but similar methodology has not been developed for use in the rat brain.

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In this study, an optimized VBM protocol has been developed for the rat brain and using this, we have undertaken an analy- PC209 sis of age-related changes in brain volume in rats. High resolution structural MRI images were obtained from Effects of Interleukin-6 on Glucagon Secretion and viability groups of young (N = 5) and aged (N = 7) rats using a Bruker 7 of the pancreatic a-cell line (a TC1-9) Tesla animal scanner (Bruker BioSpin, Ettlingen, Germany) and compared using FSL (FMRIB Software Library, 4.0) tools [2]. The M. Farrelly1,2, C. Murphy2 and P. Newsholme1 animals were anaesthetised before entering the magnet with 1 isoflurane (4% induction), delivered in oxygen. The isoflurane School of Biomedical and Biomolecular Science, UCD, Dublin, 2 was then reduced to the minimum level to keep the animal Ireland and Department of Biology, IT Tallaght, Dublin 24, Ireland asleep, with the depth of anaesthesia controlled by altering the There is a clear correlation between obesity and the develop- percentage of isoflurane in response to changes in respiratory ment of Type 2 Diabetes Mellitus1. The hallmarks of Type 2 rate. Images were skull-stripped using the brain extraction tool Diabetes Mellitus (T2DM) are insulin resistance, insulin insuf- (within MIPAV 4.0.2 software, [3]) and the grey matter parti- ficiency and hyperglycaemia. Normal endocrine regulation of tions were registered to the same stereotaxic space using an glucose homeostasis is lost in T2DM pancreatic due to β-cell in-house generic grey matter template image. The resulting and α-cell dysfunction and eventually pancreatic β-cell loss. images were averaged to create a study-specific template, to Obesity (BMI>30kg/m2)iscausedbyexcessadiposelipidstor- which the individual images were compared. Permutation- age, which is associated with a significant increase in various based non-parametric statistical analysis was undertaken. cytokines secreted by adipose tissue including IL-1β,TNF-α The data indicate that there was a significant reduction in vol- and IL-62. It is known that IL-6 is a pleiotropic cytokine that is ume in the hippocampus and cortex of aged, compared with secreted by many nucleated cells including adipocytes and is young, rats. Specifically, volume reduction was observed in area linked to insulin resistance and T2DM 3,4.Theeffectsofpro CA1 of the hippocampus, as well as in the motor areas M1 and inflammatory cytokines on nutrient metabolism in the insulin M2 of the cortex. secreting pancreatic β-cellhavebeenstudiedindepth5 in These findings indicate that this optimized VBM methodology Philip Newsholmes laboratory with results indicating a change can be used to analyse volumetric changes in the rodent brain. in β-cell function from insulin production to cell defense. Despite this there has been relatively little research into the effects of IL-6 or other pro-inflammatory cytokines on pancreatic α-cells. Exposure to the pleiotropic cytokine IL-6 or indeed other pro-inflammatory cytokines may result in loss of regulatory control of glucagon secretion in obese and T2DM patients. To determine the effects of IL-6 or β-cell secretory products on the functional integrity of pancreatic α-cell line, α TC1-9. α TC1-9 cells were incubated with various concentrations of glucose, glutamine and (sub lethal) combinations of IL-6, TNFα, IL-1β, IFN-γ, for various incubation times (from 0.5 to 24 hours). The cell media was analysed for glucose and glutamine to determine consumption. Cell contents were analysed for ATP, glutamate and GSH + GSSG as indicators to changes in metabolic function. Viability was determined by the WST-1 assay. Glucagon levels in the culture media were also determined. Coronal image of the rat brain highlighting areas of significant tissue decline α TC1-9 cells viability and function was determined at high glu- in the aged rat brain compared to young. cose levels (up to 25mM) and high glutamine levels (up to Ashburner, J. and Friston, K.J. (2000) Voxel-Based Morphometry: The 5mM). No effect on cell viability in high glucose or high gluta- Methods. Neuroimage 11: 805 821. mine was determined. IL-6 at sub lethal concentrations did not FMRIB Software Library tools freely available at alter cell viability but altered parameters of cell metabolism http://www.fmrib.ox.ac.uk/fsl and glucagon secretion Medical Imaging Processing, Analysis and Visualization, available at IL-6 alters parameters of alpha cell metabolism and function. http://www.mipav.cit.nih.gov/ Natalie Moroz, Ming Tong, Lisa Longato, Haiyan Xu, Suzanne M. de la Monte. Limited Alzheimer-Type Neurodegeneration in Experimental This study funded by Science Foundation Ireland. We acknowl- Obesity and Type 2 Diabetes Mellitus edge the assistance of colleagues in the Trinity Centre for High Journal of Alzheimer’s Disease Volume 15:1 (September 2008) Performance Computing. Leonid Roytblat, Maxim Rachinsky, Allan Fisher, Lev Greemberg, Yoram Where applicable, the authors confirm that the experiments Shapira, Amos Douvdevani and Simon Gelman described here conform with The Physiological Society ethical Raised Interleukin-6 Levels in Obese Patients requirements. Obesity Research (2000) 8, 673–675; doi: 0.1038/oby.2000.86

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Joachim Spranger, Anja Kroke, Matthias Möhlig, Kurt Hoffmann, control males, as compared to young mice. Age also diminished Manuela M. Bergmann, Michael Ristow, Heiner Boeing, and Andreas the levels of BCL2 (p<0.01), in this animals. In the case of SAM- F.H. Pfeiffer R1 animals a significantly increased BAX as found (p<0.05), as Inflammatory Cytokines and the Risk to Develop Type 2 Diabetes: compared to SAM-R1 young controls. The expressions of NFkB Results of the Prospective Population-Based European Prospective and BCL2 were similar in old and young SAM-R1 mice. The Investigation into Cancer and Nutrition (EPIC)-Potsdam Study, Diabetes expression of BAD on SAM-P8 and SAM-R1 mice, showed no 52: 812-817 significant differences between young and old animals. Exoge- Aruna D. Pradhan; JoAnn E. Manson; Nader Rifai; Julie E. Buring; Paul nous GH administration showed a significant decrease in lev- M. Ridker els of NFkB (p<0.01), BAX (p<0.01) and BAD (p<0.05). The expression of BCL2 was not significantly affected with the treat- C-Reactive Protein, Interleukin 6, and Risk of Developing Type 2 Dia- betes Mellitus, JAMA. 2001;286(3):327-334 ment in old SAM-P8. Our results suggest that the inflammatory process and some mediators of apoptosis could contribute to AoifeKiely,NevilleHMcClenaghan,PeterRFlatt,PhilipNewsholme,Pro- the observed alterations of the cardiovascular system associ- inflammatory cytokines increase glucose, alanine and triacylglycerol ated with aging and that GH may play a potential protective β utilization but inhibit insulin secretion in a clonal pancreatic -cell line effect during this process. J Endocrinol 2007 195: 113-123 Zhang Y & Herman B. (2002). Mech Ageing Dev 23, 563-565.

Abbreviations: IL, Interleukin; GSH, Glutathione; RIA, Radio Harman D et al. (1956). J Gerontol 11, 298-300. Immunoassay Chung H Y et al. (2001). An NY Acad Sci 928, 327-335.

Where applicable, the authors confirm that the experiments This work has been possible trough a RETICEF RD 06/0013, grant described here conform with The Physiological Society ethical from the Instituto Carlos III and another of SAF 2007 66878- requirements. C02-01. Katherine Forman was supported by Pre-Doctoral Fel- lowship, Chile PC210 Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Apoptosis and inflammation in cardiovascular aging: effect requirements. of chronic treatment with growth hormone in senescence- accelerated mice PC211 K. Forman1, E. Vara2, C. García2, R. Kireev1, S. Cuesta1 and J. Tresguerres1 Neonatal lipopolysaccharide exposure delays puberty and alters hypothalamic kisspeptin and kiss1r expression in the 1 Physiology, University Complutense of Madrid, Madrid, Spain and female rat 2Biochemistry and Molecular Biology, University Complutense of 1 1 1 1 1 Madrid, Madrid, Spain A. Knox , X. Li , J. Kinsey-Jones , E. Wilkinson , X. Wu , Y. Cheng1, S. Milligan1, S. Lightman2 and K. O’Byrne1 Aging induces deleterious effects in several organs, and is asso- 1Department of Anatomy and Human Sciences, King’s College ciated with complex changes in cardiovascular structure and London, London, UK and 2Henry Wellcome Laboratory for function. This process might be due to damage caused by Integrative Neuroscience & Endocrinology, University of Bristol, inflammatory mediators, free radicals and apoptosis (1-3). The Bristol, UK purpose of this study was to investigate the effect of aging on Immunologicalchallengeexperiencedinearlylifecanhavelong- different parameters related to apoptosis and NFkB (nuclear termprogrammingeffectsonthehypothalamic-pituitary-adre- factor kappa B) expression in hearts from male senescence- nal (HPA) axis which permanently influence the stress response. accelerated mice (SAM-P8) and its controls male senescence- Similarly, neonatal exposure to immunological stress enhances accelerated-resistant (SAM-R1). Also to study the influence of stress-inducedsuppressionofthehypothalamic-pituitarygonadal chronic administration of Growth Hormone (GH) on these (HPG)axisinadulthood,butmayalsoaffectearlierdevelopment, parameters in old SAM-P8 mice. Forty male mice were used. including the timing of puberty. To investigate the timing of the Animals were divided into five experimental groups, two old critical window for this programming of the HPG axis neonatal groups of 10 month of age (SAM-P8/SAM-R1), one GH group femaleratswereinjectedwithlipopolysaccharide(LPS,50μg/kg of also 10 months age (SAM-P8), that was treated with the hor- ip)orsalineonpostnataldays3+5,7+9,or14+16andmonitored mone (2 mg/kg/day/s.c.) and two young groups, of 2 months forvaginalopeningandfirstvaginaloestrusasmarkersofpuberty. of age (SAM-P8/SAM-R1) that were used as young controls. We also investigated the effects of neonatal programming on After 30 days of treatment, animals were sacrificed by decap- thedevelopmentoftheexpressionpatternsofkisspeptin(Kiss1) itation, and hearts were collected. The expression of BCL2-asso- and its receptor (Kiss1r) in hypothalamic sites known to contain ciated death promoter (BAD), BCL2-associated X protein (BAX), kisspeptin-expressing neuronal populations critical to repro- B-cell lymphoma 2 (BCL2) and NFkB, were determined by real- ductivefunction:themedialpreopticarea(mPOA)andthearcu- time reverse transcription polymerase chain reaction. Results atenucleus(ARC)inneonatally-stressedanimals.Wedetermined were submitted to a two way ANOVA statistical evaluation using that the critical period for a significant delay in puberty due to the Statgraphics program. The expression of NFkB (p<0.05) and neonatal LPS exposure is before 7 days of age in the female rat, BAX (p<0.01) were significantly increased in the old SAMP-8

188P Poster Communications and demonstrated that Kiss1, but not Kiss1r mRNA, expression in the mPOA is down-regulated in pre-pubertal females. These PC213 data suggest that the mPOA population of kisspeptin neurones play a pivotal role in controlling the onset of puberty, and that The Effect of Clothing and Cultural Practice on Bone Mineral their function can be affected by neonatal stress. Density and bone turnover markers in Kuwaiti Pre- This work was supported by the Wellcome Trust and BBSRC (UK). Menopausal Women Where applicable, the authors confirm that the experiments F.K. Alotaibi1, M. Al-Bader1, K. Al-Shoumer2, F. Al-Yatama3, described here conform with The Physiological Society ethical M. Oommen3, V. Nair2 and A. Ali2 requirements. 1physiology, kuwait university, faculty of medicine, Kuwait, Kuwait, 2Medicine, Faculty of Medicine, Kuwait University, Kuwait, Kuwait and 3Medical Laboratory Sciences, Faculty of Allied Health, Kuwait PC212 University, Kuwait, Kuwait Eicosapentaenoic acid administration stimulates muscle Low bone mass is frequently encountered than expected in the regeneration and inhibits atrogenes gene expression in the Mediterranean and Gulf countries, which are sunny most of the gastrocnemius of arthritic rats time. As Kuwait is one of these sunny countries, we aimed to E. Castillero, A. Martín, M. López-Menduiña, M. Villanúa and investigate the effect of outfitting style on bone mineral den- A. López-Calderón sity (BMD) and on markers of bone turnover in middle-aged pre-menopausal Kuwaiti women. Physiology. Faculty of Medicine, Complutense University of Madrid, Two groups of pre-menopausal single Kuwaiti females (20-35 Madrid, Spain yrs of age; 20 per study) were recruited. Written informed con- Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated sent was obtained from each subject. A questionnaire was filled fatty acid which is present in high concentration in fish oil. Adju- in by all subjects to obtain history and anthropometric data. vant-induced arthritis is an animal model of cachexia that Group I (GI) included females wearing black veil since puberty induces muscle wasting by increasing the ubiquitin-protea- and not exposed to the sun. Group II (GII) included females some pathway. The aim of this work was to elucidate whether wearing western clothes. Fasting blood samples were collected the effect of EPA on arthritis-induced muscle wasting is medi- for measurement of baseline biochemistry, minerals, and mark- ated by changes on muscle proteolysis, regeneration and ers of bone turnover (osteocalcin, N-telopeptide of type I col- inflammation. For this purpose, arthritis was induced in male lagen [sPINP] and C-telepeptide of type I collagen [sICTP]). BMD Wistar rats by an intradermal injection of Freund’s adjuvant. was measured by dual energy x-ray absortiometry (DEXA) at Three days after the injection, control and arthritis rats were the lumbar spine, femoral neck and total body. Statistical analy- divided in two experimental groups. One group was daily gav- sis was performed using SPSS 15. aged with EPA (1g/kg bw) and the other was gavaged with The two subjects were matched for age mean±SEM (GI versus coconut oil (1g/kg bw). Rats were humanly killed after 12 days GII) (22.5 ± 0.48 vs. 22.3 ± 0.49). The mean total body BMD of of treatment. EPA administration to arthritic rats ameliorated the two groups were similar (BMD1.16 arthritis score and hindpaw swelling (P<0.01). Arthritis ± 0.02 vs. 1.13 ± 0.015 g/cm2), T-score of total body (0.405 decreased skeletal muscle weight (P<0.01), whereas EPA admin- ±0.25 vs. 0.050±0.19). However, BMD of L2-L4 spine was signi- istration reduced this effect (P<0.05). EPA prevented arthritis- ficantly lower in veiled (GI) compared with non-veiled (GII) induced increase of TNF-alpha gene expression both in liver and females (T-score of L2-L4: -1.294 ±0.33 vs. -0.06 ±0.24, p<0.01). in gastrocnemius muscle (P<0.01). Arthritis increased the atro- As for bone markers, sPINP and sICTP were significantly raised genes MAFbx and MuRF-1 mRNA in gastrocnemius, and this in veiled (GI) compared with non-veiled (GII) (p<0.01 and effect was prevented by EPA (P<0.01). Both EPA and arthritis p<0.002, respectively). increased differentiation marker myogenin mRNA and protein Bone mineral density is decreased in young pre-menopausal (P<0.01). Muscular proliferating cell nuclear antigen (PCNA) Kuwaiti females wearing veils compared with those without mRNA and protein were also increased in arthritic and EPA veils. They have evidence of raised bone turnover as shown by treated rats (P<0.01). These data suggest that EPA exerts an the elevation of markers of bone formation and resorption. It antiinflammatory effect on adjuvant-induced arthritis, and it is therefore essential to assess vitamin D status in both groups attenuates muscle wasting by inhibiting proteolysis and stim- to verify its link with the partial difference in sun exposure. ulating muscle regeneration. CollegeofGraduateStudiesGrant#YM04/08,KuwaitUniversity This work was supported by CYCYT (BFU, 2006-11899)a grant Where applicable, the authors confirm that the experiments to Estibaliz Castillero (Gobierno Vasco, BFI06.31), and a grant described here conform with The Physiological Society ethical to María López-Menduiña (BES-2007-16001). requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements.

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(NHERF1). Their activation and function in vascular smooth PC214 muscle remain to be elucidated. We have investigated the activation of these proteins in intact Wistar rat aorta by using a Multiple Ca2+ stores are involved in phenylephrine phospho-ERM antibody and Western-Blot. The role of moesin stimulation of rat aortic smooth muscle and EBP50 in the regulation of vascular tone was investigated 1 1 1 2 3 in mesenteric arteries after inhibition of moesin and EBP50 L. Foia , V. Toma , D. Forna , A. Indrei and D. Haba expression by siRNA transfection. Potential partners of EBP50 1Biochemistry, University of Medicine and Pharmacy, Iasi, Romania, were screened by LC/MS-MS analysis of gel bands obtained after 2Anatomy, University of Medicine and Pharmacy, Iasi, Romania co-immunoprecipitation with EBP50. and 3Radiology, University of Medicine and Pharmacy, Iasi, Romania Stimulation with noradrenaline (NA) 1μM rapidly increased ERM phosphorylation, which was maximum at 2 min and slightly Previous studies indicate that in pulmonary and coronary arte- decreased at 10 min. ERM proteins were also phosphorylated rial smooth muscle cells beside IP3 and ryanodine sensitive by 100 mM KCl stimulation with a maximal phosphorylation Ca2+ pools, acid-filled Ca2+ store may play an important role after 30 s and a fast decrease after longer stimulation. The in vasoconstriction. The aim of this study is to characterize the inhibitor of Rho kinase (Y27632 10 μM), totally inhibited ERM Ca2+ pools involved in phenylephrine (PHE) stimulation of rat phosphorylation measured after 2 and 10 min of NA stimula- aortic smooth muscle cells. Experiments were carried out using tion but did not affect the rapid increase (30 s) in ERM phos- Ca2+-imaging technique on cultured rat aortic smooth mus- phorylation induced either by NA or KCl. The rapid increase in cle cells and force contraction of desendothelized rat aortic ERM phosphorylation induced by KCl stimulation was totally ring. Administration of PHE (1 microM) in Ca2+ free saline on inhibited by nimodipine, a blocker of voltage-gated calcium cultured aortic smooth muscle cells induces a transitory ele- channels. Furthermore, ionomycin, a calcium ionophore, vation of [Ca2+]i by 561 + 3.6 nM (n = 38 cells). Following one induced a rapid increase in ERM phosphorylation. Moreover, hour pretreatment with bafilomycin (1 microM), a well known GÖ6983, an inhibitor of calcium-activated PKC, totally inhib- disrupter of acid-filled Ca2+ stores, the [Ca2+]i diminished up ited the fast ERM phosphorylation evoked either by NA or KCl to 174 + 2.8 nM (n = 47 cells). The blockade of ryanodine recep- stimulation but not the phosphorylation induced by a longer tors with ryanodine (10 microM; 15 min preincubation), or the stimulation with NA. Knock-down of moesin potentiated the IP3 receptors with 2-APB (2-aminoethyl diphenylborate - 100 noradrenaline-evoked contraction but not the KCl-evoked con- microM; 15 min preincubation) also reduce the PHE-induced traction. A similar effect was observed in EBP50 knock-down elevated [Ca2+]i up to a level of 352 + 2.9 nM (n = 26) and 231 arteries, indicating that moesin could exert its effect through + 2.3 nM (n = 34), respectively. Meanwhile 1 h pretreatment an interaction with EBP50. Immunoprecipitation of EBP50 in with bafilomycin (1 microM) and 15 min with 2-APB (100 noradrenaline-stimulated arteries allowed to identify its inter- microM) totally abolished the elevation of cytoplasmatic Ca2+ action with moesin and several other proteins involved in induced by PHE (n = 52). Experiments carried out on cytoskeleton regulation, especially in focal adhesion formation, desendothelized rat aorta rings reveal that 1 h treatment with that did not appear in unstimulated arteries. bafilomycin (1 microM) inhibits the PHE-induced contraction These results indicate that ERM proteins are phosphorylated in with 58 + 3.2% (n = 6), while 45 min treatment with ryanodine vascular smooth muscle in response to agonist or depolarisa- (10 microM) or 2-APB (100 microM) reduce the contractions tion stimulation. This phosphorylation consists in a fast calcium elicited by PHE with 31 + 2.8% (n=6) and 47 + 3.6% (n = 6), dependent phosphorylation induced by PKC and a much slower respectively. Concomitant treatment with bafilomycin (1 calcium independent phosphorylation induced by Rho kinase. microM; 1 h), and 2-APB (100 microM; 45 min) totally abol- Moesin activation is associated with decreased NA-evoked con- ished PHE-induced contraction (n = 6). It may be thus concluded traction, probably throught interaction with EBP50 and that PHE primary recruits Ca2+ from acid-filled and IP3-sensi- cytoskeleton elements. tive Ca2+ stores which, in turn, activate ryanodine receptors through calcium-induced calcium-release mechanism. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

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PC215 Effect of ATP on interstitial cells of Cajal isolated from the rabbit corpus cavernosum Ezrin, Radixin, Moesin: from activation to vascular tone modulation G.P. Sergeant, C. Doyle, M.A. Hollywood, N.G. McHale and K.D. Thornbury N. Baeyens1, S. Horman2, D. Vertommen3, M. Rider3 and N. Morel1 Smooth Muscle Research Centre, Dundalk Institute of Technolgy, Dundalk, Ireland 1Cellular physiology, UCLouvain, Bruxelles, Belgium, 2Cardiovascular pathology, UCLouvain, Bruxelles, Belgium and 3de Interstitial cells of Cajal (ICC) are thought to be pacemakers in Duve Institute, UCLouvain, Bruxelles, Belgium the gastrointestinal tract, where they drive gastric and intestinal motility1. Similar cells have been identified in erectile tis- ERM (Ezrin, Radixin & Moesin) proteins link the actin cytoskele- sue, although their role here is unknown2. In the present study ton to membrane proteins either directly or throught EBP50 190P Poster Communications we have isolated ICC from the rabbit corpus cavernosum in order to study the effect of exogenous ATP on their membrane PC217 currents. Rabbits were killed by lethal injection with pentibar- bitone and their penises removed. The tunica albuginea was Role of Ca2+-activated Cl- channels and Ca2+ influx in opened and the corpus cavernosum extracted and cut into 1 generating spontaneous intracellular Ca2+ waves in mm3 pieces which were stirred in an enzyme mixture, followed myocytes isolated from rabbit corpus cavernosum by washing in Hanks Ca2+ free saline to release single ICC and G.P. Sergeant, V. Cagney, M.A. Hollywood, N.G. McHale and smooth muscle cells (SMC). The cells were then plated for study K.D. Thornbury using the perforated patch clamp technique, using Cs+ rich pipette solution. When cells were held at –60 mV, ATP (20 μM) Smooth Muscle Research Centre, Dundalk Institute of Technolgy, evoked large inward currents when the pipette and bath [Cl-] Dundalk, Ireland were symmetrical (135 mM). Under these conditions the ATP The corpus cavernosum restricts blood flow through the penis ± responses were reversibly reduced from 1105 325 pA to 377 to maintain penile detumescence until an erectile stimulus ± μ 97 pA by suramin (100 M, a broad spectrum purinergic causes it to relax. The detumescent state is partly maintained receptor antagonist (n=7, p <0.05). The selective P2Y recep- by a myogenic mechanism resident in the smooth muscle bun- μ tor agonist, 2-methylthio ADP (1 M), mimicked the effect of dles lining the corporal sinuses (1). A previous study found that ATP. However, while the selective P2Y1 receptor antagonist isolated rabbit corpus cavernosum myocytes fire spontaneous MRS 2500 (100 nM) effectively reduced the responses to 2- transient inward currents (STICs) mediated by Ca2+ -activated ± ± methylthio ADP (from 460 212 pA to 110 82 pA, n=5, p < Cl- channels (2). Recently, we have shown that these cells also 0.05), it had a variable effect on the ATP responses, reducing fire regular intracellular Ca2+ waves mediated by Ca2+-release them in only 2 out of 5 cells. This suggests that 2-methylthio from intracellular stores (3). The aim of the present study was ADP mediated its effects via P2Y1 receptors, but that ATP may to determine if there was any contribution of voltage-depend- act on more than one receptor subtype. ent Ca2+ channels to the Ca2+ waves in these cells. Male New μ Also, niflumic acid (100 M), a blocker of both Ca2+-activated Zealand white rabbits were humanely killed and cells were Cl- currents and non-selective cation currents reduced the ATP- freshly dispersed from the corpus cavernosum as described pre- ± ± evoked currents from -536 143 pA to -60 26 pA (n=6, p viously (2). Cells were loaded with Fluo-4 am for study of cytoso- <0.05). To test whether ATP-evoked currents were mediated lic Ca2+ signals using a spinning disk confocal microscope at by Cl- or non-selective cation channels, the effects of ATP and acquisition rates of 5-50 frames s-1 (3). Under unstimulated caffeine (previously shown to evoke a Ca2+-activated Cl- cur- conditions cells developed regular Ca2+ waves at a frequency rent in these cells) were compared. When cells were held at - of 14 ± 4.53 min-1 (mean ± s.e.m) and amplitude of 1.20 ± 0.27 60 mV in symmetrical Cl- solutions, as above, both ATP and caf- ΔF/F0, which were abolished in Ca2+-free bath solution (n = 5, feine evoked inward currents. However, when ECl was adjusted p < 0.05, paired t test). Similarly, in 6 cells, nifedipine (1 μM) to –40 mV, by reducing intracellular [Cl-], and cells then held reduced frequency from 13.83 ± 2.12 to 2.17 ± 1.37 min-1 (p at –10 to –20 mV, ATP still evoked an inward current, but the < 0.05) and amplitude from 0.99 ± 0.15 to 0.44 ± 0.23 ΔF/F0 caffeine-evoked current shifted to the outward direction (n=6). (p < 0.05). At acquisition rates of 50 frames s-1 it was possible When the external [Na+] was reduced from 130 to 13 mM (by to also discriminate Ca2+ sparks, occurring during the periods replacement with equimolar NMDG) the ATP current also between waves. Interestingly, sparks were more resistant to shifted in the outward direction (n=3), whereas the caffeine nifedipine treatment than waves. In contrast to the effect of evoked current was little affected. nifedipine, the L-type Ca2+ channel agonist, FPL-64176 (300 These data show that ATP can evoke inward currents in ICC from nM), induced a rapid rise in intracellular Ca2+, followed by a the corpus cavernosum that are likely to mediated by non-selec- series of Ca2+ oscillations in 4 of 5 cells, while in the 5th cell a tive cation channels. sustained rise in Ca2+ was observed. The Ca2+-activated Cl- Sanders KM, Ward SM. Interstitial cells of Cajal - a new perspective on channel blockers anthracene-9-carboxylic acid (9-AC) and nif- smooth muscle function. J. Physiol. 2006; 576:721-726. lumic acid both reduced the amplitudes of Ca2+ waves (9-AC HashitaniH,SuzukiH.IdentificationofinterstitialcellsofCajalincorpo- 1mM: from 0.58 ± 0.14 to 0.19 ± 0.05 ΔF/F0, n=5, p <0.05; nif- raltissuesoftheguinea-pigpenis.Br.J.Pharmacol.,2004;141:199-204. lumic acid 100 μM: from 1.92 ± 0.61 to 0.16 ± 0.10 ΔF/F0, n=5, The authors acknowledge grant support from Science Foun- p <0.05). dation Ireland (BIMF377) and Diabetes UK (0002695). In conclusion, spontaneous Ca2+ waves in rabbit corpus cavernosum myocytes appear to depend not only on store- Where applicable, the authors confirm that the experiments released Ca2+, but on Ca2+ influx via L-type Ca2+ channels. We described here conform with The Physiological Society ethical propose that release of Ca2+ from stores activates plas- requirements. malemmal Ca2+-activated Cl- channels, which then cause membrane depolarisation that results in Ca2+ influx via L-type Ca2+ channels. This mechanism is likely to contribute to the spontaneous detumescent tone widely reported in corpus cavernosum smooth muscle. Andersson K-E & Wagner G (1995). Physiology of penile erection. Phys. Rev. 75:191-218.

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Craven M, Sergeant GP, Hollywood MA, McHale NG, Thornbury KD. mN.s produced by ATP (10 μM) in paired experiments (p<0.05, (2004). Modulation of spontaneous Ca2+-activated Cl- currents in the n = 14). rabbit corpus cavernosum by the nitric oxide-cGMP pathway. J Physiol. These results suggest that ATP could act as an excitatory neu- 15;556:495-506. rotransmitter and that these effects are likely to be mediated Sergeant GP, Craven M, Hollywood MA, McHale NG, Thornbury KD. via P2Y receptors. (2008). Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Mod- Brading A. F. (1999). The physiology of the mammalian urinary out- ulation by Nitric Oxide and cGMP. J. Sex. Med. 2008 [Epub ahead of flow tract. Exp. Physiol. 84: 215-221. print] PMID: 19138373. Pinna C, Ventura S, Puglisi L, Burnstock G. A pharmacological and his- The authors acknowledge grant support from Science Foun- tochemical study of hamster urethra and the role of urothelium. Br J dation Ireland (BIMF377), Diabetes UK (0002695) and Council Pharmacol. 1996 Oct;119(4):655-62. of Directors through the National Development Plan (ROI). The authors are grateful for support from the NIH (RO1 Where applicable, the authors confirm that the experiments DK68565), HRB (PD/2005/4 & RP/2006/127). Sonia Kadima is described here conform with The Physiological Society ethical supported by a Strand 1 Award (Council of Directors, ROI). requirements. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. PC218

Excitatory effects of ATP on rabbit urethra smooth muscle PC219 G.P.Sergeant, S. Kadima, M.A. Hollywood, K.D. Thornbury and N.G. McHale Modulation of trabecular meshwork cell function by cross- Smooth Muscle Research Centre, Dundalk Institute of Technolgy, linked actin networks Dundalk, Co. Loth, Ireland S. O’Reilly, R. Williams, D. Brotchie and I. Grierson Myogenic tone in urethra smooth muscle is regulated by several neurotransmitters, including noradrenaline, acetylcholine University of Liverpool, Liverpool, UK and nitric oxide (Brading et al., 1999). ATP is also considered to Worldwide glaucoma is a leading cause of irreversible blind- be an important neurotransmitter in the urethra (Pinna et al., ness and its incidence is increasing as the population ages as it 1996), however its effects have not yet been fully characterised is an age-related disease. Its onset is insidious as it rarely causes and therefore it’s physiological role remains unclear. The pur- symptoms and many patients are not aware of any visual field pose of the present study was to characterise the effects of ATP loss. Primary Open Angle Glaucoma (POAG) is the main type of on the contractile activity of rabbit urethral smooth muscle. Glaucoma, next being secondary angle closure glaucoma and New Zealand white rabbits were humanely killed and mechan- is an economically significant disease. Chronic raised intraoc- ical recordings were made from strips of proximal urethra (8 x ular pressure leads to damage at the optic nerve head with con- 1 x 1 mm) using a multi channel Myobath system. Muscle strips sequent visual field loss and little is known about the precise were perfused with warmed Krebs solution bubbled with 95% underlying pathologic mechanism(s). Cross-Linked Actin Net- O2-5% CO2 which contained atropine (1 μM), phentolamine (1 works (CLANs) are triangular polygonal arrangements com- μM) and NG-Nitro-L-arginine (NO-ARG, 100 μM). Strips were prised of actin; these form geodesic dome arrangements. We adjusted to a tension of 2–4 mN and allowed to equilibrate for have recently demonstrated that bovine TM cells exposed to 50 min before experimentation began. dexamethasone upregulate CLAN incidence far more robustly Exogenous application of ATP (10 μM) induced robust con- compared to human cells, this gave us a model to probe func- tractions of the muscle strips. These responses typically con- tional affects of CLANs on TM cells. The aim of this study was sisted of an increase in tone, superimposed upon which were to determine the functional effects of CLANs in the bovine TM transient phasic contractions that varied in frequency and and also to determine to heat shock protein response. Primary amplitude. The contractile effects were quantified by measur- TM cells were cultured from bovine eyes (obtained from an ing the total area under the trace (mN.s). In 45 muscle strips abattoir) and we employed a collagen contraction model to ATP increased the tone from 43.6 ± 20.8 to 163 ± 40.1 mN.s examine the effect of CLANs on cell contraction. Moreover we (p<0.05). The ATP induced contractions were inhibited by the examined the effects of CLANs on cell viability and apoptosis broad spectrum purinergic inhibitor suramin (100 μM) from 95 using standard techniques. In addition we determined the ± 18 to 37 ± 16 mN.s (n=17, p<0.05) but were not affected by expression of Heat shock proteins 70, heat shock cognate 70 the P2X receptor selective antagonist NF279 (1 μM, 99 ± 31 as well as the enzyme manganese superoxide dismutase mN.s under control conditions versus 101 ± 21 mN.s in its pres- (MnSOD) using Western Blotting and the inflammatory ence, p>0.05). In contrast, the ATP effects were greatly atten- cytokine Interleukin-6 (IL-6) by E.L.I.S.A. Results showed that uated by the P2Y1 receptor antagonist MRS2500 (100 nM) and CLANs inhibited matrix contraction in the 2D model but not 3D were mimicked by the P2Y receptor agonist, 2-methylthio ADP model of contraction (P<0.01). There was no significant increase (2-MeSADP). For example, in 18 muscle strips the mean ATP in apoptosis in association with CLAN incidence. There was a evoked contraction was reduced from 118 ± 23 to 65 ± 15 mN.s significant increase in expression of inducible (ten fold change) by MRS2500 (p<0.05) whereas 2-MeSADP (1 μM), induced a Hsp70 that was time-dependant and also hsc70 (P<0.001). contraction measuring 372 ± 108 mN.s compared to 153 ± 44 MnSOD expression was also increased in response to CLANs as

192P Poster Communications was the expression of inflammatory cytokine IL-6 as measured using ELISA (P<0.01). These data demonstrate a functional impairment of TM cells in response to CLANs and that it is not related to cellular death. Moreover, there is significantly increased expression of Hsps, antioxidant enzyme and a proinflammatory cytokine, that may impinge on cellular function.

CLAN in Trabecular meshwork cell imaged by confocal microspcopy. The usual arrangemnt of F-actin is that of stress fibres of varying length.

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Clark, A.F., Wilson, K., McCartney, M.D., Miggans, S.T., Kunckle, M., blood vessels where their branches made contacts with the ves- Howe, W Glucocortcoid-induced formation of cross-linked actin net- sel wall. In the detrusor, ICC were found on the edge of smooth works in human trabecular meshwork cells Invest Ophthalmol Vis Sci muscle bundles and the contacts between SMC and ICC were 1994 35: 281-294. typically 50-100nm. ICC were frequently associated with intra- Clark, A.F., Lane, D., Wilson, K., Miggans, S.T., McCartney, M.D Inhibi- mural nerves, again typically within 100nm. tion of dexamethasone-induced cytoskeletal changes in cultured Ultrastructurally, ICC contained mitochondria, rough and human trabecular meshwork cells by tetrahydrocortisol Invest Oph- smooth endoplasmic reticulum, thin and intermediate fila- thalmol Vis Sci 1996 37: 805-813 ments, caveolae, Golgi apparatus, free ribosomes and cyto- Clark, A.F., Brotchie, D., Read, A.T., Hellberg, P., English-Wright, S., plasmic vesicles. They were distinct from fibroblasts by the pres- Pang, I.H., Ethier, C.R., Grierson, I Dexamethasone alters F-actin ence of a basal lamina and differed from smooth muscle cells architecture and promotes cross-linked actin network formation in the absence of thick filaments and dense bodies. in human trabecular meshwork tissue Cell Motil Cytoskeleton 2005 The ultrastructural characteristics of ICC in the guinea-pig blad- 60: 83-95. der were characterized with transmission electron microscopy Urayma, S., Mursh, M.W., Retsky, J., Madonna, M.B., Straus, D., Chang, and were consistent with those of gut ICC. ICC were positively E.B Dexamethasone protection of rat epithelial cells against oxidant identified with c-Kit antibody labeling in the lamina propria and injury is mediated by induction of heat shock protein 72 J Clin Invest detrusor regions and were closely associated with each other, 1998 102: 1860-1865. nerves and smooth muscle cells, consistent with previous published work (Davidson and McCloskey, 2005) at the light micro- Fight for Sight. scope level. Where applicable, the authors confirm that the experiments Cajal SR (1911) Histologie du systeme nerveaux de l’homme et des ver- described here conform with The Physiological Society ethical tebres, vol. 2. Paris: Maloine. 891-942. requirements. Sanders KM (1996) Gastroenterology 111, 492–515 Brading AF & McCloskey KD (2005). Nat Clin Pract Urol. 2: 546-554. Davidson RA & McCloskey KD (2005) J Urol 173, 1385–1390 PC221 This work was funded by the Wellcome Trust (grant Ultrastructural characterization of interstitial cells of Cajal 074591/Z/04Z). from the guinea-pig bladder Where applicable, the authors confirm that the experiments R.M. Cunningham, P. Larkin and K.D. McCloskey described here conform with The Physiological Society ethical requirements. Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, Northern Ireland, UK ICC were first discovered by Ramon y Cajal in the intestine in 1911 using methylene blue and Golgi staining techniques (Cajal, PC222 1911) and are now established as pacemakers and mediators of neurotransmission (Sanders, 1996). It was previously Investigation of the ultrastructural properties of interstitial believed that ICC were present in only the gastrointestinal tract cells of Cajal from the guinea-pig urethra but there is now convincing evidence that the urinary tract, including the urethra, urinary bladder, renal pelvis and ureters M.C. Doran, P. Larkin and K.D. McCloskey also contain populations of cells with many characteristics of Centre for Cancer Research and Cell Biology, Queen’s University ICC, though these studies are at a comparatively early stage Belfast, Belfast, UK (Brading and McCloskey, 2005). The aim of the present study was to: identify interstitial cells of Cajal (ICC) in the wall of the Interstitial cells of Cajal (ICC) have been identified in the wall guinea-pig bladder using transmission electron microscopy and of the rabbit urethra with c-Kit antibodies (Lyons et al, 2007) antibodies to the Kit receptor conjugated to nanogold parti- and share morphological characteristics with ICC from the gas- cles; to characterize the ultrastructural properties of bladder trointestinal tract. These cells exhibit spontaneous electrical ICC and to examine their relationships with nerves and smooth and Ca2+-activity (Sergeant et al, 2000) and may act to con- muscle cells. trol contractility and tone of the urethral smooth muscle. ICC Bladders were removed from guinea-pigs (n=4), killed by cer- are ultrastructurally distinct from smooth muscle cells and fibro- vical dislocation in accordance with Schedule 1, UK Home Office blasts and the aim of the present study was to examine the regulations. Bladder tissues were prepared for transmission ultrastructural properties of urethral ICC with transmission elec- electron microscopy using standard protocols. tron microscopy. Kit-positive ICC were identified in sections from the lamina pro- Bladders and urethras were removed from guinea-pigs (n=4), pria and detrusor, consistent with previous findings with con- killed by cervical dislocation in accordance with Schedule 1, UK focal microscopy (Davidson and McCloskey, 2005). In the lam- Home Office regulations. The proximal urethra was prepared ina propria, ICC formed an interconnecting network of for transmission electron microscopy using standard protocols. branched cells and were also found in close proximity to small We have previously shown that c-Kit-positive ICC are present within the muscularis of the rabbit urethra (Lyons et al, 2007). In the present study, the muscularis region of the urethra was studied with electron microscopy. Cells with the typical properties of ICC were identified in the circular and longitudinal

194P Poster Communications smooth muscle layers, both on the boundary of the smooth had the mucosal layer removed (8.1±0.9mN, 2.8±1.7 per muscle bundles and within the bundles. ICC-like cells were elec- minute, p>0.05, n=24 strips from 12 animals, unpaired t-test). tron dense, contained thin and intermediate filaments, had a Gap 27, a Cx43 gap junction blocker transiently inhibited spon- basal lamina, contained mitochondria, rough and smooth endo- taneous activity in both intact and denuded strips (n=5 from 3 plasmic reticulum, Golgi complexes and membrane caveolae. animals). The gap junction uncoupler, heptanol (300μM) com- Unlike the neighboring smooth muscle cells, the ICC-like cells pletely inhibited activity in intact and denuded strips within did not contain thick filaments or dense bodies, consistent with 6.13±0.45min and 2.87±0.78 min respectively (p<0.05, n=6 the reported inability of ICC to contract. ICC were usually strips from 2 animals, unpaired t-test). Full recovery was branched and made contacts with up to 10 smooth muscle obtained on washout. The addition of a gap junction opener, cells. Gap junctions were not encountered although this type AAP10 did not affect the amplitude or frequency of sponta- of contact cannot presently be ruled out. ICC were also often neous activity in intact or denuded strips (p>0.05, n=3) indi- found adjacent to nerves and in addition, nerve-smooth mus- cating that gap junctions are fully open under control condi- cle cell contacts were frequently observed. tions. Moreover, in intact and denuded strips, the time taken Cells with the typical ultrastructural characteristics of ICC by heptanol to inhibit activity was not significantly affected by (Komuro, 1999) were identified in sections of guinea-pig ure- pre-treatment with AAP10 (n=5, p>0.05 in each case). thra using transmission electron microscopy. These findings The findings of the present study have shown that myogenic support previous published work on urethral tissue and iso- spontaneous activity of the bladder requires functional com- lated cells with confocal microscopy. munication via gap junctions and that this activity does not Lyons AD, Gardiner TA, McCloskey KD. (2007) BJU Int. 99, 687-94. depend on the presence of the mucosal layer. Sergeant GP et al, (2000) J Physiol. 526, 359-66. Turner WH, Brading AF (1997). Pharmacol Ther. 75, 77-110. Komuro T. (1999). Microsc Res Tech 47, 267-85. This work was funded by grants from the Wellcome Trust the European Union FP7. This work was funded by grants from the Wellcome Trust the European Union FP7. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical Where applicable, the authors confirm that the experiments requirements. described here conform with The Physiological Society ethical requirements.

PC224

PC223 c-Kit-positive Interstitial Cells of Cajal in the Human Bladder L. Johnston1, S. Woolsey2, H. O’Kane2, P. Keane2 and Intercellular communication and spontaneous activity in K.D. McCloskey1 the guinea-pig bladder 1Centre for Cancer Research and Cell Biology, Queen’s University C. Moffatt, A. Kerrin and K.D. McCloskey Belfast, Belfast, Northern Ireland, UK and 2Department of Urology, Centre for Cancer Research and Cell Biology, Queen’s University Belfast City Hospital, Belfast, Northern Ireland, UK Belfast, Belfast, Northern Ireland, UK The cellular mechanisms underlying pathological bladder con- Spontaneous activity is a phenomenon common to many ditions including urge urinary incontinence are poorly under- smooth muscle tissues including the gastrointestinal tract, stood. An increasing body of evidence suggests that intersti- some blood vessels and urinary tract tissues including the blad- tial cells of Cajal (ICC) may have significant roles in normal der. Spontaneous contractility of bladder smooth muscle are bladder activity (Brading & McCloskey, 2005) and also in con- non-voiding contractions which are thought to underlie the ditions leading to overactivity (Kubota et al, 2008). Bladder ICC tone of the bladder wall during filling (Turner and Brading, have morphological similarities to ICC of the gastrointestinal 1997). While this activity is considered to be myogenic, its ori- tract and have been identified with the established marker, anti- gin and modulation is incompletely understood. The aim of the c-Kit, in animal tissues (Davidson & McCloskey, 2005). The pur- present study was to assess the contribution of the mucosal pose of the present study was to identify ICC in human blad- layer to spontaneous contractility and to examine the effect of der samples and examine their relationships with nerves and pharmacological agents which target gap junctions. smooth muscle. Bladders were removed from guinea-pigs, killed by cervical dis- Bladder biopsies were obtained with informed consent and full location in accordance with Schedule 1, UK Home Office reg- ethical approval from patients undergoing urological investi- ulations. In vitro tension recordings were made from bladder gation. Whole-mount, flat sheet tissue preparations were strips mounted in organ baths which typically developed spon- labeled with antibodies using standard immunohistochemi- taneous activity during the equilibration period. cal protocols and imaged with a confocal microscope. Single Spontaneous activity was unaffected by tetrodotoxin (0.1μM), cells were dissociated from biopsy samples using a cocktail of indicating its myogenic origin (n=19 strips from 8 animals, enzymes and viewed with bright-field microscopy. p>0.05). There was no significant difference in the amplitude Tissues labeled with anti-c-Kit had several sub-populations of or frequency of spontaneous contractions between strips which immunopositive cells (n=18 samples). c-Kit-positive cells in the were intact (7.2±0.8mN, 3.5±2.3 per minute) and those which lamina propria were branched, stellate-shaped with a central nucleus and formed frequent connections with each other.

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2008). However, the localisation of the TRPM8 protein in vas- When the biopsy samples contained underlying detrusor cular myocytes and the electrophysiological characteristics of smooth muscle bundles, c-Kit-positive cells with lateral branches its activation remain unknown. from an elongated cell body were located on the boundary of Ventral tail artery was obtained from humanely-dispatched the bundles. In some biopsies, discrete, rounded, non-branched Sprague-Dawley rats (12 weeks). Single vascular myocytes c-Kit-positive cells were present; these were deemed to be mast were then isolated by enzymatic digestion for immunocyto- cells. Double-labeling with anti-c-Kit and anti-vAChT to label chemistry and patch clamp analysis. Staining of isolated rat cholinergic nerves demonstrated close relationships between tail artery myocytes with TRPM8 rabbit antibody revealed ICC and cholinergic nerves (n=4 samples) in both the lamina TRPM8 immunoreactivity predominantly localised on the cells propria and the underlying detrusor. Vimentin-positive cells perimeter. were abundant throughout the lamina propria and formed a In voltage-clamped tail artery myocytes, held at -20 to -40 mV, network of interconnecting cells with different morphologies 300 μM menthol accelerated STOCs discharge from 2.8±0.4 s- including stellate and elongated bipolar cells (n=4 samples). 1 to 6.3±0.4 s-1 (mean ± S.E.M.; paired t-test, P<0.01; N=4 (N = Vimentin filaments are present in fibroblasts, ICC and other no. of cells)) and often resulted in a transient increase in whole mesenchymal cells and in human bladder are therefore likely cell current. In perforated patch clamp configuration, 300 μM to represent a mixed population of cell types. Enzymatic dis- menthol depolarised tail artery myocytes from a mean mem- persal of biopsy samples yielded a heterogeneous population brane potential of -56.8±3 mV by 8.3±1 mV (paired t-test, of cells, the majority of which were spindle-shaped smooth P<0.001; N=7). Most interestingly, menthol simultaneously muscle cells and a smaller number of branched cells which increased the frequency of spontaneous hyperpolarisations resembled ICC, previously investigated in guinea-pig tissues from a mean of 0.3±0.1 s-1 to 1.4±0.4 s-1 (paired t-test, P<0.05; (Johnston et al, 2008). N=6). These hyperpolarisations also increased in amplitude The human bladder contains populations of c-Kit-positive ICC from a mean of -11.8±3 mV to -22.8±3 mV (unpaired t-test, in the lamina propria and the detrusor. These ICC are struc- P<0.05; N=1, n=7-10 (n = no. of observations)). There were no turally associated with cholinergic nerves and are similarly dis- other changes observed in the characteristics of the hyperpo- tributed to the ICC of the guinea-pig bladder (Davidson and larisations, e.g. time-to-peak and decay time constant. Taken McCloskey, 2005). together these findings suggest that TRPM8 activation causes Brading AF & McCloskey KD (2005). Nat Clin Pract Urol. 2: 546-54. acceleration of elementary Ca2+ store release events (sparks), Davidson RA & McCloskey KD (2005) J Urol 173, 1385–1390 resulting in increased BKCa channel activity. Kubota Y et al. (2008). Neurourol Urodyn. 27, 330-40 We conclude that the calcium permeable cation channel, TRPM8, is located on the periphery of tail artery vascular Johnston et al. (2008). Am J Physiol Renal Physiol. 294, F645-655 2+ myocytes, and TRPM8 activation increases [Ca ]i resulting in Financial support from Action Medical Research, The Wellcome increased BKCa channel activity. TRPM8 activation produces Trust and European Union FP7 are gratefully acknowledged. two distinct patterns of electrical activity, (i) sustained membrane depolarisation due to cation influx, and (ii) brief sponta- Where applicable, the authors confirm that the experiments neous hyperpolarisations of increased size and frequency due described here conform with The Physiological Society ethical 2+ to TRPM8/Ca sparks/BKCa channel coupling. requirements. Bautista DM et al. (2007) Nature 448, 204-209. Inoue R et al. (2006) Circ Res 99, 119-131. Yang XR et al. (2006) Am J Physiol 290, L1267-L1276. PC225 Melanaphy D et al. (2008) Proc Physiol Soc 10, PC28. Borisova L et al. (2008) Proc Physiol Soc 10, PC39. TRPM8 activation causes depolarisations accompanied by high-frequency spontaneous hyperpolarisations through a We thank Queen’s University Belfast, The British Heart Foun- 2+ dation and The Physiological Society for support Ca signalling complex with BKCa channels in rat tail artery myocytes Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical D. Melanaphy1, S. Stokesberry1, M. Kustov2, C.D. Johnson1 and requirements. A.V. Zholos1 1Queen’s University, Belfast, UK and 2Bogomoletz Institute of Physiology, Kiev, Ukraine PC226 The menthol receptor, Transient Receptor Potential Melastatin Ca2+ signalling mediated by purinergic receptors in rat aortic member 8 (TRPM8), is the principal sensor of cold in primary smooth muscle cells sensory neurones (Bautista et al., 2007). However, recently it has been described as a prominent vascular TRPM protein (Inoue S. Govindan and C.W. Taylor et al., 2006, Yang et al., 2006). Previously we have shown that Department of Pharmacology, University of Cambridge, Cambridge, TRPM8 mRNA is expressed in a number of rat arteries includ- Cambridgeshire, UK ing aorta, ventral tail, mesenteric and femoral artery (Melana- Vascular smooth muscle cells form most of the walls of blood phy et al., 2008). In aortic, pulmonary and tail arterial smooth vessels and their contractile ability determines blood flow and muscle TRPM8 activation increases cytosolic [Ca2+] and trig- blood pressure. During vascular injury (eg. atherosclerosis), gers myocyte contraction (Yang et al., 2006; Borisova et al.,

196P Poster Communications they can also proliferate and exhibit the synthetic phenotype. laminitis.ThisstudyaimstocharacteriseATPsignallinginequine Elevations of intracellular Ca2+ initiate contraction, while dur- digital vessels, before investigating ATP signalling in laminitic ing vascular injury they may promote cell growth. cAMP has digital vessels, where abnormal ATP signalling may play a role. opposing effects on smooth muscle cell activity and interac- Equine digital arteries (EDA) and veins (EDV) were collected tions between Ca2+ and cAMP are clearly important. Here, we from horses killed in an abattoir. Limbs were collected within have characterised Ca2+ signals evoked by the purinergic recep- 10 min of slaughter and iced Krebs solution was infused into tor agonist, ATP in cultured rat aortic smooth muscle cells the digital circulation to remove blood. Sections of isolated EDA (RASMCs). We have investigated whether increases in cytoso- and EDV were used in organ-bath experiments, whereby elec- lic Ca2+ concentration affect adenylyl cyclase (AC) activity. For tric field stimulation (EFS) in the absence and presence of antag- 2+ 2+ α Ca measurements, cells were loaded with the Ca indicator onists to P2R and 1-adrenoceptors was examined. Concen- fluo-4 and fluorescent measurements were made in a 96-well tration-response curves were constructed to NA and ATP on population based flexstation assay. All results are expressed low and raised tone. Immunofluorescent localization of P2R as means + S.E. ATP mobilized Ca2+ in a concentration-depend- subtypes were performed. Data are mean±s.e.m. μ ent manner (EC50 = 3.5 + 1.1 M, n = 3) and the peak response On low tone preparations EFS (28 V, 0.3 ms, 4-32 Hz) induced was unchanged in the absence of extracellular Ca2+. Involve- vasoconstriction in EDA and EDV; in both cases this was abol- ment of P2Y purinergic receptors was implicated because the ished by tetrodotoxin (1 μM; n=6). In EDA, vasoconstriction was response to ATP was completely abolished by the phospholi- partially inhibited (by 28±9%) by the P2R antagonist suramin μ μ μ pase C inhibitor, U73122 (IC50 = 1.7 + 0.27 M, n = 3) and there (10 M; n=6), but not by the P2R antagonist PPADS (10 M; was no response to the P2X receptor agonist, α,β-methyl ATP. n=6), and predominantly inhibited (by 78±12%) by phento- cAMP measurements were made using radioimmuno assay and lamine (10 μM; n=6). In EDV, vasoconstriction was partially all AC stimulations were made in the presence of the phos- inhibited by suramin (10 μM; n=6) and PPADS (10 μM; n=6) to phodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). a similar degree (by 43±18% and 39±7%, respectively), and to ATP (100μM) inhibited forskolin-stimulated AC by 24 + 1.8% a lesser extent (by 18±6%) by phentolamine (10 μM; n=6). (n = 4, p = 0.001, student’s t-test). However, this inhibition per- Exogenous NA and ATP mimicked EFS in EDA; immunostain- 2+ sisted in the absence of extracellular Ca (31 + 1.02%, n = 3, p ing for P2X1, P2X2 and P2X3 receptor subunits was seen in vas- 2+ = 0.0022) and after intracellular Ca stores were emptied using cular smooth muscle (SM) and P2X1, P2X2 and P2X3 subunits thapsigargin (1μM) (29 + 2.04%, n = 3, p = 0.005). This effect in endothelium. Denuding the EDA of endothelium did not alter was abrogated after treatment of cells with pertussis toxin (n EFS- or ATP-evoked vasoconstriction. Exogenous NA and ATP 2+ = 3). To summarise, ATP mobilized intracellular Ca via P2Y mimicked EFS in EDV; immunostaining for P2X1 and P2X7 recep- receptors and inhibited forskolin-stimulated AC activity in tor subunits was seen in vascular SM. ATP failed to induce vasodi- RASMCs. The inhibition of AC activity was independent of lation on raised tone preparations in either EDA or EDV. increases in cytosolic [Ca2+] and is likely to be mediated by Gi ATP and NA are cotransmitters in sympathetic nerves supply- G-protein. ing the equine digital vasculature, NA being the dominant partner in arteries and ATP being the dominant partner in veins. ATP SupportedbytheMRCandtheCambridgeCommonwealthTrust. did not produce either endothelium-dependent or -independent vasodilatation; P2X , P2X and/or P2X , or, P2X recep- Where applicable, the authors confirm that the experiments 1 2 1/2 1 tors are likely to mediate vasoconstriction on EDA and EDV, described here conform with The Physiological Society ethical respectively. We will now use this information to investigate requirements. whether ATP signalling is altered in the digital vasculature of horses with laminitis. Burnstock G. Dual control of vascular tone and remodelling by ATP released from nerves and endothelial cells. Pharm. Reports 60:12- PC227 20, 2008 Alice Fordham and Silvia Janska contributed equally. ATP signalling in equine digital vessels Where applicable, the authors confirm that the experiments 1 1 1 2 1 A. Fordham , S.E. Janská , C. Crawford , H. Zerpa , Y. Berhane , described here conform with The Physiological Society ethical 1 3 1 C.M. Peppiatt-Wildman , G.E. Knight and S.S. Wildman requirements. 1Urinary System Physiology Unit, Royal Veterinary College, London, UK, 2Biomedical Department, Central University of Venezuela, Maracay, Venezuela and 3Autonomic Neuroscience Centre, Royal Free and University College Medical School, London, UK

ExtracellularATPplaysaroleinthemaintenanceofvasculartone 1. It is released from perivascular nerves as a cotransmitter with noradrenaline (NA), and from endothelial cells in response to changes in blood flow and hypoxia. The role and distribution of ATP-activatedP2receptors(P2R)ischaracterisedformanyblood vessels but not in equine digital vessels. Haemodynamic distur- bancesleadingtoischaemicandreperfusioninjuryoftheequine digit are thought to be involved in the debilitating condition

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