APHAR 2007 Abstract Preview for publication in BMC Pharmacology

Meeting abstracts 13th Symposium of the Austrian Pharmacological Society (APHAR) Joint meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria 22–24 November 2007

AUSTRIAN PHARMACOLOGICAL SOCIETY A2 A1 Restricted expression between parvalbumin and

Neurokinin 1 receptor antagonism promotes active substance P receptor NK1 in interneurones of the stress coping via enhanced septal 5-HT lateral amygdala transmission Hari Kishore Sreepathi and Francesco Ferraguti Georg M Singewald1, Karl Ebner1, Nigel Whittle1, Department of Pharmacology, Medical University of Francesco Ferraguti2 and Nicolas Singewald2 Innsbruck, Austria 1Department of Pharmacology and Toxicology, Center E-Mail: [email protected] for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Austria Increasing evidence suggests that substance P (SP) and its receptor, namely the neurokinin 1 receptor (NK -R), play an 2Department of Pharmacology, Medical University of 1 important role in the modulation of stress-related, affective Innsbruck, Austria and/or anxious behaviours. Both SP and NK1-R are E-Mail: [email protected] expressed in brain regions critically involved in stress, fear and affective responses such as the amygdala, Antagonists of the substance P (SP) preferring neurokinin 1 hippocampus and frontal cortex. In this study we aimed at receptor (NK1-R) represent a promising novel class of drugs identifying the types of NK -R immunoreactive neurones in for the treatment of stress-related disorders including 1 the basolateral complex of the amygdala according to their depression and anxiety disorders. The underlying neuronal content of other neurochemical markers by dual or triple mechanisms involved in the effects of these drugs, however, labelling immunofluorescence. The basolateral amygdaloid are poorly understood. By using in vivo microdialysis we complex consists of the lateral (LA) and basolateral (BL) observed increased SP, but reduced serotonin (5-HT) nuclei, which are believed to be cytoarchitectonically similar. release during forced swim stress (FST) in the rat lateral Our study reveals that in the rat LA, 38.7 ± 6.7% of NK -R septum (LS), a key area in processing emotions and stress 1 immunopositive interneurones (124 ⁄ 331) co-express the responses. Acute administration of the selective high affinity calcium binding protein parvalbumin (PV), representing 15.2 NK1-R antagonist L-822429 injected either systemically or ± 3.4% (124 ⁄ 820) of PV+ neurones. Conversely, in the BL locally into the LS reversed the FST-induced decrease in no coexistence between NK -R (293 neurones counted) and 5-HT efflux and facilitated active coping strategies during the 1 parvalbumin (2385 neurones counted) expressing FST. Increased active coping in the FST was attenuated by interneurones was detected. These results suggest that intraseptal 5-HT1A-R blockade with WAY100635, indicating interneurones in the LA and BL differentially express that the behavioural effect during NK1-R blockade is molecules involved in cell signalling and indicate a distinct mediated by enhanced intraseptal serotonergic transmission organization in local interneurones. The BL resemble the acting on 5-HT1A-R. Taken together, our findings identify the hippocampal region CA1, in which NK -R-expressing LS as an important brain area for the modulation of stress 1 neurones do not coexist with PV [1]. responses by the SP/NK1-R system. NK1-R blockade References resulted in behaviorally significant enhancement of 5-HT 1. Acsády L, Katona I, Gulyás AI, Shigemoto R and transmission. We show for the first time that this modulation Freund TF. Immunostaining for substance P receptor does not necessarily involve interaction with neuronal firing labels GABAergic cells with distinct termination at the cell body level of 5-HT neurons as previously patterns in the hippocampus. J Comp Neurol 1997, postulated, but can be elicited in a terminal region of these 378:320–336. neurons. APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A3 A5

GPR55 is a novel cannabinoid receptor SAP102, a novel interaction partner of the A2A Nariman Balenga1, Andrew J Irving2 and adenosine receptor Maria Waldhoer1 Ingrid Gsandtner, Nicole Ferstl, Michael Freissmuth 1Department of Experimental and Clinical and Jürgen Zezula Pharmacology, Medical University of Graz, Austria Department of Pharmacology, Medical University of 2Neurosciences Institute, Division of Pathology and Vienna, Austria Neuroscience, University of Dundee, UK E-Mail: [email protected] E-Mail: [email protected] Adenosine receptors are G protein-coupled receptors and Cannabinoids exert their effects by binding to G protein- are implicated in several neurological and psychiatric coupled receptors (GPCRs). To date, two cannabinoid disorders such as Parkinson’s disease, schizophrenia and receptors have been cloned. The cannabinoid CB1 receptor Alzheimer’s disease. These receptors can be distinguished is one of the most abundant GPCRs in the central nervous by their affinity for adenosine analogues and by their system and plays an important role in pain transmission, preferred signal transduction pathway. The A2A receptor has feeding and the rewarding effects of cannabis, whereas the an unusually long intracellular carboxyl terminus. We

CB2 receptor is predominantly found in immune cells. identified SAP102 (synapse-associated protein of 102 kDa) However, some effects of cannabinoids (especially in the as a novel interaction partner of the adenosine A2A receptor. vascular system) could not be attributed to either CB1 or CB2 SAP102 belongs to the family of MAGUK (membrane- receptor function. Here we present GPR55 as a putative associated guanylate kinase-like domain) proteins. These novel cannabinoid receptor, since GPR55 signals, binds to proteins have an established function in synaptic and internalizes in the presence of synthetic cannabinoid organization, which is reflected by their modular structure. ligands. Our data demonstrate that the A2A receptor binds to C-terminal domains of SAP102. Furthermore we identified A4 the responsible binding motif consisting of 5 amino acids in The hCMV chemokine receptor US28 prevents the receptor’s C-terminus. In hippocampal neurons we melanoma growth observed a co-localization of both proteins especialy in Elisabeth Moser1, Joshi Shripad2, Ping Quan2, punctuate structures along the neurite extensions that presumably represented dendritic spines. In the next step Helmut Seidl1,2, Sasa Frank3, Zhao-Jun Liu4, we will use several fluorescence-based techniques in order Mizuho Fukunaga4, Ronan McDaid4, Helmut Kerl2, to investigate the influence of SAP102 on the mobility and Meenhard Herlyn4, Helmut Schaider2 and targeting of the A2A receptor. Maria Waldhoer1 1 Departments of Experimental and Clinical A6 2 Pharmacology, Dermatology, and of Heterologous expression of membrane proteins in 3 MedicalBiochemistry and Microbiology, Medical cardiac myocytes University of Graz, Austria Miroslav Radenkovic, Halyna Pankevych, 4 The Wistar Institute, Philadelphia, PA, USA Oliver Kudlacek, Ingrid Gsandtner and E-Mail: [email protected] Karlheinz Hilber Department of Pharmacology, Center of Biomolecular The human cytomegalovirus (hCMV) encodes the G protein- coupled receptor (vGPCR) US28. This receptor signals Medicine and Pharmacology, Medical University of constitutively and interacts with a broad range of Vienna, Austria chemokines, which are crucial to the pathophysiological E-Mail: [email protected] significance and immunregulatory aspects of this receptor. The cardiac isoform of the Na+ channel (Na 1.5) is known to Chemokines and their receptors have been shown to be key V accumulate in the endoplasmic reticulum (ER). This determinants of tumor growth and formation of metastases. retention presumably reflects quality control in the ER. In US28 (and mutants thereof, i.e. US28R129A and order to understand the underlying mechanism, we US28Δ317) exert anti-tumorigenic effects in various heterologously expressed the human orthologue of the Na+ melanoma cell lines by scavenging chemokines from the channel (Na 1.5) in neonatal primary rat and murine tumor environment. Here we show that in contrast to all V cardiomyocytes, in a cardiomyoblast cell line (H9c2) and in other previously studied cell lines, US28 is expressed on the HEK293 cells (internal control). In HEK293 cells, Na 1.5 cell surface in the melanoma cell lines Sbcl2 and 451Lu. We V readily accumulated at the cell surface and gave rise to suggest that GASP – the G protein-coupled receptor- functional channels with the expected electrophysiological associated sorting protein – which sorts US28 and many properties. In contrast, in cardiomyocytes and H9c2 cells, other GPCRs to the lysosomes is absent in melanoma cells. the Na 1.5 accumulated in the ER regardless of the The absence of GASP might effect the tumor suppressing V transfection method employed (lipofection, nucleofection). properties of US28. As a positive control, we employed G protein-coupled

β1-adrenergic, A1 and A2A adenosine receptors. In HEK293 cells, export of the A2A receptor is known to be enhanced by APHAR 2007 Abstract Preview for publication in BMC Pharmacology the deubiquinating enzyme USP4. Accordingly, we also co- A8 expressed USP4 and the A2A receptor in the different Selective serotonin reuptake inhibitors: a new cardiomyocyte preparations. However, in all instances, the modality for the treatment of membrane proteins were trapped within intracellular lymphoma/leukaemia? compartments. This was, in particular true for the NaV1.5, Christian Schuster1, Nora Fernbach2, Uwe Rix2, which, in many instances, accumulated in circular bodies, Giulio Superti-Furga2, Marion Holy1, which are reminiscent of calnexin-rich organized smooth ER Michael Freissmuth1, Harald H Sitte1 and structures. Based on these findings, we conclude that 1 (i) membrane proteins undergo stringent quality control in Veronika Sexl 1Department of Pharmacology, Center of Biomolecular cardiac myocytes and (ii) ER-export of the NaV1.5 is limited by the availability of additional cardiomyocyte-specific Medicine and Pharmacology, Medical University of components. Vienna, Austria Acknowledgements 2Research Center for Molecular Medicine, Austrian Supported by FWF M989-B09 (MR) and ÖAW DOC-fFORTE Academy of Sciences, Vienna, Austria 22255 (IG). E-Mail: [email protected]

A7 Selective serotonin reuptake inhibitors (SSRIs) have PI3Kδ: a double-edged sword in leukemia recently been reported to specifically kill malignant cells of formation B-lymphoid origin. However, we found that all cell lines Eva-Maria Zebedin1, Olivia Simma1, investigated underwent apoptotic cell death when exposed to SSRI concentrations exceeding 10 µM, regardless of Christian Schuster1, Eva Eckelhart1, whether the cell lines were derived from B- or T-lymphoid Wolgang Warsch1, Dagmar Stoiber2, Eva Weisz2, 3 4 tumors or other sources. The structure-activity relationship Winfried F Pickl , Roland Piekorz , readily distinguished the pro-apoptotic and growth-inhibitory 1 1 Michael Freissmuth and Veronika Sexl effect of SSRIs from their eponymous action: acetylation of 1 Department of Pharmacology, Medical University of the SSRIs fluvoxamine and paroxetine abrogated the ability Vienna, Austria of these compounds to inhibit 5-HT uptake, but did not 2Ludwig Boltzmann Institute for Cancer Research, impair their cytotoxic action. Based on these data we Vienna, Austria conclude that (i) SSRIs inhibit growth of transformed cells, 3Department of Immunology, Medical University of but that (ii) this effect is neither specific for malignant cells Vienna, Austria nor specific for any particular cellular subset. (iii) The pro- 4Institute of Biochemistry and Molecular Biology II, apoptotic effect of SSRIs (at µM concentrations) is unrelated Heinrich-Heine-University Düsseldorf, Germany to their principal pharmacological action, i.e. inhibition of serotonin uptake (at nM concentrations). SSRIs or improved E-Mail: [email protected] versions thereof are therefore unlikely to represent useful The PI3Kδ isoform is a candidate drug target in leukemia. lead compounds for inducing apoptosis in B-cell derived Here, we explored its role in Abelson-induced leukemia. tumors. Frank leukemia emerges if the tumor cells have managed to outwit the immune system. The absence of PI3Kδ affected A9 both the tumor cells and the NK cells. Abelson-transformed Duramycin effects on voltage-gated ion channels: PI3Kδ−/− cells induced leukemia in RAG2−/− animals with a a QT-prolongation risk? significantly increased latency, implicating PI3Kδ in tumor Eva-Maria Zebedin, Xaver Koenig, progression. NK cell function, however, was also contingent Miroslav Radenkovic, Halyna Pankevych, −/− on PI3Kδ. PI3Kδ NK cells failed to lyse target cells. Hannes Todt, Michael Freissmuth and Karlheinz Hilber Capacitance measurements revealed the underlying defect: −/− Center of Biomolecular Medicine and Pharmacology, in PI3Kδ NK cells lytic granules did not fuse with the cell Institute of Pharmacology, Medical University of membrane. Accordingly, transplanted leukemic cells killed Vienna, Austria PI3Kδ−/− animals more rapidly, both in syngeneic (PI3Kδ−/−) −/− −/− E-Mail: [email protected] or immunocompromised (RAG2 PI3Kδ ) animals. Our observations define a dual function of PI3Kδ in leukemia and Drug-induced prolongation of the QT interval has become a document that the action of PI3Kδ in the NK compartment is major safety concern during drug development. Duramycin, as relevant to the survival of the mice as the delayed tumor a peptide antibiotic, is in clinical development for the progression. treatment of cystic fibrosis. It becomes deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change biophysical membrane properties and perturb ion channel function. Thus, its application possibly carries the risk to elicit a QT- prolongation. Here, we tested the effects of duramycin on currents through voltage-gated hERG potassium, sodium and calcium channels of various mammalian cell types in whole cell patch clamp studies. We found that duramycin APHAR 2007 Abstract Preview for publication in BMC Pharmacology bath concentrations between 1 nM and 0.1 µM did not A11 generate any effects on these currents. Concentrations ≥0.3 C-terminal splicing reveals intramolecular gating µM, however, reduced the amplitudes of all the investigated 2+ modulation in CaV1.3 L-type Ca channels currents. Moreover, sodium current fast inactivation kinetics Anamika Singh1, Mathias Gebhart1, Reinhard Fritsch2, was slowed in the presence of duramycin. The described Jean-Charles Hoda1, Martina Sinnegger-Brauns1, effects exhibited concentration-dependency. A further rise in Christoph Romanin2, Jörg Striessnig1 and duramycin bath concentration (≥3.3 µM) induced a leak Alexandra Koschak1 current consistent with pore formation. The reported effects 1 of duramycin on ion channel function may be generated by Pharmacology and Toxicology, Department of disruption of biophysical membrane properties rather than Pharmacy, University of Innsbruck 2 by specific interaction with ion channel proteins. Our data Department of Biophysics, University of Linz, Austria suggest that, under therapeutic conditions (i.e. E-Mail: [email protected] administration via inhalation), duramycin is unlikely to elicit a Neuronal excitability and pace-making in the sinoatrial node QT-prolongation. 2+ are controlled by low-voltage activated CaV1.3 L-type Ca A10 channels. We recently found that in related CaV1.4 channels a highly-structured distal C-terminal motif (CTM) modulates Anticonvulsant and neuroprotective actions of voltage- and Ca2+-dependent gating (CDI). In Ca 1.3, endogenous dynorphin V C-terminal splicing leads to a full-length (CaV1.3L) and at Christoph Schwarzer1, Stephan Loacker1, least 1 short (CaV1.3S) splice form. If a CTM would also Eduard Schunk1, Mohammad Sayyah1 and modulate CaV1.3 gating it would be present in CaV1.3L but 2 Herbert Herzog not CaV1.3S variants. We therefore compared the 1 Department of Pharmacology, Medical University of biophysical properties of CaV1.3L or CaV1.3S coexpressed Innsbruck, Austria with β3 + α2δ-1 in tsA-201 cells using the whole-cell patch- 2 Garvan Institute of Medical Research, Darlinghurst, clamp technique. CaV1.3S channels activated at more Sydney, Australia negative potentials compared to CaV1.3L (~ −10 mV, E-Mail: [email protected] p < 0.0001), inactivated faster (p < 0.01) and showed more CDI (p < 0.01). These changes resulted in a decreased Anticonvulsant actions of dynorphin were proposed since window current shifted to more hyperpolarized potentials more than 2 decades, however till now, we do not have likely to cause a reduction in the channels’ dynamic range. information on the functions of endogenous dynorphin. Removal of the C-terminal 158 (CaV1.3Δ158) or 76 amino Thus, we investigated prodynorphin knockout (KO) mice in acids was sufficient to induce gating properties similar to different models of seizures and epilepsy. Seizure threshold CaV1.3S. FRET experiments revealed interaction of the last was investigated by tail-vein infusion of 100 µg 158 amino acids (C158) to a proximal C-terminal domain in pentylenetetrazole (PTZ)/ml at a rate of 100 µl/min until mice CaV1.3L. Coexpression of C158 with CaV1.3Δ158 completely displayed generalized clonic seizures. Wild-type mice restored CaV1.3L gating properties confirming this protein showed clonic seizures at 39.2 ± 1.88 (mean ± SEM; n = 5) interaction. Thus CaV1.3 channel gating is under control of mg PTZ/kg body weight. KOs displayed a significantly the distal C-terminus allowing alternative splicing to fine- reduced seizure threshold of 32.7 ± 1.17 (n = 6) mg PTZ/kg. tune channel activity and adapt channel function to This phenotype could be rescued entirely by the κ opioid physiological needs. receptor specific agonist U-50488, but not the µ opioid receptor specific agonist DAMGO. The δ opioid receptor A12 specific agonist SNC80 decreased seizure threshold in both Mapping the interaction sites of the viral encoded genotypes. Pre-treatment with the κ-selective antagonist chemokine receptor US28 and the sorting protein GNTI completely blocked the rescue effect of U-50488. After GASP-1 injection of kainic acid into stratum radiatum of CA1 of the Gerald P Parzmair, Wolfgang Platzer and dorsal hippocampus, KO mice displayed increased neuronal Maria Waldhoer loss along the rostro-caudal axis of the ipsi- and partially the Department of Experimental and Clinical contralateral hippocampus at early time points after treatment. Thus, marked neurodegeneration was seen Pharmacology, Medical University of Graz, Austria already 1 week after treatment in KO mice. The differences E-Mail: [email protected] faded with time, being almost gone 5 weeks after kainate. US28 is a chemokine receptor encoded by the human Our data clearly indicate that endogenous prodynorphin cytomegalovirus (hCMV). It has been shown to be crucial for derived peptides have anticonvulsant and neuropotective viral propagation. For instance, it is present on the viral properties. envelope where it may serve as a cell-to-cell attachment Acknowledgments factor through binding to the membrane-bound chemokine This work was supported by the FWF (P20701-B05 and fractalkine. Most notably, US28 is a constitutively signaling P18471-B05). and endocytosing receptor, thus playing a key role in the early reprogramming of the host cell. It has further been suggested that during viral propagation, US28 is integrated into the viral envelope in the lysosomes of host cells. A APHAR 2007 Abstract Preview for publication in BMC Pharmacology candidate protein for sorting many G protein-coupled A14 receptors to lysosomes is the G protein-coupled receptor- Experiments to localize the site for the anxiogenic associated protein-1 (GASP-1). Here we show that GASP-1 action of NPY mediated by Y2 receptors in the interacts with US28 in vitro and set out to map the mouse brain interaction sites of US28 and GASP-1 by using in vitro Ramon O Tasan1, Stefan Weger2, Regine Heilbronn2, translation binding assays. Ngoc K Nguyen3, Nicolas Singewald3, Herbert Herzog4 and Günther Sperk1 A13 1Department of Pharmacology, Medical University of State-dependent dissociation of HERG channel Innsbruck, Austria inhibitors 2Institute of Virology, Charité, Free University of Berlin, Daniela Stork1, Andreas Windisch1, Evgeny N Timin1, Germany Annette Hohaus1, Manfred Auer3, Gerhard Ecker2 and 3Department of Pharmacology and Toxicology, Steffen Hering1 University of Innsbruck, Austria 1Department of Pharmacology and Toxicology, 4Garvan Institute of Medical Research, Darlinghurst University of Vienna, Austria Sydney, Australia 2Department of Medicinal Chemistry, University of E-Mail: [email protected] Vienna, Austria 3 Novartis Institute for Biomedical Research, Vienna, Neuropeptide Y (NPY) is abundant in the nervous system. It

Austria acts through Y1, Y2, Y4 and Y5 receptors and is involved in a E-Mail: [email protected] variety of brain functions. When applied locally into the amygdala, NPY exerts an anxiolytic action, presumably Inhibition of HERG channels prolongs the ventricular action mediated by Y1 receptors. Depletion of Y2 receptors induces potential and correspondingly the QT-interval with the risk of an anxiolytic phenotype, possibly by abolishing the release- torsade de pointes arrhythmias and sudden cardiac death. inhibiting action of presynaptic Y2 receptors. In the present Many drugs induce stronger HERG channel inhibition when study we aimed to find the exact site of the presumed the cell membrane is depolarised frequently. The anxiogenic action mediated by Y2 receptors. We conducted dependence of inhibition on the pulsing rate may yield lox/lox site-specific deletions of Y2 receptors in Y2 mice by local different IC50 values at different frequencies and thus affect injection of an AAV-Cre vector into the hippocampus and the the quantification of HERG channel block. We compared the amygdala. As controls, an AAV-GFP vector was injected in kinetics of HERG channel inhibition and recovery from block lox/lox Y2 littermates at the same sites. Expression of Cre and by 9 known blockers and several new propafenone GFP was verified by in situ hybridization and derivatives at different frequencies. HERG channels were immunohistochemistry. Deletion of Y2 receptors was expressed heterologously in Xenopus oocytes and currents visualized by receptor autoradiography and in situ were measured with the two-electrode voltage-clamp hybridization. After bilateral injection of an AAV-Cre vector technique. Frequency-dependent block was observed for into the basolateral amygdala, mice revealed a tendency amiodarone, cisapride, droperidol, haloperidol and the towards an anxiolytic phenotype in the light-dark test (LDT). propafenone derivative GPV-0576 (group 1) whereas When deletion of Y2 receptors was confined to the central bepridil, domperidone, E-4031, terfenadine and propafenone nucleus of the amygdala, an anxiolytic phenotype was (group 2) induced similar pulse-dependent block at all observed in the elevated plus maze and the LDT. Moreover, frequencies. With the group 1 compounds, HERG channels a better stress coping ability was demonstrated in the tail recovered from block in the presence of drug. No substantial suspension test. In contrast, no anxiolytic effect was recovery from block was observed with the second group of detected after intrahippocampal injections. The experiments compounds. Washing out of bepridil, domperidone, E-4031 indicate that the anxiolytic and antidepressant-like effects of and terfenadine was substantially augmented by frequent Y2 receptor deletion may be generated in certain subnuclei pulsing. Our data suggest that apparently “trapped” drugs of the amygdala. (group 2) dissociate from the open channel state whereas Acknowledgments group 1 compounds dissociate from open and resting states. Grant support: FWF S102004.

A15 Interaction between the selectivity filter and the fast inactivation machinery in the voltage-gated Na+ channel René Cervenka, Touran Zarrabi, Xaver Koenig, Eva-Maria Zebedin, Karlheinz Hilber and Hannes Todt Centre for Biomolecular Medicine and Pharmacology, Medical University of Vienna, Austria E-Mail: [email protected]

In the voltage-gated Na+ channel the central pore is believed to be lined by the S6 segments of all four domains. APHAR 2007 Abstract Preview for publication in BMC Pharmacology

Conformational changes of these S6 segments are thought Acknowledgments to give rise to channel opening, closing and fast inactivation This work was supported by the GEN-AU Dragon Project to (FI). Whereas FI most likely occurs by an occlusion of the M.H. intracellular part of the pore, the selectivity filter of the channel is located in the extracellular vestibule. We sought A17 to investigate possible interactions between the selectivity Cell adhesion-dependent trafficking and targeting filter and the intracellular part of the domain IV S6 segment of TRPC4 channels in human vascular which is known be involved in FI. To this end, a critical endothelium residue within the selectivity filter of the rNaV1.4 channel, Annarita Graziani, Martin Krenn, Michael Poteser and K1237 was replaced by the negatively charged glutamate Klaus Groschner (K1237E). This mutation was combined with serial cysteine Institute of Pharmaceutical Sciences, Pharmacology & replacements of amino acids in the S6 segment of domain IV. In K1237E the midpoint of FI (V05) was shifted to the Toxicology, University of Graz, Austria hyperpolarized direction relative to wild-type (−60 ± 13 vs. E-Mail: [email protected] −47 ± 11 mV, n = 6, p < 0.01). Mutations of 16 residues in Introduction: TRPC4 has been suggested as a prominent the domain IV-S6 produced inconsistent changes of V05. Ca2+ signaling element of vascular endothelial cells, which However, when these mutations were combined with governs endothelial permeability. In an attempt to identify K1237E, V05 was shifted to more negative values in all but mechanism that link TRPC4 to cell adhesion, we tested the one double mutants (mean shift −13 mV), irrespective of the hypothesis that TRPC4 communicates with adherens direction and the amount of shift produced by the single S6 complexes by mechanisms other than generation of global mutation. Conclusion: The selectivity filter of the voltage- cellular Ca2+ signals. gated Na+ channel is coupled to the machinery of FI. Results: In human microvascular endothelial cells (HMEC), Acknowledgments TRPC4 was found to co-precipitate with VE-cadherin, Support: Austrian Science Fund P17509-B11. indicating a physical coupling between TRPC4 and components of junctional complexes. Membrane A16 presentation of TRPC4 as determined by surface Activation of RhoB in simvastatin-induced biotinylation was found dependent on the formation of cell- apoptosis of differentiated human skeletal muscle cell contacts. Membrane presentation of TRPC4 was cells divergently affected by pro-inflammatory stimuli in cell 1 2 3 Timm Zörgiebel , Lukas Weigl , Harald Genth and displaying cell-cell contacts as compared to cells lacking Martin Hohenegger1 contacts. At >90% confluency, epidermal growth factor 1Institute of Pharmacology, Medical University of (EGF) reduced membrane presentation of both VE-cadherin Vienna, Austria and TRPC4. By contrast, enhanced EGF-induced surface 2Department of Anesthesiology and Critical Care recruitment of TRPC4 was observed when cell-cell contacts Medicine, Medical University of Vienna, Austria were lacking. Cell adhesion-dependent targeting of TRPC4 3Institute of Toxicology, Medical School Hannover, was further analyzed by fluorescence imaging of channel localization and cellular Ca2+ signals in HMEC as well as in Germany HEK293 expression system. E-Mail: [email protected] Conclusion: Our results suggest recruitment of TRPC4 into Statins are well-tolerated cholesterol lowering drugs. a junctional signaling microdomain of the vascular However, in rare cases myotoxicity or even rhabdomyolysis endothelium. occur. We have recently shown that in vitro statins are able Acknowledgments to induce apoptosis in differentiated human skeletal muscle Supported by FWF P19280. cells. In particular, simvastatin triggers the mitochondrial pathway of apoptosis via translocation of Bax from the A18 cytosol to the mitochondrial membrane. Accordingly, Sensitization of cAMP formation in a neuron-like caspase 9 and caspase 3 were consecutively activated. cell line Here we show that an early event in simvastatin action is up- Edin Ibrišimović1, Martin Bilban2 and Christian Nanoff1 regulation of RhoB on mRNA level which is paralleled by the 1Department of Pharmacology, Medical University of downstream Krüppel-like factor-2 (KLF-2) and KLF-6. Vienna, Austria Moreover, the level of active, GTP-bound RhoB was 2Clinical Institute for Laboratory Diagnostics, Medical increased in simvastatin-treated cells in a concentration- University of Vienna, Austria dependent manner as determined by Rhotekin pull-down E-Mail: [email protected] assay. Up-regulation of RhoB on mRNA and protein level was dependent on HMG-CoA reductase inhibition. Differentiation into a nerve cell-like phenotype and growth Interestingly, the up-regulation of RhoB was paralleled by arrest of SH-SY5Y neuroblastoma cells went along with caspase 2 activation, which might be directly associated with increased cAMP formation. Both receptor-dependent as well RhoB and therefore be required for the cytotoxic effect of as direct activation of adenylyl cyclase by forskolin were simvastatin. enhanced by at least twenty-fold. Since cAMP controls many processes in nerve cell function and development we have APHAR 2007 Abstract Preview for publication in BMC Pharmacology investigated the causal factors and mechanism of A20 sensitization in SH-SY5Y cells. The degree of sensitization Differential anti-inflammatory properties of depended on pre-incubation of the cells with retinoic acid; peroxisome proliferator-activated receptors however, maximizing the extent of sensitization required the (PPAR) α and γ in experimental pancreatitis withdrawal of serum from the culture medium. This was Thomas Griesbacher, Veronika Pommer, necessary for the cells to secrete endogenous substances Rufina Schuligoi and Bernhard A Peskar into the culture supernatant. Because sensitization was Institute of Experimental and Clinical Pharmacology, blocked by inhibitors of gene transcription we surmised that the autocrine factors were relevant for sensitization and Medical University of Graz, Austria were generated by de novo protein synthesis. A gene E-Mail: [email protected] expression screen revealed several factor candidates Perfluorooctanoic acid (PFOA) has anti-inflammatory effects (including dkk1, EphB2, NPY, VEGFB); our preliminary data in models of cutaneous inflammation, possibly via activation indicated that a combination of these may be needed to of PPAR-α and PPAR-γ. We have therefore investigated induce full sensitization. Our data further suggest that whether PFOA has similar effects in a model of acute sensitization was not due to up-regulation of stimulatory G oedematous pancreatitis and whether such effects could be proteins or adenylyl cyclase. Rather, the immediate cause explained by agonism at PPAR-α or PPAR-γ. Acute may be clustering of the catalyst and its activator G . This s pancreatitis was induced in anesthetized rats by i.v. infusion interpretation is consistent with the effect caused by altering of the cholecystokinin analogue, caerulein. The PPAR-α the membrane lipid composition which enhanced and agonist clofibrate or the PPAR-γ agonist rosiglitazone were reduced cAMP formation in undifferentiated and injected s.c. before the experiments. Pancreatic oedema, differentiated cells, respectively. neutrophil activation and production of prostaglandin (PG) E and prostacyclin (via 6-keto-PGF ) were assessed in the A19 2 1α pancreas. Acute pancreatitis caused significant oedema Mapping the binding sites for accessory proteins formation, neutrophil activation as assessed by on the A2A adenosine receptor myeloperoxidase activity in the tissue, and increased Ivana Ostrouska, Ingrid Gsandtner, synthesis of pro-inflammatory prostanoids. Neutrophil Halyna Pankevych, Christian Nanoff and activation was unaffected by clofibrate but was abolished by Oliver Kudlacek rosiglitazone. In contrast, prostanoid synthesis was Department of Pharmacology, Medical University of unaffected by rosiglitazone but was inhibited by clofibrate. In Vienna, Austria conclusion, our data demonstrate that activation of PPAR-α E-Mail: [email protected] and PPAR-γ has differential anti-inflammatory effects in acute interstitial-oedematous pancreatitis. Neutrophil Among the four receptors for the purine nucleoside activation is sensitive to inhibition by PPAR-γ activation adenosine the A2A receptor subtype is the only one with an while the production of pro-inflammatory prostanoids can be extended cytoplasmic domain and this is due to a attenuated by activation of PPAR-α. Thus, the anti- remarkably long carboxyl terminal tail (C-tail). We have inflammatory potential of PPAR-α and PPAR-γ ligands previously identified from an adult human brain cDNA library should be further investigated. potential interaction partners which bind to the receptor C-tail and suggest that they impinge on receptor biology. APHAR SECTION OF CLINICAL PHARMACOLOGY (i) ARNO, the activator of the small GTP-binding protein ARF6, was shown to propagate sustained receptor signalling A21 to ERK (extracellular signal regulated kinase); (ii) the Daptomycin elimination by continuous ubiquitin-splitting protease USP4 controls receptor turnover; venovenous hemofiltration: in vitro evaluation of (iii) SAP (synapse-associated protein) 102 may form an factors influencing sieving and membrane anchor localizing the receptor in nerve cells. While ARNO adsorption binds to the membrane proximal portion, USP4 and SAP102 Claudia Wagner1, Ilka Steiner2 and Markus Zeitlinger1 recognize more distal segments of the C-tail. In the case of 1 SAP102 the recognition sequence could be narrowed down Department of Clinical Pharmacology, Medical to a stretch of five amino acid residues (DVELL) which is University of Vienna, Austria 2 conserved between species. A receptor where the DVELL Institute of Pharmacology, Center for Biomolecular sequence was changed to RVRAA has been characterized Medicine and Pharmacology, Medical University of upon stable transfection of HEK293 cells (which express Vienna, Austria SAP102). Surface expression, radioligand binding and E-Mail: [email protected] receptor-dependent stimulation of the effector adenylyl cyclase were similar to the wild type. In contrast, the The present in vitro study set out to determine for the first agonist-dependent activation of ERK was attenuated in cells time the continuous venovenous hemofiltration (CVVH) stably expressing the mutant receptor. Hence, SAP102 may clearance of the novel lipopetide antibiotic, daptomycin, from be necessary to establish a signalling complex including human whole blood. Factors influencing daptomycin sieving ERK and the receptor as has been previously observed for and membrane adsorption were investigated in an in vitro the NMDA receptor in nerve cells. setting. A recirculation model was established and daptomycin was added to the simulated blood circuit at APHAR 2007 Abstract Preview for publication in BMC Pharmacology different concentrations and in different solvent systems. after 0 and 15 mg/kg of TQD were 0.23 and 3.15, The concentration of daptomycin over time in the modelled respectively. blood compartment and in ultrafiltrate was measured by Conclusion: Our data suggest that TQD is a potent inhibitor HPLC. Mean sieving coefficients (SCs) of daptomycin over of P-gp at the rat BBB. Moreover, VPM PET appears to be a time were 1 ± 0.05, 0.3 ± 0.02 and 0.4 ± 0.03 in Ringer useful tool for in vivo dose finding of novel P-gp inhibitors in lactate, Ringer lactate containing human albumin and animals and humans. plasma, respectively. The CVVH clearance of daptomycin References from whole blood exceeded the physiological clearance in 1. Choo EF, Kurnik D, Muszkat M, Ohkubo T, Shay SD, an individual with normal renal function. Adsorption of Higginbotham JN, Glaeser H, Kim RB, Wood AJ and daptomycin to synthetic surfaces proved moderate, resulting Wilkinson GR. Differential in vivo sensitivity to in loss of around 20% of the initial dose at 1 hour after the inhibition of P-glycoprotein located in lymphocytes, start of CVVH. Since mean SCs of daptomycin in protein- testes, and the blood-brain barrier. J Pharmacol Exp containing media were higher than the free fraction in Ther 2006, 317:1012–1018. plasma, our results suggest that besides protein binding, unknown factors such as intracellular drug partitioning A23 influence sieving of daptomycin during hemofiltration. Due to Metabolism of (R)-[11C]verapamil in epilepsy the high in vitro CVVH clearance of daptomycin we patients determined from human whole blood, we recommend Aiman Abrahim1,5, Gert Luurtsema6, Martin Bauer1, monitoring of daptomycin concentrations in patients Rudolf Karch2, Christoph Baumgartner3, Kurt Kletter4, undergoing hemofiltration. Markus Müller1 and Oliver Langer1,5 Departments of 1Clinical Pharmacology, 2Medical A22 Computer Sciences, 3Neurology, and 4Nuclear In vivo dose finding of tariquidar using Medicine, Medical University of Vienna (R)-[11C]verapamil µPET 5Department of Radiopharmaceuticals, ARC GmbH, Oliver Langer1,2, Jens Bankstahl1, Claudia Kuntner1, Seibersdorf, Austria Aiman Abrahim1,2, Rudolf Karch3, Johann Stanek1, 6Department of Nuclear Medicine and PET Research, Thomas Wanek1, Maria Zsebedics1, Kurt Kletter4, VU University Medical Center, Amsterdam, Wolfgang Löscher5, Markus Müller2 and The Netherlands Herbert Kvaternik1 E-Mail: [email protected] 1Department of Radiopharmaceuticals, ARC GmbH, Seibersdorf, Austria Introduction: (R)-[11C]Verapamil (VPM) is a new positron 2 3 Departments of Clinical Pharmacology, Medical emission tomography (PET) tracer to measure Computer Sciences, and 4Nuclear Medicine, Medical P-glycoprotein (P-gp)-mediated transport at the blood-brain University of Vienna, Austria barrier (BBB). Owing to the lack of a suitable reference 5Department of Pharmacology, Toxicology and region in brain that is devoid of P-gp, a metabolite-corrected Pharmacy, School of Veterinary Medicine Hannover, arterial input function is required for quantitative analysis of Germany VPM PET data [1]. The aim of this study was to compare E-Mail: [email protected] metabolism of VPM in epilepsy patients and healthy volunteers. Introduction: Tariquidar (TQD, Xenova, UK) is a third- Methods: Selected arterial blood samples from 9 patients, generation inhibitor of the multidrug efflux transporter who underwent VPM PET, were analyzed for radiolabeled P-glycoprotein (P-gp) with potential applications in neurology metabolites by a previously described combined solid-phase and oncology in order to increase drug exposure of tissues extraction/HPLC assay [2]. targeted for treatment. We used small-animal positron Results: VPM metabolism was significantly faster in emission tomography (µPET) with the P-gp substrate patients as compared to healthy volunteers [1] (unchanged (R)-[11C]verapamil (VPM) in order to measure in vivo the VPM at 60 min after injection: 26.1 ± 6.4 vs. 49.0 ± 13.4%, degree of P-gp inhibition at the rat blood-brain barrier (BBB) p < 0.05, t-test). after administration of different doses of TQD. Conclusion: Faster metabolism of VPM in epilepsy patients Methods: Wistar Unilever rats received intravenous doses may be caused by CyP450 enzyme induction by antiepileptic of 0, 1, 3, 5, 7.5 and 15 mg/kg of TQD followed by a 1-hour drugs. Based on these data caution is warranted when using VPM µPET scan recorded at 2 hours after TQD an averaged arterial input function derived from healthy administration. Brain-to-plasma radioactivity ratios were volunteers for the analysis of patient data. As radiolabeled fitted to a sigmoidal dose-response curve. metabolites of VPM are known to cross the BBB [1], different Results: TQD inhibited P-gp-mediated efflux of VPM across kinetic modeling parameters obtained in patients and the BBB with an apparent half-maximum effective dose healthy volunteers might be at least partly attributed to

(ED50) of 6.6 mg/kg (95% confidence interval: 4.9–8.2 different rates of tracer metabolism rather than to differences mg/kg) which was in good agreement with previous data in cerebral P-gp activity. reported in mice for another P-gp substrate (loperamide, References

ED50: 5.7 mg/kg) [1]. Brain-to-plasma radioactivity ratios 1. Lubberink M, Luurtsema G, van Berckel BN, Boellaard R, Toornvliet R, Windhorst AD, Franssen EJ and APHAR 2007 Abstract Preview for publication in BMC Pharmacology

Lammertsma AA. Evaluation of tracer kinetic models A25 for quantification of P-glycoprotein function using mTORC1 inhibition with rapamycin or LY294002 11 (R)-[ C]verapamil and PET. J Cereb Blood Flow Metab alone but not in combination leads to AKT 2007, 27:424–433. phosphorylation via mTORC2 in melanoma cells 2. Luurtsema G, Molthoff CF, Schuit RC, Windhorst AD, Johannes Werzowa1,2, Daniel Cejka2, Lammertsma AA and Franssen EJ. Evaluation of Bettina Dekrout1, Thorsten Fuereder2, (R)-[11C]verapamil as PET tracer of P-glycoprotein Christiane Thallinger2, Hubert Pehamberger1, function in the blood-brain barrier: kinetics and 2 1 metabolism in the rat. Nucl Med Biol 2005, 32:87–93. Volker Wacheck and Barbara Pratscher 1Department of Dermatology, Medical University of A24 Vienna, Austria 2 P-Glycoprotein inhibition at the blood-brain barrier Department of Clinical Pharmacology, Medical visualized with (R)-[11C]verapamil µPET University of Vienna, Austria Claudia Kuntner1, Jens Bankstahl5, Aiman Abrahim1,2, E-Mail: [email protected] Rudolf Karch3, Johann Stanek1, Thomas Wanek1, 1 4 5 Inhibition of mTOR complex 1 (mTORC1) with rapamycin Maria Zsebedics , Kurt Kletter , Wolfgang Löscher , leads to phosphorylation of AKT in some cancer cells with 1 2 Herbert Kvaternik , Markus Müller and unknown biological consequences. A mechanism involving 1,2 Oliver Langer insulin receptor substrate (IRS) and phosphatidylinositol 1 Department of Radiopharmaceuticals, ARC GmbH, 3-kinase (PI3K) leading to increased AKT phosphorylation Seibersdorf, Austri has been described. For melanoma, it is unknown, whether Departments of 2Clinical Pharmacology, 3Medical this feedback loop plays a role, although preliminary clinical Computer Sciences, and 4Nuclear Medicine, Medical data indicate poor activity of rapalogues in melanoma. Here, University of Vienna, Austria we report that treatment of melanoma cells with rapamycin 5Department of Pharmacology, Toxicology and resulted in strong and long lasting AKT phosphorylation, but Pharmacy, School of Veterinary Medicine Hannover, had little or no effects on cell viability, cell cycle arrest or apoptosis. Combined PI3K/mTOR inhibition with LY294002 Germany had strong effects on these parameters, but also led to E-Mail: [email protected] increased phospho-AKT levels after prolonged treatment. In Introduction: Inhibition of the multidrug efflux transporter contrast to the single treatments, combination of rapamycin P-glycoprotein (P-gp) at the blood-brain barrier (BBB) is plus LY294002 was able to suppress AKT phosphorylation considered a promising strategy in order to increase even after prolonged treatment. Inhibition of mTOR intracerebral penetration of therapeutics, such as complex 2 (mTORC2) using RNAi led to reduced levels of p- antiepileptic and anticancer drugs. The aim of this study was AKT, also under conditions when mTORC1 was inhibited. to evaluate the usefulness of (R)-[11C]verapamil (VPM) and PTEN mutant melanoma cells showed slightly increased small-animal positron emission tomography (µPET) to levels of AKT phosphorylation compared to PTEN wild-type measure P-gp inhibition at the BBB following administration cells. Thus, our results indicate that mTORC1 inhibition with of the third-generation P-gp inhibitor tariquidar (TQD, specific inhibitors such as rapamycin as well as with multi- Xenova, UK). target inhibitors such as LY294002 can lead to AKT Methods: Five Wistar Unilever rats underwent paired VPM phosphorylation in melanoma cells via mTORC2, but without µPET scans, one baseline scan followed by i.v. significant influence on treatment efficacy in vitro. administration of TQD (15 mg/kg) and a second PET scan at 2 hour after TQD administration. Arterial blood sampling was A26 performed along with analysis of metabolism and plasma Monitoring platelet activation with two different protein binding of VPM. shear-dependent function tests: application of Results: Following TQD administration, the brain-to-plasma PFA-100 and the Impact-R ARA ratio of radioactivity was increased by a factor of 11–16 as Ghazaleh Gouya1, Michael Wolzt1, Marianne Niel2, compared to baseline scans, whereas VPM metabolism and Bernd Jilma1 and Simon Panzer2 plasma protein binding were left unaffected. 1Department of Clinical Pharmacology, Medical Conclusion: Our pilot data suggest that VPM PET is a University of Vienna, Austria sensitive tool to quantitatively visualize P-gp inhibition at the 2Department of Blood Group Serology, Medical animal and human BBB. University of Vienna, Austria E-Mail: [email protected]

To analyze the aspirin effect we compared two methods, the PFA-100 and the Impact-R to determine their sensitivity and specificity. In this prospective double-blind single centre trial 37 healthy male volunteers (median age 27, range ± 7) were randomized to take aspirin 160 mg per day for 7–10 days or placebo. The platelet function was determined under close to physiological and high shear. At study entry and after 2 APHAR 2007 Abstract Preview for publication in BMC Pharmacology weeks we determined the closure time (CT) of resistance increased at baseline and during squatting after collagen/epinephrine cartridges obtained by the PFA-100 administration of the α2 agonists (p < 0.01) in both groups to (CEP-CT), and the surface coverage (SC), as a measure of a comparable degree (p = 0.56). platelet adhesion, with and without pre-incubation with Conclusions: Alpha-2 agonists may induce changes in arachidonic acid (ARA). By the PFA-100 CEP-CT was choroidal blood flow even after single topical administration. median 210 ± 62 before aspirin, and >300 sec in all Long-term studies are required to study potential effects of individuals taking aspirin for 2 weeks (p < 0.0001). The SC brimonidine and clonidine in the clinical setting. pre-incubated with ARA was median 4.6 ± 2.5 before study entry and median 6.8 ± 2.7 after two weeks of aspirin A28 (p < 0.0001). Changes were not significant in controls. In Pharmacokinetics of amphotericin B colloidal order to normalize for the broad variation of the SC from one dispersion in liver failure individual to the next, we determined the relative change of Stefan Weiler, Rosa Bellmann-Weiler, the SC due to the addition of ARA. Again, aspirin Michael Joannidis and Romuald Bellmann significantly inhibited ARA induced reduction of adhesion Department of Internal Medicine, Medical University of (p = 0.014). Sensitivity and specificity to detect aspirin was Innsbruck, Austria 100% and 50% with the PFA-100, 100% and 42% with the Impact-R. E-Mail: [email protected] Conclusion: The PFA-100 (collagen/epinephrine) and the Introduction: Clinical studies on the elimination of Impact-R are useful to estimate the individual response to amphotericin B (AMB) lipid formulations in liver failure have aspirin. While a variety of factors may contribute to been lacking so far. Therefore, the pharmacokinetics of AMB prolonged CEP-CT by the PFA-100, the Impact-R ARA test colloidal dispersion (ABCD) was assessed in critically ill is specific for aspirin. patients with cholestatic liver failure. Patients and Methods: Time-concentration profiles were A27 determined in critically ill patients with cholestatic liver failure Effects of topical clonidine vs. brimonidine on and in critically ill patients with normal hepatic function choroidal blood flow and intraocular pressure requiring ABCD for invasive fungal infections. Lipid- during isometric exercise associated and liberated AMB were separated by solid Günther Weigert1,2, Hemma Resch1, phase extraction and quantified by high performance liquid Gerhard Garhöfer1,2, Gabriele Fuchsjäger-Mayrl1,2 and chromatography. Leopold Schmetterer1,3 Results: Three patients with impaired and two patients with 1Department of Clinical Pharmacology, Medical normal hepatic function (one patient on day 1, one patient University of Vienna, Austria on day 5 of therapy) were enrolled so far. After a single dose 2Department of Ophthalmology, Medical University of of ABCD, the AMB half life was similar in patients with impaired and normal liver function. The AMB clearance was Vienna, Austria slower in liver failure (0.15 vs. 0.54 L/h/kg for total AMB, 3Center of Biomedical Engineering and Physics, 0.22 vs. 0.55 L/h/kg for the liberated AMB fraction and 0.52 Medical University of Vienna, Austria vs. 35.6 L/kg for lipid-associated AMB) and the apparent E-Mail: [email protected] volume of distribution was smaller (2.2 vs. 10.9 L/kg for total AMB, 3.1 vs. 11.2 L/kg for liberated and 8.2 vs. 154.5 L/h/kg Purpose: Clonidine and brimonidine, two α agonists, have 2 for lipid-associated AMB). been shown to reduce intraocular pressure in glaucoma Conclusions: The elimination of ABCD appears to be patients. Little is, however, known about the exact role of delayed in cholestatic liver failure. More pharmacokinetic alpha receptors in the control of ocular blood flow in the data are required to establish reliable dose posterior pole of the eye. Hence, we set out to investigate recommendations for ABCD in patients with liver failure. the effects of topical clonidine vs. topical brimonidine on choroidal blood flow and intraocular pressure during isometric exercise. Methods: We performed a randomized double-masked, controlled two-way cross-over study. Twelve healthy male non-smoking volunteers, aged between 19 and 35 years were included in the study. Two drops of clonidine or brimonidine were administered in the subjects’ study eyes. Continuous measurement using the compact laser Doppler Flowmeter was done during a 6 minutes squatting period, to assess choroidal blood flow regulation during an increase in ocular perfusion pressure. Results: IOP decreased significantly after administration of the α2 agonists (p < 0.001 vs. each baseline, p = 0.8 between groups). Choroidal blood flow was significantly reduced by both drugs at baseline (p < 0.01 each) as well as during isometric exercise (p < 0.01 each). Vascular APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A29 prednisolone on optic nerve head blood flow in patients with Pharmacokinetics and pharmacodynamics of the acute optic neuritis. dual FII/FX inhibitor BIBT 986 in endotoxin-induced Methods: Nine patients with acute optic neuritis were coagulation included in the study. One gram of prednisolone was infused Judith M Leitner1, Bernd Jilma1, Florian B Mayr1, intravenously over 30 minutes on three consecutive days. On each study day optic nerve head blood flow was Francesco Cardona1,2, Alexander O Spiel1, measured using laser Doppler flowmetry. The ocular Christa Firbas1, Karin Rathgen3, Hildegard Stähle3, 3 3 hemodynamic measurements were performed on the Uwe Schühly and Eva U Graefe-Mody contralateral eye of the patients with optic neuritis before 1 Department of Clinical Pharmacology, Division of and immediately after cessation of the infusion. Intraocular Haematology and Immunology, Medical University of pressure and systemic blood pressure was measured before Vienna, Austria and after the infusion on each study day. Data was analyzed 2Department of Pediatrics, Division of Neonatology using a 3–2 repeated measures ANOVA model. and Intensive Care, Medical University of Vienna, Results: Prednisolone increased optic nerve head blood Austria flow in the patients under study (p < 0.05). No significant 3Boehringer Ingelheim Pharma GmbH & Co. KG, change in mean arterial pressure (p = 0.14) or intraocular Biberach, Germany pressure (p = 0.91) could be detected in the patients treated E-Mail: [email protected] with high dose prednisolone. Conclusions: High dose prednisolone showed a small but Introduction: BIBT 986 is a dual inhibitor of factors Xa and significant increase in optic nerve head blood flow. Further IIa. The aim of this study was to compare with placebo the studies are required to study whether this effect contributes effect of three doses of BIBT 986 on coagulation, platelet to the therapeutic efficacy of cortisone in patients with optic activation and inflammation. neuritis. Methods: This was a prospective, randomized, double- blind, placebo-controlled, parallel-group dose escalation trial A31 in 48 healthy male volunteers. Participants received one of Influence of the Duffy genotype on three doses of BIBT 986 or placebo intravenously together pharmacokinetics and pharmacodynamics of with a bolus infusion of 2 ng/kg lipopolysaccharide (LPS). recombinant monocyte chemoattractant protein Results: BIBT 986 dose-dependently changed global (MCP-1) in vivo coagulation parameters and in vivo markers of thrombin Florian B Mayr1, Alexander O Spiel1, Judith M Leitner1, generation and action: BIBT 986 doses, which prolonged Christa Firbas1, Janet Schnee2, James Hilbert2 and activated partial thromboplastin time by 100%, completely Bernd Jilma1 suppressed the LPS-induced increases in prothrombin 1Department of Clinical Pharmacology, Medical fragment, thrombin-antithrombin complexes and D-dimer, which were 6.1, 14.5, and 3.5-fold in the placebo group, University of Vienna, Austria 2 respectively. BIBT 986 did not influence inflammation, Boehringer Ingelheim, Ridgefield, CT, USA fibrinolysis, or platelet activation. E-Mail: [email protected] Conclusion: BIBT 986 is a potent anticoagulant in the Monocyte chemoattractant protein-1 (MCP-1) binds to the human endotoxemia model. Duffy antigen (Fy) on erythrocytes, which may act as a sink for several chemokines including MCP-1. We hypothesized A30 that infusion of MCP-1 could result in different Effects of high dose prednisolone on optic nerve pharmacokinetics of MCP and possibly altered head blood flow in patients with acute optic pharmacodynamics between Duffy positive and negative neuritis individuals. The primary aim of this trial was to compare 1,2 1,2 Hemma Resch , Günther Weigert , pharmacokinetics of MCP-1 between Duffy positive and 1,2 2 2 Gerhard Garhöfer , Karl Kircher , Guido T Dorner , Duffy negative individuals under infusion of recombinant Andreas Reitner2 and Leopold Schmetterer1,3 human MCP-1. This was a randomized, double-blinded, 1Department of Clinical Pharmacology, Medical placebo-controlled dose escalation trial in 36 healthy University of Vienna, Austria volunteers. Subjects received infusions of 0.02–2.0 µg/kg 2Department of Ophthalmology, Medical University of MCP-1 or placebo for one hour. MCP-1 displayed linear Vienna, Austria pharmacokinetics. Duffy negative individuals reached 3Center of Biomedical Engineering, Medical University maximal plasma levels earlier, but plasma concentration profiles were not altered. MCP-1 markedly increased of Vienna, Austria monocyte counts, and estimated EC values were 10-fold E-Mail: [email protected] 50 higher in Duffy positive than Duffy negative subjects. Purpose: In the present study patients with optic neuritis Increased monocyte counts were associated with decreased were treated with high dose prednisolone. Little information surface expression of intercellular adhesion molecule 1 is available about the effects of this treatment on ocular (ICAM-1, CD54). In contrast, MCP-1 neither altered CCR-2 blood flow. We set out to investigate the effects of high dose or CD11b surface expression nor markers of platelet or endothelial activation, inflammation and coagulation. MCP-1 acts as a highly selective chemoattractant for monocytes in APHAR 2007 Abstract Preview for publication in BMC Pharmacology humans. The Duffy antigen had minimal effects on isolated rat brain stem which allowed to measure the pharmacokinetics of MCP-1, but may affect EC50 values. enhancer effect of (−)-deprenyl and to detect how the releasing effect of methamphetamine hinders the recognition HUNGARIAN SOCIETY FOR EXPERIMENTAL AND of its enhancer effect. We fill up the intraneuronal transmitter CLINICAL PHARMACOLOGY stores of the noradrenergic neurons in an isolated rat brain stem with [3H]noradrenaline and thereafter continuously A32 measure the amount of the labeled transmitter released from the organ. Electrical stimulation increases the amount of The selective enhancer substance (−)-BPAP 3 counteracts the histological and functional [ H]noradrenaline released into the bathing fluid. Due to its enhancer effect, 2 µg/ml (−)-deprenyl significantly increases consequences of an experimental stroke in rats the amount of labeled transmitter to the same stimulation. László Dénes and Ildikó Miklya The addition of 2 µg/ml amphetamine or methamphetamine Department of Pharmacology and Pharmacotherapy, to the bathing fluid in the resting state rapidly leads to the Semmelweis University, Budapest, Hungary continuous release of a high amount of [3H]noradrenaline E-Mail: [email protected] into the organ bath and electrical stimulation is unable to further increase the amount of the labeled transmitter, thus The effect of the newly developed, up to the present most hindering the detectability of the enhancer effect. potent and selective enhancer substance, R-(−)-1- Acknowledgments (benzofuran-2-yl)-2-propylaminopentane [(−)-BPAP], was This work was supported by the Health Scientific Council studied in rats exposed to an experimental stroke. (ETT 140/2003, ETT 606/2006). Permanent focal ischemia was induced by cortical photothrombosis in Wistar rats with Rose Bengal (20 mg/kg, A34 i.v.), irradiated by a cold light source for 10 min. Rats were New method for detecting ATP release from rat treated daily with (−)-BPAP (0.2 mg/kg, i.p.) for 3 days. The infarction was visualized with 2% TTC. The lesion size was hippocampal slices evaluated by morphometry using image analysis software Attila Heinrich, E Sylvester Vizi and Beáta Sperlágh (AxioVison/Zeiss). The effect of (−)-BPAP on the learning Laboratory of Molecular Pharmacology, Institute of ability of rats was measured in the shuttle box. The Experimental Medicine, Hungarian Academy of acquisition of a two-way conditioned avoidance reflex (CAR) Sciences, Budapest, Hungary was analyzed during 5 consecutive days. The rats were E-Mail: [email protected] trained with 100 trials per day. The light-irradiation for 10 min after injection of Rose Bengal caused well-demarcated Chemical signaling has a key role in neural and non-neural tissue damage in the cerebral cortex and decreased the function; however, only a few techniques are available to performance of the lesioned rats in the shuttle box. measure the concentrations of neurotransmitters and (−)-BPAP treatment caused a statistically significant modulators directly in the extracellular space. A recent reduction of the infarct size (from 1.190 ± 0.096 mm2 to advance in this area is the development of the multi- 0.256 ± 0.057 mm2; n = 18) and improved the performance enzymatic microelectrode biosensor technique whereby we of the rats (45.75 ± 15 to 75.67 ± 7.2; n = 12). The data are can measure the concentration of different neurotransmitters in excellent harmony with the series of papers which and modulators around single neurons or synaptic structures described the protective effect of (−)-BPAP in various types in the brain with high temporal and spatial resolution. Micro- of enhancer-sensitive cultured cells. biosensors are also ideal for detecting real-time Acknowledgments neurotransmitter release in the central nervous system in This work was supported by the Health Scientific Council vivo because they are minimally invasive. The ATP (ETT 140/2003, ETT 606/2006). biosensor is formed by coating a Pt microelectrode with an ultrathin biolayer containing glycerol kinase and glycerol-3- A33 phosphate oxidase, which results in the production of The basic difference in the pharmacological electroactive H2O2, detected by amperometry. The null spectrum between methamphetamine and its sensor, which possesses the silicate layer but lacks the enzymes, shows near-identical responses to the derivative, the enhancer substance (−)-deprenyl interferences potentially allowing the use of differential Ildikó Miklya recordings to remove almost all of the interfering signals. We Department of Pharmacology and Pharmacotherapy, used the ATP microelectrode biosensor to demonstrate the Semmelweis University, Budapest, Hungary release of ATP from rat hippocampal slices in vitro. The E-Mail: [email protected] basal ATP levels have been estimated to be as low as a few nmol/L (25 ± 5 pA). The ATP sensor exhibited a rapidly Beta-phenylethylamine (PEA), the parent compound of both + increasing current during K depolarization which reached methamphetamine and (−)-deprenyl, has two effects: it is a 341 ± 170 pA (peak concentration 0.79 ± 0.2 µmol/L). This releaser of catecholamines and a natural enhancer + effect was significantly decreased by the Na channel substance. Methamphetamine, the closest mother 2+ blocker TTX (0.17 ± 0.08 µmol/L) and by Ca -free medium compound of (−)-deprenyl, acts like PEA. (−)-Deprenyl was (0.22 ± 0.1 µmol/L). the first PEA derivative which lost the releasing property but preserved the enhancer effect. We elaborated a method on APHAR 2007 Abstract Preview for publication in BMC Pharmacology

Acknowledgments following the injection of PTZ. The following parameters This work was supported by the grant from the Hungarian were evaluated: survival rate, duration of survival, seizure Research and Development Fund (NKFP1A/002/2004). latency, seizure level (from 0 to 4) and seizure duration. The activity score for each mouse was defined as the seizure A35 duration time-weighted average of the seizure levels. The P2 receptor-mediated modulation of noradrenaline significance of the survival rate was assessed by the Chi- release by electrical field stimulation and ischemic square test, for the other parameters we applied one-way conditions in superfused rat hippocampus slices ANOVA. mCPP (1, 5 and 25 mg/kg s.c.) significantly Cecília Csölle, Attila Heinrich and Beáta Sperlágh increased the rate and the duration of survival and changed the pattern of seizure levels. These effects followed a bell- Laboratory of Molecular Pharmacology, Institute of shaped dose-response curve and were completely abolished Experimental Medicine, Hungarian Academy of by 5 mg/kg of SB 242084, although the antagonist alone did Sciences, Budapest, Hungary not modify the seizure parameters. These results indicate E-Mail: [email protected] that the anticonvulsant effects of mCPP might be mediated by activation of 5-HT receptors. However, the lack of the In this study the regulation of the release of noradrenaline by 2C effect of the selective antagonist SB 242084 (1, 2.5 and 5 P2 receptors was investigated in hippocampus slices mg/kg s.c.) on the seizure parameters suggests that the preincubated with [3H]NA. Electrical field stimulation (EFS; 2 function of this 5-HT receptor subtype depends on the level Hz, 240 shock, 1 ms) enhanced the release of noradrenaline of 5-HT tone. in a [Ca]o-dependent manner. In contrast, the excess release in response to ischemic-like conditions (combined A37 oxygen and glucose deprivation) was [Ca]o-independent. The Na+ channel blocker tetrodotoxin (1–3 µM) abolished Ionotropic neurotransmitter receptors: activation both EFS-evoked and in vitro ischemia-evoked release of and allosteric modulation 1 2 tritium. The P2 receptor agonists ATP, ADP and 2-MeSADP Gábor Maksay and László Fodor 1 concentration-dependently decreased the tritium overflow Department of Molecular Pharmacology, Institute of with the potency order of ADP > 2-MeSADP > ATP. The Biomolecular Chemistry, CRC-HAS, Budapest, inhibition by ATP (300 µM) was prevented by the P2 Hungary 2 receptor antagonist PPADS (30 µM), by the P2Y1 receptor Department of Pharmacology, G. Richter Plc., antagonist MRS2179 (10 µM) and by the P2Y12/13 receptor Budapest, Hungary antagonist 2-MeSAMP (10 µM). Under ischemic-like E-Mail: [email protected] conditions the P2X1 receptor antagonist PPNDS (1 µM) 3 inhibited the outflow of [ H]NA, whereas MRS2179 (10 µM) Introduction: GABA and glycine are major inhibitory significantly increased the tritium outflow. PPADS and neurotransmitters. GABAA and glycine receptors (GlyR) form 3 2-MeSAMP did not affect ischemia-evoked [ H]NA efflux. pentameric chloride channels and belong to the Cys-loop

RT-PCR analysis revealed that mRNA encoding P2Y12 and receptor superfamily with 5-HT3 serotonin and nicotinic P2Y13 receptor subunits were expressed in the brainstem receptors. Homology modelling has revealed distinctive including locus coeruleus. The pharmacological profile of the binding interactions of antagonists and agonists in the underlying receptor subtype resembles the P2Y1 and P2Y13 interface of 5-HT3A receptors leading to ligand translocation, receptor phenotype, and the endogenous activation of P2X1 closure of the binding cavity and ionophore activation. and P2Y1 receptors contribute to the modulation of Allosteric modulation of ionotropic receptors enables the noradrenaline efflux upon ischemic-like conditions. pharmacological fine-tuning of neurotransmission. Methods: Radioligand binding of [3H]EBOB and 3 A36 [ H]strychnine to native GABAA and recombinant GlyRs,

Further evidence for the involvement of 5-HT2C respectively, and whole-cell patch clamp electrophysiology receptors in the pentylenetetrazole model of in cultured rat cerebellar granule cells. epilepsy Results: A 17β-alkenyl derivative of the neurosteroid Pál Riba, György Bagdy and Valéria Kecskeméti allopregnanolone antagonized the potentiating effects of Department of Pharmacology and Pharmacotherapy, allopregnanolone selectively on a cerebellar (α6βδ) population of GABA receptors with nanomolar potency. Semmelweis University, Faculty of Medicine, A Nortropine esters exerted bidirectional allosteric modulation Budapest, Hungary of GlyRs: nor-O-zatosetron had the highest affinity reported E-Mail: [email protected] for GlyRs. The anaesthetic propofol restored the potency of glycine impaired by a point mutation R271L of GlyR α1 To clarify the role of 5-HT2C receptors in the generation of subunits leading to hyperekplexia, an inherited neurological epilepsy this study investigated the effects of the 5-HT2C disorder. receptor-preferring agonist 1-(m-chlorophenyl)-piperazine Conclusions: Some of these allosteric modulators have (mCPP) and the subtype-selective 5-HT2C receptor nanomolar potencies and serve as promising leads for antagonist SB 242084 in pentylenetetrazole (PTZ)-evoked subunit-selective modulation of ionotropic receptors. seizures in mice. All drugs were given subcutaneously (s.c.) Acknowledgments 60 minutes before PTZ injection (100 mg/kg s.c.). The Support: OTKA K-62203. seizure patterns were recorded during a 60 min period APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A38 passive nursing, littering and behaviours out of the nest) was Evidence against a separate high affinity binding checked on the 2nd, 3rd, 5th, 7th and 9th PDs. Analgesic affect of MO was measured in tail-flick test in the offspring site on the P2X3 receptor Robert Karoly1, Arpad Mike1, Zoltan Gerevich2, on PD 23. Conditioned place preference (CPP) inducing Peter Illes2 and E Sylvester Vizi1 effect of MO was checked in adult offspring. Results: (1) Chronic MO treatment significantly impaired the 1Institute of Experimental Medicine, Budapest, MB. (2) Analgesic effect of MO was less marked, the CPP- Hungary 2 inducing effect of MO, however, was more marked in Rudolf Boehm Institute of Pharmacology and offspring. Toxicology, Leipzig, Germany Conclusions: Perinatal MO exposure alters the MO E-Mail: [email protected] sensitivity in the offspring. The enhanced sensitivity to the reinforcing effect of MO in CPP, however, may be the It has been proposed that P2X receptors possess a unique 3 consequence of altered maternal behaviour, too. mechanism of agonist-induced conformational transitions. Acknowledgments Recovery from ATP-induced desensitization was found to be This work was supported by Hungarian grants OTKA K- very slow; during this period a special agonist binding site 60999 and ETT-441/2006. was supposed to be formed which should bind the agonist with high affinity and promote desensitization without A40 activation. The authors supposed that this high affinity Electrophysiological effects of heat shock proteins binding site is absent from non-activated receptors. The theory was supported by an unexpected outcome of an and dofetilide on cardiac preparations of experiment in which a low concentration of agonist was streptozotocin-induced diabetic rats applied at different phases during recovery from Éva Virágh, Andrea Szebeni and Valéria Kecskeméti desensitization. The inhibition by a low concentration of Department of Pharmacology and Pharmacotherapy, agonist was stronger when it was applied during the early Semmelweis University, Budapest, Hungary phase of recovery when more desensitized receptors were E-Mail: [email protected] present. The authors used different agonists for initial desensitization and for prolonged perfusion at low Vaccination with different heat shock proteins (HSP60, concentration. We repeated the experiment on HEK 293 HSP65) prevented the development of low-dose cells expressing human P2X3 receptors with the same streptozotocin (STZ)-induced autoimmune diabetes in results. However, when we used the same agonist at both C57BL/KSJ mice. The aim of our work was to examine concentrations the inhibition was stronger when the low whether the vaccination with HSP65 can prevent the well- concentration was applied during the late phase. known cardiac electrophysiological characteristics of Simulations revealed that formation of high affinity binding diabetes. The electrophysiological effects of dofetilide (a sites does not require any unique mechanism and can be class III antiarrhythmic drug) were also studied. Diabetes readily described by an allosteric mechanism. Furthermore, was induced by a low dose of STZ (3 × 30 mg/kg i.p.), the they predict that the unexpected phenomenon can only vaccination with HSP65 (100 μg/animal) occurred 7 days occur when a rapidly dissociating drug is replaced by a before STZ treatment. The parameters of transmembrane slowly dissociating drug on the receptor. action potentials (APs) of right ventricular papillary muscles in rats were studied by the microelectrode technique. The A39 previously observed prolongation of AP duration in diabetes Changes in the action of morphine challenge induced by a high dose of STZ was recorded in the case of following perinatal morphine exposure in rats low dose of STZ too. The vaccination with HSP65 could not modify the increase of AP duration. Dofetilide (1–10 μM) did Melinda Sobor1,2, Julia Timár1, Susanna Gyarmati1 not cause any significant alterations in the parameters of AP and Susanna Fürst1,3 1 either in diabetic or in the HSP-treated animals. Our results Department of Pharmacology and Pharmacotherapy, with dofetilide differ from published data. This result could Semmelweis University, Budapest, Hungary be due to the fact that the ion current characteristic of the rat 2 National Institute of Pharmacy, Budapest, Hungary ventricular repolarization phase highly differs from that of 3 HAS-SE Neuropsychopharmacology Research other species. We need further experiments to find out Group, Budapest, Hungary additional explanations of our results. E-Mail: [email protected] Acknowledgments Supported by Grant ETT 578/2006 of the Ministry of Health. Objective: In our previous experiments decreased adaptability in rats exposed to perinatal morphine (MO) was observed 2 days after weaning. The aim of the present study was to examine how chronic MO treatment of dams influences the maternal behaviour (MB) and the sensitivity toward MO in offspring. Methods: Pregnant Wistar rats were treated daily with MO (10 mg/kg s.c.) from the day of mating until weaning (post partum day [PD] 21). Spontaneous MB (active nursing, APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A41 shown [1]. The two major hypotheses that explain use- and Exploring the heterogeneity of use-dependent state-dependent inhibition are the MRH [2] and the GRH [3]. sodium channel inhibitor drugs. I: Fast- vs. slow- In this study we investigated which hypothesis better inactivated state preference describes inhibition by various drugs, and whether drugs can Nora Lenkey, Robert Karoly, E Sylvester Vizi and be classified based on this aspect of their mechanism of action. We assumed that the two hypotheses are not Arpad Mike mutually exclusive, and speculated that both altered affinity Institute of Experimental Medicine, Budapest, Hungary (as predicted by the MRH) and altered accessibility (as E-Mail: [email protected] predicted by the GRH) to the inactivated state can be expressed as changes in association and dissociation rates Certain drugs evidently exert their therapeutic effects via upon conformational transition. We developed a method to sodium channel inhibition: local anesthetics, class I test the relative contribution of affinity and accessibility in the antiarrhytmics and certain anticonvulsants. Novel sodium effect of the drugs based on the degree of inhibition channel inhibitor compounds are actively investigated for (reflecting affinity) and the time constant of the onset of other indications involving stroke, ischemia, various inhibition (reflecting accessibility) as a function of changes in neurodegenerative conditions and pain syndroms. All these the voltage protocol. We tested the method by simulations drugs cause voltage- and use-dependent inhibition of and found that original parameters of a simulated drug can sodium channels. However, biophysical properties of be deduced using the method. Experiments using 12 fast- inhibition can differ widely even between use-dependent inactivated state-preferring use-dependent sodium channel sodium channel inhibitors which seem to act similarly. inhibitors suggest significant differences in this aspect of the Recently it has been proposed that the mechanism of mechanism. inhibition can be more important than potency or isoform References selectivity regarding the therapeutical potential of the drugs 1. Lenkey N, Karoly R, Kiss JP, Szasz BK, Vizi ES and (e.g. [1]). One important question is which conformational Mike A. The mechanism of activity-dependent sodium state is preferred by the drug. In this study we attempted to channel inhibition by the antidepressants fluoxetine discriminate preference to fast- vs. slow-inactivated and desipramine. Mol Pharmacol 2006; 70:2052–2063. conformations. Slow association to fast-inactivated state and 2. Hille B. Local anesthetics: hydrophilic and fast association to slow-inactivated state cannot be hydrophobic pathways for the drug-receptor distinguished using traditional protocols. We have recently reaction. J Gen Physiol 1977; 69:497–515. developed a protocol to test fast- vs. slow-inactivated state 3. Starmer CF, Grant AO and Strauss HC. Mechanisms of preference using electrophysiology only, i.e. without use-dependent block of sodium channels in excitable mutagenesis or enzymatic treatment experiments. We tested membranes by local anesthetics. Biophys J 1984; 28 use-dependent sodium channel inhibitors of different 46:15–27. chemical structure and therapeutic indication using this protocol, and found that the mechanisms primarily overlap with the latter. A43 References Electrophysiological characteristics of heart 1. Ilyin VI, Pomonis JD, Whiteside GT, Harrison JE, ventricular papillary muscles from histidine Pearson MS, Mark L, Turchin PI, Gottshall S, Carter RB, decarboxylase knockout and wild-type mice: Nguyen P, Hogenkamp DJ, Olanrewaju S, Benjamin E effects of rosiglitazone and Woodward RM. Pharmacology of 2-[4-(4-chloro-2- Andrea Szebeni and Valéria Kecskeméti fluorophenoxy)phenyl]-pyrimidine-4-carboxamide: A Department of Pharmacology and Pharmacotherapy, potent, broad-spectrum state-dependent sodium Semmelweis University, Budapest, Hungary channel blocker for treating pain states. J Pharmacol E-Mail: [email protected] Exp Ther 2006, 318:1083–1093. Background: Thiazolidinediones (troglitazone, A42 rosiglitazone), synthetic peroxisome proliferator-activated Exploring the heterogeneity of use-dependent receptor agonists, act as insulin sensitizers but beyond their sodium channel inhibitor drugs. II: Drugs antidiabetic actions improve cardiac function in experimental described by the modulated receptor hypothesis animals. (MRH) and/or the guarded receptor hypothesis Aim and Methods: Our first aim was to characterise the (GRH) electrophysiological parameters of right ventricular papillary muscles from histidine decarboxylase knockout (HDC KO) Arpad Mike, Nora Lenkey, Robert Karoly and mice compared with those of wild-type (WT) by standard E Sylvester Vizi microelectrode technique. Furthermore we investigated the Institute of Experimental Medicine, Budapest, Hungary effects of rosiglitazone (1, 3, 30 μM) on transmembrane E-Mail: [email protected] action potentials. Results: In KO mice statistically significant prolongation of All therapeutically used sodium channel inhibitor drugs seem action potential duration (APD) and decrease in maximum to act similarly: in a use-dependent and state-dependent rate of rise of depolarisation phase (V , dV/dt) can be manner. These properties, however, can be caused by max observed. Rosiglitazone caused a concentration-dependent multiple mechanisms, as we and others have previously APHAR 2007 Abstract Preview for publication in BMC Pharmacology

shortening of APD in both types of mice but reduced Vmax A45 only in WT mice. 14-Methoxy-metopon: a highly potent µ opioid Conclusions: The most important difference in the agonist on rat vas deferens electrophysiological parameters (APD, Vmax) between HDC Kornél P Király1, Pál Riba1, Tamás Friedmann1, KO and WT mice could be due to the fact that HDC KO mice Mahmoud Al-Khrasani1, Helmut Schmidhammer2 and are more susceptible for hyperglycaemia. The results also Susanna Fürst1 suggest that rosiglitazone might act on K+ channels and this 1Department of Pharmacology and Pharmacotherapy, effect might take part in the protective effect of rosiglitazone in ischemia/reperfusion injury observed in rats, but further, Semmelweis University, Budapest, Hungary 2 direct ionic current measurements need to support this Department of Pharmaceutical Chemistry, Institute of explanation. Pharmacy, University of Innsbruck, Austria Acknowledgments E-Mail: [email protected] Supported by Grant ETT 578/2006 of the Ministy of Health. Morphine, the opioid analgesic most abundantly used for A44 severe acute and chronic pain, is associated with several adverse effects such as respiratory depression, tolerance Potential role of nociceptin and nocistatin in and dependence. The intensive search to find new opioids central regulation of gastric mucosal defense 1 1 2 possessing a better pharmacological profile has resulted in Zoltán S Zádori , Nashwan Shujaa , Kornélia Tekes 14-O-methyloxymorphone (14-O-MOX) and 14-methoxy- 1 and Klára Gyires metopon (MET), a highly µ-selective and very potent opioid 1 Department of Pharmacology and Pharmacotherapy, agonist. Our aim was to test the compounds in the rat vas Faculty of Medicine, Semmelweis University, deferens (RVD) bioassay system in order to further analyze Budapest, Hungary their µ receptor efficacy and elucidate the structure-activity 2Department of Pharmacodynamics, Faculty of relationship between the compounds and oxymorphone Pharmacy, Semmelweis University, Budapest, (OX), their parent molecule. RVD was prepared, mounted Hungary and stimulated as described by Ronai et al. [1]. Opioid E-Mail: [email protected] actions were measured by determining the inhibitory effects on the electrically evoked twitches. Antagonist activity was Nociceptin (NOC) and Nocistatin (NS) are two determined by the single-dose method. MET exerted full neuropeptides derived from pre-pro-nociceptin. NOC is the agonist activity in RVD, unlike OX and 14-O-MOX which endogenous ligand of the NOP receptor which exhibits were only partial agonists. Naltrexone, the reference µ marked structural analogy with opioid receptors. NOC has receptor antagonist, antagonized the inhibitory actions on been reported to exert multiple effects in the gastrointestinal the electrically evoked twitches of the test compounds with a tract. The aims of this study were to compare the similar potency than in mouse vas deferens indicating the gastroprotective effects of NOC and NS and to analyse the presence of µ opioid receptors in RVD. We found that MET mechanism of their gastroprotective action. Gastric mucosal is a highly efficacious µ receptor agonist and that the damage was induced by acidified ethanol in rats. The 5-methyl substitution may be responsible for the full agonist compounds were given i.c.v. Both NOC and NS (0.2–5 activity. We could not prove the distinction between rat vas nmol/rat) induced gastroprotective effects. Pre- deferens and other µ receptor-containing organs. administration of NS significantly decreased the effect of References NOC, as well as the competitive antagonist J-113397 (70 1. Rónai AZ, Berzétei IP and Kurgyis J. Opioid effects in nmol/rat) and vagotomy. The effect of both neuropeptides developing rat vas deferens. Cell Mol Neurobiol 1981, was reduced by naloxone (27 nmol/rat), naltrindole (4.8 1:335–342. nmol/rat), norbinaltorphimine (10 µg/rat) and β-FNA (20 nmol/rat). In conclusion, both NOC and NS initiate centrally A46 a series of events which result in gastric mucosal defense. Changes in stereotyped behaviour following acute The gastroprotective effect of NOC and NS is likely to be or repeated methylenedioxy-methamphetamine mediated by endogenous opioids and conveyed to the (MDMA) treatment periphery by a vagal-dependent mechanism. Susanna Szelényi1, Ágnes Bajnogel-Orosz1, Acknowledgments Julia Timár1, Susanna Gyarmati1, Melinda Sobor1,2 This work was supported by ETT 529/2006. and Susanna Fürst1,3 1Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary 2National Institute of Pharmacy, Budapest, Hungary 3HAS-SE Neuropsychopharmacology Research Group, Budapest, Hungary E-Mail: [email protected]

Objective: In our previous experiments a decrease of the MDMA-induced 5-HT syndrome (HTS) was observed following repeated administration of the dextrorotatory (d) APHAR 2007 Abstract Preview for publication in BMC Pharmacology enantiomer of MDMA. The aim of the present study was to A48 examine how the dopamine agonist-induced stereotyped The possible link between insulin resistance and behaviour (SB) alters following single or repeated d-MDMA increased cardiovascular mortality treatment. Barna Peitl1, László P Drimba1, Róbert Döbrönte2, Methods: Wistar male rats were treated with single or József Németh1, Réka Zs Sári1, Csaba Pankucsi1, repeated (4 times with 2 hours intervals) doses (10 mg/kg) Angelika Varga1, László Fésüs3, József Tőzsér3 and of d-MDMA. Three, 7 and 14 days later the intensity of Zoltán Szilvássy1 d-MDMA-induced HTS and SB was examined. The SB- 1 inducing effect of d-amphetamine (AM) and apomorphine Department of Pharmacology and Pharmacotherapy, (APO) was also checked. University of Debrecen, Hungary 2 Results: (1) Following repeated administration of d-MDMA Department of Anesthesiology and ICU, Petz Aladár the intensity of MDMA-induced HTS decreased while that of County Hospital, Győr, Hungary SB increased. (2) Single dose pretreatment with d-MDMA 3Department of Biochemistry and Molecular Biology, did not influence the d-MDMA-induced HTS but enhanced University of Debrecen, Hungary the SB. (3) The d-AM-induced SB also increased while the E-Mail: [email protected] APO-induced SB did not change. Conclusion: A single dose of d-MDMA, which, in contrast to Introduction: Hyperinsulinaemia and insulin resistance are the repeated treatment, did not alter the 5-HT-related considered as independent risk factors of ischemic heart behaviour, may already result in development of DA disease. We sought whether hyperinsulinaemia per se is of sensitization. significant influence on cardiac arrhythmia generation in Acknowledgments conscious rabbits. This work was supported by Hungarian grants OTKA Methods: Chronically instrumented conscious rabbits were K-60999 and ETT-441/2006. equipped with a right ventricular electrode catheter for pacing and recording the intracavitary electrogram as well A47 as with arterial and venous catheters for blood sampling, blood pressure monitoring and for insulin and glucose The role of cannabinoid CB1 receptor agonists in gastric mucosal protection in rats and mice infusions, respectively. Hyperinsulinaemia was produced by Nashwan Shujaa1, Zoltán S Zádori1, 2-step hyperinsulinaemic (35.7 ± 7.4 and 103.2 ± 10.5 µU/ml) euglycaemic (5.5 ± 0.5 µU/ml) glucose clamping. Mahmoud Al-Khrasani1, Susanna Fürst1, Programmed electrical stimulation (PES) was applied for Tibor Wenger2 and Klára Gyires1 1 ventricular effective refractory period (VERP) determination Department of Pharmacology and Pharmacotherapy, and arrhythmia generation. Semmelweis University, Budapest, Hungary Results: The VERP shortened from 110.4 ± 3.7 to 2 Department of Human Morphology, Semmelweis 104.8 ± 2.9 ms, (p < 0.05) and from 109.3 ± 2.9 to University, Budapest, Hungary 101.4 ± 1.7 ms (p < 0.05) in animals with 35 and 103 µU/ml E-Mail: [email protected] clamped hyperinsulinaemic euglycaemia, respectively. The incidence of ventricular premature beats, non-sustained CB1 receptor agonists inhibit stimulated gastric acid ventricular tachycardia and sustained ventricular tachycardia secretion and exert an anti-ulcer activity in acid-dependent induced by PES increased from control 11, 0, 0% to 24 ulcer models. The aims of this study were to investigate the (p < 0.05), 5, 0%; and 56, 44 (p < 0.001 for each), 0% in gastroprotective effect of cannabinoids in an acid- animals with 35 and 103 µU/ml clamped hyperinsulinaemic independent ulcer model and to analyze the role of opioid euglycaemia, respectively. and vanilloid receptors in this effect. Gastric mucosal Conclusion: The results provide evidence for the “sui damage was induced by acidified ethanol in rats and in generis” proarrhythmic effect of hyperinsulinaemia in +/+ −/− CB1 and CB1 mice. Anandamide, methanandamide and otherwise healthy rabbits. The results also suggest that this WIN-55,212-2 inhibited the ethanol-induced gastric mucosal is underpinned by a hyperinsulinaemia-induced reduction of damage significantly after peripheral and central VERP. administration, and their effects were reversed by the CB1 receptor antagonist SR141716A. The gastroprotective effect of cannabinoid agonists was significantly decreased by naloxone and partially by capsazepine (TPRV1 receptor antagonist). The gastroprotective effect of opioid peptides –/– DAGO and deltorphin II was significantly reduced in CB1 mice. In conclusion, cannabinoid CB1 receptors are likely to be involved in gastric mucosal defense. The effect seems to be central, and correlation between opioid and cannabinoid system in gastric mucosal protection may be raised. Acknowledgments This work was supported by ETT 529/2006. APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A49 A50 Nitrate tolerance-induced deterioration of the The peripheral antinociceptive effect of DAMGO ischemic adaptability of the heart and 6β-glycine-substituted 14-O- Zoltán Szilvássy1, János Szaszkó2, Róbert Döbrönte3, methyloxymorphone (HS-731) after systemic József Németh2, Réka Zs Sári2, Csaba Pankucsi2, administration in a mouse visceral pain model Angelika Varga2, József Tőzsér4, László Fésüs4 and Mahmoud Al-Khrasani1, Mariana Spetea2, Barna Peitl1 Tamás Friedmann1, Pál Riba1, Kornél P Király1, 1CERAMED Research Group, University Debrecen, Helmut Schmidhammer2 and Susanna Fürst1,3 Hungary 1Department of Pharmacology and Pharmacotherapy, 2Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary University of Debrecen, Hungary 2Department of Pharmaceutical Chemistry, Institute of 3Department of Anesthesiology and ICU, Petz Aladár Pharmacy and Center for Molecular Biosciences, County Hospital, Győr, Hungary University of Innsbruck, Austria 4Department of Biochemistry and Molecular Biology, 3HAS-SE Neuropsychopharmacology Research University of Debrecen, Hungary Group, Budapest, Hungary E-Mail: [email protected] E-Mail: [email protected]

Introduction: We tested whether postconditioning limited Inhibition of nociception through activation of peripheral µ infarct size in rabbits with haemodynamic nitrate tolerance. opioid receptors (MOR) avoiding the central adverse effects Methods: Male rabbits made tolerant to the hypotensive of opioids has added a new possibility to manage pain response to 30 µg/kg intravenous nitroglycerin (NG) by a control. Here we report the antinociceptive effects of the preceding one-week exposure to transdermal NG (0.07 peripherally restricted MOR agonist HS-731, the peptide mg/kg/h) were subjected to 35 min coronary occlusion (test DAMGO, and morphine in the writhing test in mice. S.c. and ischemia) followed by 3 h of reperfusion with the following i.c.v. HS-731 dose-dependently and completely inhibited additional interventions: no intervention (NI); writhing, being 24–598 times more potent than the two postconditioning pacing (PPC): five cycles of 5 min periods MOR-selective agonists DAMGO and morphine. However, of rapid ventricular pacing (500 b.p.m.), or postconditioning extremely high s.c./i.c.v. potency ratios were calculated for coronary occlusion (PCO): five cycles of 5 min coronary HS-731 and DAMGO and much lower for morphine. occlusion with 10 min interpacing/interocclusion intervals, Remarkably, a long duration of action was induced by applied after the end of the test ischemia. These protocols HS-731 and much shorter by morphine and DAMGO. The were applied in both nitrate-tolerant and non-tolerant antinociceptive effects of systemic opioids were reversed by animals. Infarct size expressed as a percentage of area at s.c. naloxone while i.c.v. administration of the MOR selective risk (I/R) was determined by triphenyltetrazolium chloride antagonist CTAP significantly abolished the antinociceptive staining, left ventricular cyclic nucleotides were determined effect of s.c. morphine but completely failed to antagonize by radioimmunoassay from samples out of the area at risk, the effects of systemic HS-731 or DAMGO. In addition, in 75 min after the test ischemia. the rat vas deferens HS-731 and DAMGO, but not morphine, Results: In non-tolerant animals both PPC and PCO showed high intrinsic efficacy and naltrexone-sensitive reduced the I/R compared to the NI group. When animals agonist response at MOR by depressing electrically-evoked had been made nitrate-tolerant, the I/R was significantly contractions of this organ. These data demonstrate that the higher in the NI group compared with non-tolerant animals selectivity and high efficacy of HS-731 and DAMGO at and the beneficial effect of the PPC or PCO on the I/R peripheral MOR as well as their inability to cross the blood disappeared. brain barrier are a cornerstone for producing peripheral Conclusions: We conclude that (i) nitrate tolerance blocks antinociception after systemic administration. postconditioning induced by either PPC or PCO, (ii) PPC is Acknowledgments more effective post-conditioning challenge than PCO, and This work was supported by grants from the European (iii) nitrate tolerance per se reduces the capability of the Committee (QLK6-0999; EPILA), Hungarian research grants heart to tolerate an ischemic insult. (OTKA K-60999 and ETT-441/2006) and the Austrian Science Foundation (P15481). APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A51 AUSTRIAN SOCIETY OF TOXICOLOGY Ion channels alteration in dilated cardiomyopathy in human heart A52 Viktoria Szűts1, István Baczkó1, Dalma Ménesi4, Spin trapping experiments with ethyl-substituted Zoltán Varga-Orvos4, János Zs Kelemen4, EMPO derivatives (EEMPO) László G Puskás4, Zoltán Bódi1, Lajos Csincsik1, Klaus Stolze1, Nataliya Rohr-Udilova2, Nazamin Houshmand1, Miklós Bitay2, Thomas Rosenau3, Andreas Hofinger3 and Hans Nohl1 Miklós Csanády2, Norbert Jost1,3, László Virág1, 1Molecular Pharmacology and Toxicology Unit, Julius Gy Papp1,3 and András Varró1 Department of Natural Science, University of 1Department of Pharmacology and Pharmacotherapy, Veterinary Medicine, Vienna, Austria University of Szeged, Hungary 2Division of Gastroenterology and Hepatology, Clinic 22nd Department of Internal Medicine, Cardiology of Internal Medicine III, Medical University of Vienna, Center, Cardiac Surgery, University of Szeged, Austria Hungary 3Department of Chemistry, University of Natural 3Division of Cardiovascular Pharmacology, Hungarian Resources and Applied Life Sciences (BOKU), Academy of Sciences, Szeged, Hungary Vienna, Austria 4Laboratory of Functional Genomics, BRC, Hungarian E-Mail: [email protected] Academy of Sciences, Szeged, Hungary Free radicals in biological systems play a major role in the E-Mail: [email protected] onset of many diseases, e.g. oxygen-centered radicals such The complex mechanism by which genes are mostly as hydroxyl or superoxide radicals. In order to identify and involved in cardiomyopathy (clinical heart failure) is not localize these radicals a series of four novel spin traps have resolved. Cardiac ion channels are shown as a mixture of been developed and their structure fully characterized by 1 13 homomeric and heteromeric tetramers composed of closely [ H]- and [ C]-NMR spectroscopy as well as mass related subunits from homo- or heteromeric assembly of α spectrometry. The novel compounds can be described as and β subunits, and underlie the main part of the currents. ethyl-substituted EMPO derivatives, namely 5- The aim of this study was to compare the expression of the ethoxycarbonyl-3-ethyl-5-methyl-pyrroline N-oxide (3,5- ion channels and regulatory genes in ventricular muscle EEMPO), 5-ethoxycarbonyl-4-ethyl-5-methyl-pyrroline N- between failing and non-failing human heart. We also tested oxide (4,5-EEMPO), 5-ethoxycarbonyl-5-ethyl-3-methyl- the marker genes of inflammation, fibroblast, neuronal pyrroline N-oxide (5,3-EEMPO) and 5-ethoxycarbonyl-5- tissues and vascular vessels. Therefore, we have quantified ethyl-4-methyl-pyrroline N-oxide (5,4-EEMPO). Their spin the pore-forming α and auxiliary β subunit-coding mRNAs of trapping behaviour towards a series of different oxygen- and carbon-centered radicals is described. All compounds were ICa, INa, Ito, IKr, IKs, IK1 channels and regulatory genes by real- time qPCR (BioTrove) in left ventricle of heart tissues. obtained in two different stereochemical forms (cis and Coding genes for sodium ion channels have marked trans), but only 3,5-EEMPO and 5,3-EEMPO could be differences in failing and control tissues of left ventricular separated into the different diastereomers using muscle. Our results suggest that coding genes for Na+/Ca2+ conventional chromatographic procedures. The cis- and exchange current is mostly upregulated in dilated trans-forms exhibited considerably different spectral parameters and stabilities of the respective superoxide cardiomyopathies patients heart while NaV1.5 is much less. Potassium ion channel α and β subunits exhibit different adducts (ranging from about 12 to 35 min). In addition, spin distribution in the failing ventricle tissues versus control. adducts obtained from different carbon-centered radicals These findings may provide insight into a mechanism derived from methanol, ethanol, formic acid and linoleic acid responsible for hearth failure, DCM, HCM and RCM due to hydroperoxide have also been characterized. several regulatory genes-related up- or down-regulation of sodium, calcium, chloride and potassium ion channels. A53 Acknowledgments In situ monitoring of the genotoxic effects in the This work was supported by grants from OTKA 5K-522 and vicinity of a petrochemical plant with the 5W022. Tradescantia micronucleus (Trad-MCN) and with pollen abortion test Miroslav Mišík1, Karol Mičieta2, Martina Solenská2, Nataša Markusková2, Katarína Mišíková2 and Siegfried Knasmüller1 1Institute of Cancer Research, Medical University of Vienna, Austria 2Department of Botany, Faculty of Natural Sciences, Comenius University Bratislava, Slovakia E-Mail: [email protected] APHAR 2007 Abstract Preview for publication in BMC Pharmacology

The aim of this study was to monitor the genotoxic effects of presence and absence of superoxide dismutase which polluted air in Bratislava (Slovakia). In situ monitoring was catalyzes the dismutation of primarily produced superoxide carried out at a location polluted with emissions from a city radicals to H2O2. Amplex Red turned out to be a more incinerator and of a petrochemical plant. The Tradescantia sensitive analytic tool for H2O2 detection than HVA. micronucleus (Trad-MCN) test and pollen abortion assays with wild life plants (Chelidonium majus, Clematis vitalba, A55 Cichorium intybus, Linaria vulgaris) were used in this study. Toxicological investigation of nanoparticles: The experiments were carried out during two sampling effects on human cells periods i.e. 1997–2000 and 2003–2006. Compared to the Letizia Farmer1, Alexander Graff3, Sandra Szameit2, effects seen in Tradescantia plants exposed in a rural area Eva Valic4 and Helga Tuschl1 with low pollution levels, the MCN frequencies in pollen 1Toxicology Unit and 2Molecular Diagnostics Unit, tetrads were significantly higher in specimens from the Austrian Research Centers GmbH – ARC, urban site during the first period whereas no significant differences were found during the second sampling period. Seibersdorf, Austria 3 The results of the pollen abortion assays showed a similar Österreichische Staub- und pattern as that observed in the Trad-MCN bioassays, i.e. Silikosebekämpfungsstelle, Leoben, Austria 4 pronounced effects were only seen between 1997 and 2000. Austrian Worker`s Compensation Board (AUVA), The differences of the results obtained in the two sampling Vienna, Austria periods can be explained by reduction of the pollution of the E-Mail: [email protected] urban air due to new production technologies and installation of filter systems which led to a substantial The aim of the present study was the establishment of an in improvement of air quality parameters such as particulate vitro test system to reveal the potential risk to human health matter, organic volatiles and SO2 emissions. These results of nanoparticles at the workplace. The essential advantage confirm the sensitivity of the Tradescantia MCN test and of of in vitro investigations is to be non-invasive, the employees pollen abortion assays for the detection of air pollution and don't have to be bothered and the work routine doesn't have that both biomonitoring models reflect improvement of air to be intercepted. At occupational settings test cells on ® quality parameters. Transwell inserts were exposed to the workplace atmosphere or to particle filtered air for 1 to 3 hrs using a ® A54 CULTEX System. 2 types of co-cultures were tested: In the first type differentiated macrophages were exposed and H2O2 detection with 10-acetyl-3,7- dihydroxyphenoxazine: comparison with post-incubated with human lung epithelial cells. In the second type differentiated macrophages were seeded on homovanillic acid human lung epithelial cells and the co-culture was exposed. Katrin Staniek As endpoints for particle exposure cell viability (WST-1 Molecular Pharmacology and Toxicology Unit, assay), oxidative stress (DHR-Assay) and pro-inflammatory Department of Natural Sciences, University of cytokines (BDTM CBA-Assay) were evaluated. Cell viability Veterinary Medicine, Vienna, Austria testing showed a negative effect at high exposure. In cells E-Mail: [email protected] exposed to the workplace atmosphere an increased oxidative burst was detected compared to cells exposed to H2O2 is assumed to be produced and involved in several particle filtered air. Exposure of co-cultures resulted in (patho-)physiological processes. For the determination of significantly enhanced TNF-α, IL-6, IL-1β and IL-8 levels. low amounts of H2O2 formed in biological systems sensitive We could show that our in vitro exposure system is very well and reliable assays are necessary. Exploring suitable adapted for the assessment of adverse effects of detection systems for mitochondrial H2O2 production nanoparticles at the workplace. Our results indicate that different enzyme-catalyzed redox reactions were tested. By nanoparticles involve an occupational risk and further horseradish peroxidase (HRP) and H2O2, homovanillic acid experiments will be performed to analyse additional (HVA) and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex endpoints. Red) were enzymatically oxidized to the fluorescent HVA dimer (λex = 312 nm; λem = 420 nm) and resorufin (λex = 563 nm; λem = 587 nm), respectively. The specificity of the assays was confirmed by catalase which dose-dependently inhibited the H2O2-induced fluorescence increase. Amplex Red and HVA were applied to the H2O2-generating glucose/glucose oxidase system and compared for their sensitivity. Albumin, which is frequently added to mitochondrial or cell incubation media, significantly decreased the fluorescence intensity of the Amplex Red oxidation product while the fluorescence intensity of the HVA dimer was significantly increased. This effect was observed in 0.3 M sucrose and was lacking in 0.15 M KCl. The mitochondrial H2O2 formation was studied in antimycin A- inhibited succinate-respiring rat heart mitochondria in the APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A56 found to be upregulated. For functional studies we chose Submitochondrial particles from Saccharomyces primary co-cultures of normal and premalignant (GSTp+) rat cerevisiae to screen compounds for antiprotozoal hepatocytes. DNA synthesis was significantly higher in activity GSTp+ than in normal hepatocytes indicating an inherent Carmen Streicher1, Gerlinde Wiesenberger2, growth advantage of the premalignant cell population. FGF-8 and FGF-18 stimulated DNA preferentially in GSTp+ Thomas Rosenau3 and Lars Gille1 hepatocytes, while FGF-17 exhibited no effect. In HCC- 1Department of Natural Sciences; University of derived cell lines FGF-8, FGF-17 and FGF-18 stimulated Veterinary Medicine Vienna, Austria growth, which involved phosphorylation of ERK1/2 and S6. 2 Max F. Perutz Laboratories, University of Vienna, Conclusions: FGF-8 and FGF-18 induced preferential growth Austria of (pre)malignant hepatocytes and are highly upregulated in 3 Department of Chemistry, University of Natural liver tumors indicating auto- and/or paracrine stimulation Resources and Applied Life Sciences, Vienna, Austria during formation and progression of the tumors. The results E-Mail: [email protected] also show considerable similarities in the role of these FGFs in rat and human hepatocarcinogenesis implying that the The development of atovaquone (ATQ)-resistant plasmodia molecular mechanisms underlying chemically induced rat strains suggests a need of new drugs for the mitochondrial hepatocarcinogenesis and human hepatocarcinogenesis are bc1 complex as drug target in protozoa. It was the aim of the often identical. study to establish a model consisting of submitochondrial particles (SMP) from yeast (mimicking the protozoal drug A58 target) and bovine heart for the screening of compounds. Role of mesenchymal liver cells in mediating Saccharomyces cerevisiae wild-type strains (W303-1A, hepatic toxicity and carcinogenesis DBY747) were grown on YPD-media at 30°C for three days. Sandra Sagmeister, Wolfram Parzefall, After harvesting the cells their cell wall was digested by Christine Gauglhofer, Christopher Gerner, zymolyase and mitochondria were isolated. From these organelles SMP where prepared by sonication. The Rolf Schulte-Hermann and Bettina Grasl-Kraupp inhibition of the quinol:cyt c oxidoreductase activity Department of Medicine I, Institute of Cancer catalyzed by the mitochondrial bc1 complex of yeast and Research, Medical University of Vienna, Austria bovine heart SMP was measured for a nonspecies-specific E-Mail: [email protected] inhibitor stigmatellin and for the anti-malaria drug ATQ. IC50 values for stigmatellin in both SMP preparations confirmed Mesenchymal liver cells (Kupffer cells, KC; sinusendothelial its nonselectivity while ATQ inhibited the yeast complex cells, EC) are considered to play a role in the response of activity about 16 times stronger than the activity of bovine the liver to pro-inflammatory stimuli. We studied whether heart SMP. This confirms the functionality of yeast SMP as genotoxic and non-genotxic carcinogens are also capable to model for protozoa. A new lead structure TMC2O activate these mesenchymal liver cells. Liver cell suspensions were separated into hepatocytes, KC and EC. demonstrated an IC50 value of 808 µM and 112 µM in yeast and bovine heart SMP, respectively. This suggests that Cells were incubated with lipopolysaccharid (LPS), the structural adjustments are required to increase the inhibition genotoxic N-nitrosomorpholine (NNM) or non-genotoxic in yeast/protozoa. The yeast model in context with yeast carcinogens like nafenopin, cyproterone acetate, strains mimicking mutated protozoa provides an efficient phenobarbital, or arsenic. LPS and NNM incuced a release way to develop drugs for ATQ-resistant plasmodia strains. of TNF-α and superoxide while the other compounds showed mostly minor effects on KC and EC. To assess the A57 impact of activated mesenchymal cells on hepatocarcinogenesis a co-culture model of unaltered and Role of fibroblast growth factors in preneoplastic hepatocytes was used. DNA synthesis was hepatocarcinogenesis of humans and rats significantly higher in preneoplastic than unaltered cells and Christine Gauglhofer, Sandra Sagmeister, was further increased by supernatant of LPS-stimulated KC Wolfram Parzefall, Rolf Schulte-Hermann and and EC. The supernatant effect was greatly abrogated by Bettina Grasl-Kraupp antisera neutralizing heparin-binding epidermal growth Department of Medicine I, Institute of Cancer factor-like growth factor (HB-EGF). HB-EGF itself was a Research, Medical University of Vienna, Austria potent inducer of DNA synthesis and mitosis preferentially in E-Mail: [email protected] the preneoplastic hepatocytes. In conclusion, KC and EC, activated by pro-inflammatory stimuli, may contribute to The fibroblast growth factors (FGF) FGF-1 and FGF-2 are carcinogenesis via release of growth factors for often deregulated in hepatocellular carcinoma (HCC). preneoplastic hepatocytes. Whether this cell activation may Information is missing on the role of other FGF-family be caused by non-genotoxic carcinogens requires further members for hepatocarcinogenesis. We therefore investigations. determined the function of FGF-8, FGF-17 and FGF-18 in the development and progression of liver cancer. Half of the rat liver tumors studied showed enhanced expression of FGF-18. In about 50% of human HCC, FGF-8, FGF-17 and FGF-18 and the corresponding FGF-receptors 3 and 4 were APHAR 2007 Abstract Preview for publication in BMC Pharmacology

A59 blood cells of EW, who are exposed to vanadium pentoxide New epithelial and mesenchymal cell lines from via inhalation and in a matched control group (CG, n = 54). primary liver cancer to study cell interactions in Median level of vanadium serum concentrations of EW (2.23 hepatocarcinogenesis µg/l) was found to be 7-fold increased compared to CG (0.31 Sandra Sagmeister, Maria Eisenbauer, µg/l). In the standard single cell gel electrophoresis (SCGE) assay with leucocytes, no differences were detected Christine Pirker, Klaus Holzmann, Wolfram Parzefall, between EW and CG, but increased levels of oxidised DNA Christopher Gerner, Rolf Schulte-Hermann and bases (detected with FPG and ENDO III) were found Bettina Grasl-Kraupp (p < 0.05). Pretreatment of leukocytes with bleomycin Department of Medicine I, Institute of Cancer resulted in a higher extent of DNA migration (27%, Research, Medical University of Vienna, Austria p < 0.001) and reduced capacity of DNA repair (by 42%, E-Mail: [email protected] p < 0.001) in EW. Furthermore, the cytochalasin blocked micronucleus (CBMN) assay was carried out with peripheral To study cell interactions in tumor development, new lymphocytes (n = 24 per group). EW showed a 2.5-fold epithelial and mesenchymal cell lines were established from higher MN frequency as CG and a 7-fold increase of human hepatocellular carcinoma by spontaneous outgrowth nucleoplasmic bridges (which reflect formation of dicentric in culture. We obtained several hepatocarcinoma (HCC), chromosomes) was seen. Nuclear buds (attributable to gene B-lymphoblastoid (BLC) and myofibroblastoid (MF) lines. In- amplification events) were 3-fold higher in EW (p < 0.001 for depth characterization included cell kinetics, genotype, all events). Apoptosis rates did not differ significantly tumorigenicity, expression of cell-type specific markers and between the two groups, whereas necrosis rates doubled in proteome patterns. Many functions of cells of origin were EW (p < 0.001). Taken together, our results show increased found to be preserved. Thus, HCC cells secrete albumin and genetic damage in individuals which inhale vanadium dust, α1-antitrypsin, BLC cells phagocytose and release TNF-β, possibly indicating increased risk for cancer. other cytokines and reactive oxygen species upon Acknowledgments stimulation, while MF cells express fibulin-2, vimentin and This project was financed by a grant from the Austrian hepatocyte growth factor (HGF). We studied the impact of Social Insurance for Occupational Risks (AUVA), Vienna, the mesenchymal lines on hepatocarcinogenesis by in vitro Austria. assays. BLC and MF supernatants strongly increased DNA replication of premalignant hepatocytes. The stimulation by A61 MF lines was mainly attributed to HGF secretion. In HCC Influence of an Ironman triathlon on sister cells, MF supernatant had only minor effects on cell growth chromatid exchanges and high frequency cells but enhanced migration. MF lines also stimulated neoangiogenesis via vEGF release. BLC supernatant Stefanie Reichhold, Marlies Meisel, Oliver Neubauer induced dramatically death of HCC cells, which could be and Karl-Heinz Wagner largely abrogated by neutralizing the supernatant with Department of Nutritional Sciences, University of TNF-β-antiserum. In conclusion, the new cell lines reveal Vienna, Austria stage-specific stimulatory and inhibitory interactions E-Mail: [email protected] between mesenchymal and epithelial tumor cells. They offer new tools to unravel the role of the microenvironment during Introduction: Regular moderate exercise and its beneficial hepatocarcinogenesis. influence on health have already been well-investigated. However, information on high volume exercise which might A60 lead to increased oxidative stress and DNA damage is still Investigation of genotoxic effects of inhalative very limited. The purpose of the present study was to investigate the effect of an Ironman triathlon race (3.8 km occupational exposure to vanadium: results of a swim, 180 km cycle, 42 km run) on genomic stability and a multiple endpoint study 1 1 possible DNA damage. Veronika Ehrlich , Armen Nersesyan , Materials and Methods: The sister chromatid exchange 1 1 1 Christine Hoelzl , Franziska Ferk , Julia Bichler , (SCE) assay is a cytogenic biomarker, which can be 1 2 3 Kambis Atefie , Eva Valic , Andreas Schaffer , influenced by various factors such as oxidative stress that 1 1 Rolf Schulte-Hermann and Siegfried Knasmüller occurs during extreme endurance exercise. Within this study 1Institute of Cancer Research, Department of SCEs and high frequency cells (HFCs) were measured in Medicine I, Medical University of Vienna, Austria peripheral blood lymphocytes of nine well trained male 2 Austrian Social Insurance for Occupational Risks triathlets (age 38 ± 6 years; VO2 peak 55.59 ± 2.97 (AUVA), Vienna, Austria ml/kg/min; height 179.1 ± 4.19 cm; weight 76.3 ± 4.27 kg). 3Department of Medicine II, Medical University of Blood samples were collected 48 h before and 24 h post Vienna, Austria race. Results: The mean SCE frequency in the Ironman triathlets E-Mail: [email protected] 2 days before the race was 6.55 ± 2.71 per metaphase, Animal experiments showed that inhalative exposure to which was significantly higher than post race (5.69 ± 2.60 vanadate causes lung cancer. To assess DNA damage in SCEs per metaphase, p < 0.05). Additionally the mean of exposed workers (EW, n = 52) of a metal factory, we HFCs significantly decreased from 11.06 ± 2.66 per monitored DNA migration and formation of micronuclei in APHAR 2007 Abstract Preview for publication in BMC Pharmacology metaphase before the race to 9.42 ± 3.74 per metaphase Since a long line of years we have studied liver growth post race (p < 0.05). regulation (cell proliferation, apoptosis) in vivo. One of the Conclusion: The significant decrease of SCEs and HFCs prominent findings was that TGF-β1 constitutes a major after the Ironman triathlon race provides an indication of death signal in rat liver, acting in concert with nutritional endogenous repair mechanisms or counterregulations, factors to maintain liver cell number homeostasis. which seem to prevent DNA damages probably through Toxicological tests with rodents in vivo showed that liver releasing antioxidants. tumor promoter (e.g. phenobarbital) favour cell multiplication and inhibit apoptosis, thereby accelerating A62 hepatocarcinogenesis. To elucidate their mode of action β-Sitosterol oxidation products failed to show (MOA), along with the general need for alternative test mutagenic potential in the Ames test models in toxicology, we searched for a liver cell culture Karin Koschutnig1, Suvi Kemmo2, Anna-Maija Lampi1, system yielding a high concordance with our in vivo findings. Vieno Piironen1 and Karl-Heinz Wagner1 Here we report on a newly established cell line HCC-1.2 [Grasl-Kraupp et al., submitted] exhibiting the following 1Department of Nutritional Sciences, University of features: (1) a high sensitivity towards the anti-proliferative Vienna, Austria 2 and pro-apoptotic action of TGF-β1; (2) apoptosis is Department of Applied Chemistry and Microbiology, mediated via the intrinsic pathway as demonstrated by University of Helsinki, Finland caspase analysis; (3) inhibition of TGF-β1-induced apoptosis E-Mail: [email protected] by liver tumor promoter, as exemplified by phenobarbital; (4) glucose withdrawal exerts an additive effect to the pro- Background: Over the past few years phytosterols have apoptotic action of TGF-β1, all of which agree with our received great attention due to their serum cholesterol- previous in vivo observations on liver growth regulation. lowering effect. As a consequence a growing number of Thus, our observations suggest that the HCC-1.2 cells functional foods are fortified with phytosterols and their constitute a valid test system as it meets well with esters. In structure phytosterols are similar to cholesterol. prerequists for further studies tackling the MOA of liver Both contain an unsaturated ring structure and are therefore tumor promoter, along with their interaction with nutritional prone to oxidation. While possible health implications of factors. cholesterol oxidation products (COPs) have been well documented, data on phytosterol oxidation products (POPs) A64 are still rare. First data with different cultured mammalian Cancer and nutrition: role of amino acids (AA) for cells show for POPs similar toxicity like COPs. Objective: Therefore we investigated for the first time the regulation of hepatocellular apoptosis 1 2 1 possible mutagenic and pro-oxidative effects of two common Miriam Mayer , Adolf Ellinger , Anneliese Karwan , 1 1 oxidation products of β-sitosterol, 7-keto-sitosterol and 7β- Rolf Schulte-Hermann and Wilfried Bursch 1 OH-sitosterol, in the Ames test. Different Salmonella Department of Medicine I, Institute of Cancer thyphimurium strains, TA 98, 100, 102, were used. For Research, Research Unit Toxicology and Prevention, metabolic activation the oxidation products were treated with Medical University of Vienna, Austria a rat liver enzyme mixture (S9). To further investigate the 2Center for Anatomy and Cell Biology, Department of anti-/pro-oxidative effects the oxidant tBOOH was used. Cell Biology and Ultrastructure Research, Medical Results: In general neither 7-keto-sitosterol nor 7β-OH- University of Vienna, Austria sitosterol could increase the revertant colony numbers E-Mail: [email protected] beyond the doubled negative control, which was set as threshold for mutagenic activity. No dose-dependent Since decades nutrition is known to play a central role in increase could be observed. Since these two criteria must development of cardiac diseases, cancer and diabetes. be fulfilled in order to identify a compound as a possible However, the causative nutritional factors and their mode of mutagen our tests showed no increased risk by the two action are not well understood. Accumulating evidence investigated POPs. suggest that certain nutrients (including glucose and AA) may regulate cell signalling events/gene expression A63 independently of hormones, possibly acting through specific Control of hepatocellular apoptosis by cytokines nutrient “sensor” mechanisms [1]. Previous in vivo studies (TGF-β1), liver tumor promoter (phenobarbital) and by ourselves on the regulation of rat hepatocyte proliferation nutritional factors (glucose): approach to validate and apoptosis revealed that the lack of glucose and/or AA the hepatoma cell line HCC-1.2 as cell culture blocks the initiation of DNA synthesis (G1/S-transition). model Furthermore, feed restriction favoured apoptosis in rat liver. Anneliese Karwan, Julia Dornetshuber, Here we report on the role of AA for apoptosis of human hepatoma cells (HCC-1.2). The following changes in AA Rolf Schulte-Hermann and Wilfried Bursch composition – as compared to standard RPMI 1640 medium Department of Medicine I, Institute of Cancer – exerted a pro-apoptotic action, without and with TGF-β1 Research, Research Unit Toxicology and Prevention, treatment: (1) deprivation of branched chain AA, with Medical University of Vienna Val > Leu > Ile when tested individually; (2) deprivation of E-Mail: [email protected] Phe, Trp, Lys, Met, Thr, either as group or, with the exception of Met, when tested individually; (3) deprivation of APHAR 2007 Abstract Preview for publication in BMC Pharmacology

His and Gln seemed less effective than the other individual in comparison with DDT. Toxicology 2000; 142:135– AA tested; (4) occurrence of autophagy was demonstrated 143. light- and electron-microscopically. In summary, this first 3. Heder AF, Hirsch-Ernst KI, Bauer D, Kahl GF and series of experiments revealed HCC-1.2 cells sensitive to Desel H. Induction of cytochrome P450 2B1 by the AA deprivation, rendering this model suitable for pyrethroids in primary rat hepatocyte cultures. studying pharmacological actions of AA. Biochem Pharmacol 2001; 62:71–79. References 1. Kimball SR and Jefferson LS. New functions for amino A66 acids: effects on gene transcription and translation. Influence of dissolved organic matter on the Am J Clin Nutr 2006, 83:500S–507S. bioavailability of hydrophobic organic pollutants in sediments investigated by bioassays A65 Klaus Wruss, Dragana Todorovic and Impact of stereochemistry on biological effects of Andrea P Loibner permethrin: induction of apoptosis in human Department of Environmental Biotechnology, IFA hepatoma cells (HCC-1.2) and primary rat Tulln, Austria hepatocyte cultures E-Mail: [email protected] Julia Dornetshuber1, Wolfgang Bicker2, Michael Lämmerhofer2, Wolfgang Lindner2, Only little attention has been paid to hetero polycyclic Anneliese Karwan1 and Wilfried Bursch1 aromatic hydrocarbons (N-, S- or O-substituted PAHs), a 1Department of Medicine I, Institute of Cancer group of substances which is found together with PAHs. Research, Research Unit Toxicology and Prevention, Hetero PAHs typically show a higher water solubility and a Medical University of Vienna, Austria higher bioavailability than the structurally corresponding PAHs. The focus was to study the toxicity of fluorene and 2Institute of Analytical Chemistry and Food Chemistry, the similar hetero PAHs as carbazole, dibenzothiophene and University of Vienna, Austria dibenzofuran. Bioassays were carried out with Heterocypris E-Mail: [email protected] incongruens (Ostracod Toxkit F) and Vibrio fischeri (Lumistox). H. incongruens show the highest sensitivity to Stereochemistry plays a crucial role in determining the carbazole, V. fischeri are most sensitive to dibenzofuran toxicological profile of many chiral xenobiotics, e.g. the from the studied substances. In the presence of natural insecticidal action of mixtures containing the four sediment toxicity was dramatically reduced. This effect can stereoisomers of permethrin is essentially brought about by be deduced to the presence of organic matter (e.g. humic the (1R,cis)- and (1R,trans)-forms. Primarily non-ion channel acids) in solid and dissolved form. A aqueous mix of all related mammalian effects like induction of cytochrome tested PAHs showed different toxicity results compared to P450 enzymes and inhibition of mitochondrial complex I – the tested single substances. An explanation for this result is relevant endpoints in elucidating a chemical’s mode of presented. No additive toxicity effects could be observed. action and thus toxicological risk assessment – were elucidated in studies with four-isomer mixtures of permethrin only [1, 2, 3]. Therefore, we initiated a project to shed light A67 on the stereoselectivity of permethrin effects in mammals, Lipid hydroperoxide, an intermediate product of using human hepatoma cells (HCC-1.2) and primary rat oxidative stress, induces tumour progression- hepatocyte cultures as test models. Here we report (1) a associated genes in hepatocarcinoma cells commercially available four-isomer mixture of permethrin Nataliya Rohr-Udilova1, Klaus Stolze2, (cis-racemate/trans-racemate ~ 25:75) exhibited a dose- Bettina Grasl-Kraupp3, Rolf Schulte-Hermann3 and dependent (2–50 µM) pro-apoptotic activity; (2) the Markus Peck-Radosavljevic1 physiological death signal TGF-β1 (10 ng/ml) and 1Division of Hepatology and Gastroenterology, permethrin exerted an additive pro-apoptotic effect; Department of Medicine III, Medical University of (3) purified permethrin stereoisomers, i.e. (1R,cis), (1S,cis), Vienna, Austria (1R,trans), (1S,trans), exhibited – in contrast to their 2Department of Natural Science, Veterinary University insecticidal action – no significant differences in their pro- of Vienna, Austria apoptotic action as compared to the four-isomer mixture; 3 (4) the pro-apoptotic potency of permethrin was lost upon Institute of Cancer Research, Department of metabolism to permethrinic acid, 3-phenoxybenzyl alcohol, Medicine I, Medical University of Vienna, Austria and 3-phenoxybenzoic acid. E-Mail: [email protected] References Hepatocellular carcinoma often develops in the liver under 1. Gassner B, Wüthrich A, Scholtysik G and Solioz M. The chronic inflammation. Oxygen radicals, produced during pyrethroids permethrin and cyhalothrin are potent inflammation, attack membrane lipids and form a number of inhibitors of the mitochondrial complex I. oxidized metabolites including lipid hydroperoxides. The J Pharmacol Exp Ther 1997; 281:855–860. addition of linoleic acid hydroperoxide (LOOH) to the 2. Kostka G, Palut D, Kopeć-Szlezak J and Ludwicki JK. medium of recently established human hepatocarcinoma cell Early hepatic changes in rats induced by permethrin line (HCC-1.2) caused dose-dependent cell loss and enhanced LDH-release. Under subtoxic conditions LOOH APHAR 2007 Abstract Preview for publication in BMC Pharmacology induced intracellular hydrogen peroxide production and a genotoxic and cytotoxic events. Ethanol is a widely decrease of cellular GSH content. Elevated expression of consumed hepatotoxic agent and has been shown to protooncogene c-myc and a catalytic subunit of telomerase increase hepatocarcinogenesis in humans. Both compounds hTERT were observed under LOOH exposure. Myc can activate Kupffer cells to produce cytotoxic reactive activation is sufficient to induce cell cycle entry in the oxygen species (ROS) and growth promoting cytokines. absence of growth factors. Accordingly, the cells were Therefore, p47-NADPH oxidase knockout (phox−/−) mice pushed into the S- and G2/M-phase by LOOH. An increased were thought to be protected from hepatocarcinogenesis. expression of c-fos, c-jun, the antiapoptotic enzyme heme We found that tumor formation in phox−/− took place, oxygenase 1 (HO-1) and the proinflammatory angiogenic although to a lower extent than in wild type mice. interleukin-8 (IL-8) was detected under LOOH exposure. Objectives and Methods: To examine whether reactive Pre-treatment of cells with antioxidant N-acetylcystein or nitrogen species alternatively could be responsible for with selenite, which induces the LOOH-detoxifying enzyme carcinogenesis in these livers. Experimental model: glutathione peroxidase, partially inhibited the expression of diethylnitrosamine (DEN) [1]; quantitative RT-PCR of iNOS LOOH-induced genes implicating the involvement of mRNA; sum of nitric oxides by Griess reaction. oxidative stress. Application of SnPPIX, a HO-1 inhibitor, Results: iNOS expression was increased in phox−/− as decreased the viability of HCC-1.2 cells indicating the compared to wt mice. This is in agreement with a higher protective role of HO-1 induction. These results show that level of iNOS protein 24 hrs after DEN-treatment in phox−/− lipid hydroperoxides may be an important driving force for mice. However, total nitric oxide levels did not differ carcinogenesis in the liver. significantly between both strains. Correlation analysis of iNOS mRNA and NO levels on a per liver basis did not A68 reveal significant associations. iNOS expression and levels of nitric oxide in a Discussion: We conclude that although iNOS appears to be −/− hepatocarcinogenesis model of p47-NADPH induced in phox mice the resulting nitric oxides may no knockout mice longer be accessible for biochemical measurements at later Constanze Freiler, Eveline Kainzbauer, times after treatment. Acknowledgments Rolf Schulte-Hermann and Wolfram Parzefall Grant support by Herzfelder’sche Familienstiftung is Research Unit Toxicology and Prevention, Institute of gratefully acknowledged. Cancer Research, Department of Medicine I, Medical References University of Vienna, Austria 1. Freiler C, Brink A, Lutz WK, Kainzbauer E, Schulte- E-Mail: [email protected] Hermann R and Parzefall W. Hepatocarcinogenesis by diethylnitrosamine (DEN) in NADPH knock-out mice Background: Nitrosamines occur in traces in food or may and their wild-type counterparts. Pharmacology 2006, be generated endogenously. They are one chemical factor 78:155(T7). involved in hepatocarcinogenesis, a process mediated by

Author Index

Numbers refer to Abstract No.

Abrahim A … A22, A23, A24 Bicker W … A65 Dornetshuber J … A63, A65 Fodor L … A37 Al-Khrasani M … A45, A47, Bilban M … A18 Drimba LP … A48 Frank S … A4 A50 Bitay M … A51 Ebner K … A1 Freiler C … A68 Atefie K … A60 Bódi Z … A51 Eckelhart E … A7 Freissmuth M … A5, A7, A8, Auer M … A13 Bursch W … A63, A64, A65 Ecker G … A13 A9 Baczkó I … A51 Cardona F … A29 Ehrlich V … A60 Friedmann T … A45, A50 Bagdy Gy … A36 Cejka D … A25 Eisenbauer M … A59 Fritsch R … A11 Bajnogel-Orosz A … A46 Cervenka R … A15 Ellinger A … A64 Fuchsjäger-Mayrl G … A27 Balenga N … A3 Csanády M … A51 Farmer L … A55 Fuereder T … A25 Bankstahl J … A22, A24 Csincsik L … A51 Ferk F … A60 Fukunaga M … A4 Bauer M … A23 Csölle C … A35 Fernbach N … A8 Fürst S … A39, A45, A46, Baumgartner C … A23 Dekrout B … A25 Ferraguti F … A1, A2 A47, A50 Bellmann R … A28 Dénes L … A32 Ferstl N … A5 Garhöfer G … A27, A30 Bellmann-Weiler R A28 Döbrönte R … A48, A49 Fésüs L … A48, A49 Gauglhofer C … A57, A58 Bichler J … A60 Dorner GT … A30 Firbas C … A29, A31 Gebhart M … A11 APHAR 2007 Abstract Preview (update: 21.09.2007)

Genth H … A16 Kuntner C … A22, A24 Puskás LG … A51 Streicher C … A56 Gerevich Z … A38 Kvaternik H … A22, A24 Quan P … A4 Striessnig J … A11 Gerner C … A58, A59 Lämmerhofer M … A65 Radenkovic M … A6, A9 Superti-Furga G … A8 Gille L … A56 Lampi A-M … A62 Rathgen K … A29 Szameit S … A55 Gouya G … A26 Langer O … A22, A23, A24 Reichhold S … A61 Szaszkó J … A49 Graefe-Mody EU … A29 Leitner JM … A29, A31 Reitner, A … A30 Szebeni A … A40, A43 Graff A … A55 Lenkey N … A41, A42 Resch, H … A27, A30 Szelényi S … A46 Grasl-Kraupp B … A57, A58, Lindner W … A65 Riba P … A36, A45, A50 Szilvássy Z … A48, A49 A59, A67 Liu Z-J … A4 Rix U … A8 Szűts V … A51 Graziani A … A17 Loacker S … A10 Rohr-Udilova N … A52, A67 Tasan RO … A14 Griesbacher T … A20 Loibner AP … A66 Romanin C … A11 Tekes K … A44 Groschner K … A17 Löscher W … A22, A24 Rosenau T … A52, A56 Thallinger C … A25 Gsandtner I … A5, A6, A19 Luurtsema G … A23 Sagmeister S … A57, A58, Timár J … A39, A46 Gyarmati S … A39, A46 Maksay G … A37 A59 Timin EN … A13 Gyires K … A44, A47 Markusková N … A53 Sári RZs … A48, A49 Todorovic D … A66 Heilbronn R … A14 Mayer M … A64 Sayyah M … A10 Todt H … A9, A15 Heinrich A … A34, A35 Mayr FB … A29, A31 Schaffer A … A60 Tőzsér L … A48, A49 Hering S … A13 McDaid R … A4 Schaider H … A4 Tuschl H … A55 Herlyn M … A4 Meisel M … A61 Schmetterer L … A27, A30 Valic E … A55, A60 Herzog H … A10, A14 Ménesi D … A51 Schmidhammer H … A45, Varga A … A48, A49 Hilber K … A6, A9, A15 Mičieta K … A53 A50 Varga-Orvos Z … A51 Hilbert J … A31 Mike A … A38, A41, A42 Schnee J … A31 Varró A … A51 Hoda J-C … A11 Miklya I … A32, A33 Schühly U … A29 Virág L … A51 Hoelzl C … A60 Mišík M … A53 Schuligoi R … A20 Virágh É … A40 Hofinger A … A52 Mišíková K … A53 Schulte-Hermann R … A57, Vizi ES … A34, A38, A41, Hohaus A … A13 Moser E … A4 A58, A59, A60, A63, A64, A42 Hohenegger M … A16 Müller M … A22, A23, A24 A67, A68 Wacheck V … A25 Holy M … A8 Nanoff C … A18, A19 Schunk E … A10 Wagner C … A21 Holzmann K … A59 Németh J … A48, A49 Schuster C … A7, A8 Wagner K-H … A61, A62 Houshmand N … A51 Nersesyan A … A60 Schwarzer C … A10 Waldhoer M … A3, A4, A12 Ibrišimović E … A18 Neubauer O … A61 Seidl H … A4 Wanek T … A22, A24 Illes P … A38 Nguyen NK … A14 Sexl V … A7, A8 Warsch W … A7 Irving AJ … A3 Niel M … A26 Shripad J … A4 Weger S … A14 Jilma B … A26, A29, A31 Nohl H … A52 Shujaa N … A44, A47 Weigert G … A27, A30 Joannidis M … A28 Ostrouska I … A19 Simma O … A7 Weigl L … A16 Jost N … A51 Pankevych H … A6, A9, A19 Singewald GM … A1 Weiler S … A28 Kainzbauer E … A68 Pankucsi Cs … A48, A49 Singewald N … A1, A14 Weisz E … A7 Karch R … A22, A23, A24 Panzer S … A26 Singh A … A11 Wenger T … A47 Karoly R … A38, A41, A42 Papp JGy … A51 Sinnegger-Brauns M … A11 Werzowa J … A25 Karwan A … A63, A64, A65 Parzefall W … A57, A58, Sitte HH … A8 Whittle N … A1 Kecskeméti V … A36, A40, A59, A68 Sobor M … A39, A46 Wiesenberger G … A56 A43 Parzmair GP … A12 Solenská M … A53 Windisch A … A13 Kelemen JZs … A51 Peck-Radosavljevic M … A67 Sperk G … A14 Wolzt M … A26 Kemmo S … A62 Pehamberger H … A25 Sperlágh B … A34, A35 Wruss K … A66 Kerl H … A4 Peitl B … A48, A49 Spetea M … A50 Zádori ZS … A44, A47 Király KP … A45, A50 Peskar BA … A20 Spiel AO … A29, A31 Zarrabi T … A15 Kircher K … A30 Pickl WF … A7 Sreepathi HK … A2 Zebedin E … A7, A9, A15 Kletter K … A22, A23, A24 Piekorz R … A7 Stähle H … A29 Zeitlinger M … A21 Knasmüller S … A53, A60 Piironen V … A62 Stanek J … A22, A24 Zezula J … A5 Koenig X … A9, A15 Pirker C … A59 Staniek K … A54 Zörgiebel T … A16 Koschak A … A11 Platzer W … A12 Steiner I … A21 Zsebedics M … A22, A24 Koschutnig K … A62 Pommer V … A20 Stoiber D … A7 Krenn M … A17 Poteser M … A17 Stolze K … A52, A67 Kudlacek O … A6, A19 Pratscher B … A25 Stork D … A13