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Nociception in the Hypertensive Rat
A thesis submitted to the University of Adelaide in fulfilment of the requirements of the degree.ofPhD ,n
The Department of Clinical and Experimental Pharmacology, University of Adelaide.
by
Rodney Jamos Irvine
April 1996 ii
ABSTRACT vll
DECLARATION viii
PUBLICATIONS IN SUPPORT OF THIS TFIESIS ix
ACKNOWLEDGMENTS X
ABBREVIATIONS xi
CHAPTER T I
GENERAL INTRODUCTION I
l.l Hypertension I
1.2 Animal models of hypertension 3 1.2.1 Renal J 1.2.2 Genetic 4 1.2.3 Spontaneously hypertensive rat (SHR) 4 1.2.4 Sub-strains of the SHR 5
1.3 Blood pr$sure mechanisms 6
I . 3. I Baroreceptor-reflex-arc 6 1.3.2 Sympathetic nervous system 7 1.3.3 Renin-angiotensin system (RAS) 9 1.3.4 Opioids l3 1.3.5 Central mechanisms l8
1.4 P¡in mechrnisms l9 1.4.1 Definition of pain 19 1.4.2 Nociception 20 1.4.3 Afferent pain pathwaYs 20 1.4.4 Opioids 2l 1.4.5 Endogenous pain control mechanisms 23
1.5 Pain perception in hypertension 24 1.5.1 Animals 24 1.5.2 Humans 25 1.ó Adaptation to stress 26 1.6.1 Definition and occurrence 26 I .6.2 Physiological mechanisms 26 I,6. 3 Behavioural factors 29
1.7 Introduction summary 30
l.E Aims 3l
CHAPTER 2 33
GENERAL METHODS 33
2.1 Animals 33 2.1.1 Source of strains and housing conditions 33 2.1 .2 Surgscal procedures 34 2.1 .3 Drug administration 34
2.2 Blood pressure measurement 36 2.2.1 lndirect blood pressure measurements 36 2.2.2 Direct blood pressure measurements 37
2.3 Nociception tests 39 2. 3. I .Hotplate measurements 39 2.3 .2 T atl-flick measurements 4l
2.4 Locomotor activity measurement 42
2.5 Statistical analyses 42
CHAPTER 3 43
STUDIES ON T}VO STRAINS OF RAT DERIVED FROM THE SHR AND THE INFLUENCE OF GENDER
3.1 Introduction 43 3. l.l WK-HA and WK-HT rats 43 3.1.2 Gender 45
3.2 Methods 47 3.2.1 Animals 47 3.2.2. Blood pressure, hotplate and LMA measurements 48
3.3 Results 48 3.3 I WK-HA and \ryK-HT strains 48 3.3.2 Gender 50 lv
3.4 Discussion 53
CHAPTER 4 55
THE EFFECT OF ANTIHYPERTENSTVE DRUGS ON NOCICEPTION IN THE SHR AND \ilKY RAT.
4.1 Introduction 55
4.2 Methods 59 4.2.1 Animals and drugs 59 4.2.2 Blood pressure and behavioural measurements 59
4.3 Results 60 4.3.1 Effects of injected drugs 60 4.3 .2.Dru9 doses consumed 60 4.3.3 Effects of oral captopril 62 4.3.4 Effects of oral hydralazine 62 4.3.5 Effects of oral losartan 62 4.3.6 Effects of oral verapamil 67
4.4 Discussion 67
CEAPTER 5 7l
THE EFFECTS OF PERIPHERALLY ADMINISTERED ANGIOTENSIN II AND NOREPINEPHRINE ON BLOOD PRESSURE AND NOCICEPTION IN }VISTAR AND WI(Y RATS.
5.1 Introduction 7l
5.2 Methods 73 5.2.1 Animals 73 5 .2.2 Drug administration 73 5.2.3 Surgery 73 5.2.4 Blood pressure, behavioural tests and water consumption 74
5.3 Results 74 5.3.1 Angiotensin in WKY animals 74 5.3.2 Angiotensin in outbred Wistar rats 74 5.3.3 Norepinephrine in IWKY rats 79 5.3.4 Water consumption 79
5.4 Discussion 79 v
CHAPTER 6 83
THE EFFECTS OF CENTRAL ADNTINISTRATION OF ANGIOTENSIN AND LOSARTAN ON BLOOD PRESSURE AND NOCICEPTION IN }VKY AND SHR RATS.
6.1 Introduction 83
6.2 Methods 85 6.2.1 Animals 85 6.2.2 Surgcal procedures 85 ó.2.3 Blood pressure and behavioral tests 87
6.3 Results 87 6.3.1 Infusions of angiotensin in WKYs 87 6.3.2 Infusions of losartan in SHRs 91 6.3.3 Water consumption 9l
6.4 Discussion 91
CHAPTER 7 94
RADIOTELEMETRIC BLOOD PRESSURE IVTONITORING IN THE SHR
7.1 Introduction 94
7.2 Methods 96 7.2.1 A¡nmals and drug administration 96 7.2.2 Surgery for radiotelemetric implants 97 7 .2.3 T r ansducer/transmitter 97 7.2.4 Receivers 100 7.2.5 Sampling parameters and data storage 100 7 .2.6 Baseline blood pressures 100 7.2.7 Carotid blood pressures 103
7.3 Results 103 7.3.1 Twenty four hour readings 103 7.3.2 Influence of brief handling 104 ' 7 .3.3 Unrestrained readings t04 7 3.4 Readings in anaesthetised animals 104 7.3.5 Telemetry vs tail-cuffin conscious animals ll0 7.3.6 Drug effects 110
7.4 Discussion 111 vl
CHAPTER 8 l14
PAIN PERCEPTION IN HUMAN NOR}TOTENSIVE AND HYPERTENSIVE SUBJECTS; EFFECTS OF DRUG TREATMENT.
8.1 Introduction t14
8.2 Methods ll6 8.2. I Experimental subjects l16 8.2.2 Blood pressure and heart rate ll6 8.2.3 Cold pressor test rt7
8.3 Results tt7 8.3.1 Age and resting blood pressures tt7 8.3.2 Cold pressor test t20
8.4 Discussion 120
CHAPTER 9 r23
ALCOHOL CONSUMPTION IN TIIE SHR
9.1 Introduction r23
9.2 Experiment l: The influence of antihypertensive drugs t26 9.2.lMethods 127 9.2.1.1 Animals 127 9.2.1.2 Two bottle choice test t27 9.2.1.3 Drug treatment and blood pressures r27 9.2.2 Results & Discussion 127
9.3 Experiment 2: The Influence of age l3l 9.3.1 Methods t32 9.3.2 Results 132
9.4 Discussion 135
CHAPTER IO 138
DISCUSSION 138
APPENDIX 1,44
BIBLIOGRAPHY t45 vll
ABSTRACT
The relationship between nociceptive responses, blood pressure and locomotor activity was studied in spontaneously hypertensive ( SHR ) and normotensive Wistar-Kyoto ( \vKy) rats. No gender differences were observed and when two strains derived from the SHR were examined, , the analgesic trait was linked to the hypertensive (HT) and not the hyperactive (HA) strain.
Administration of the antihypertensive drugs to SHRs showed that the pain responses could be returned to normal in the SHR by treatment with drugs which influence the renin-angiotensin-system (RAS), but not by antihypertensive drugs which work through other mechanisms.
Subcutaneous administration of angiotensin II to WKY rats increased blood pressure and nociceptive thresholds such that they were similar to untreated SHRS. This did not occur when blood pressure was raised with norepinephrine. Icv infusions of angiotensin did not influence nociception and icv angiotensin receptor blockade did not influence blood pressure.
Human hypertensives treated with beta blockers and with blood pressures identical to normotensive controls had a reduced sensitivity to pain. However, those treated with ACE inhibitors had identical pain sensitivity to normotensives which is in concert with the animal data.
Radiotelemetric blood pressure recording was investigated as an improved method in this area of research *it"rr the reduction of stress is important. Heart rates and blood pressures were lower in the telemetered animals compared to those tested via the tail- òuffmethod and the effect of antihypertensive drugs was altered.
In view of the role of opioids in hypertension, pain and consummatory behaviours, alcohol consumption wasìt.rdi"d. Alcohol consumption in the \ryKY was lower than in the SHR and this difference was abolished by captopril treatment. This pattern was shown to alter with the age of the animal.
Overall, the studies showed that the SHR has a decreased sensitivity to nociceptive stimuli which is not directly linked to blood pressure or central angiotensin levels. peripheral angiotensin, at an unknown site, modulates pain perception in the SHR.
Hypoalgesia in human hypertensives is influenced by ACE inhibitors in a manner similar to the rat model.
Radiotelemetry will be the method of choice for blood pressure monitoring in this area ofresearch.
The SHR may provide a useful model for investigation of the self administration of drugs of abuse. vlll
DECLARATION
I declare this thesis to be based on originøl data obtained while I was enrolled as a PhD. candidote in the Depørtment of Clinical ond F-vperimental Pharmacologt al the University of Adelaide. To the best of my htowledge this thesis contains no material which has been previously accepted for the awarcl oÍ ony degree or diploma al any universitlt, nor øny material previously published hy any person, except where due reference is cited in the lext. The aulhor consenls to the thesis being made wøilable for loan or photocopying.
Rodney James lrvine 996
DA IX
PUBLICATIONS IN SUPPORT OF THIS THESIS
Irvine RI, White J Head RJ " The renin angiotensin system and nociception in spontaneously hypertensive rats" Life Sci. 56.13.1073-1078. 1995
Irvine RJ, White J "The effects of central and peripheral administration of angiotensin on hypertension and nociception in rats" Pharmacology Biochemistry and Behaviour 1996 In press
PRESENTATIONS TO LEARNED SOCIETIES
R.J.Irvine, J.M.White & R.J.Head."The effect of captopril on locomotor activity and nociceptive behaviour in the spontaneously hypertensive rat" Clinical and Experimental Pharmacology and Physiolory Suppl. 18, 28,1991
R.J.Irvine, J.M.White & R.J.Head." Alcohol consumption in the spontaneously hypertensive rat" Clinical and Experimental Pharmacology and Physiology Suppl. 21,31, 1992
The renin system and nociception in the R.J.Irvine, J.M.White & R.J.Head." angiotensin- spontaneousþ hypertensive rat" Canadian J of Physiolory and Pharmacology 72, Suppl. l, 360,1994
R.J. Iwine, T. Nunan & A. Tonkin. " Pain perception in normal and hypertensive subjects" Proceedings of the Australian Society of Clinical and Experimental Pharmacologists and Toxicologists. l, 61, 1994
R.J Irvine, N Toop & JM White, "Comparison of tail-cuffand telemetered blood pressure measurements in spontaneously hypertensive rats." Proceedings of the Australian Society of Clinical and Experimental Pharmacologists and Toxicologists. 2, 87, 1995 X
ACKNOWLEDGMENTS
Science is about sharing and I have been fortunate to have many friends and colleagues over the years who have either directly or indirectly contributed to this thesis. I will mention a few and apologise for any omissions.
' :/- r) Jason White and Richard Head who¡q acted as exemplary supervisors.
All members of the Department of Clinical and Experimental Pharmacology, University of Adelaide for their tolerance and valuable discussions.
Natasha Toop for her help with many of the animal studies.
Anne Tonkin and Tim Nunan for their substantial contribution to the human studies.
Peter Howe and Lina irúò;JÉ, for their gift of animals used in chapter 3.
Gordon Crabb who has kept my computer healthy.
The members of my family who have generously given their love and support and without which this project would have been rather pointless.
Finally I would like to thank Felix Bochner who encouraged me to pursue this thesis and who has always been diligent and understanding in his role as my boss. xr
ABBREVTATIONS USED IN THIS THESIS
ACE angiotensin converting enzyme ACTH adrenocorticotrophic hormone AI angiotensin I AII angiotensin II AIII angiotensin III AT1 angiotensin II recePtor, tYPe I AT2 angiotensin II recePtor, tYPe 2 BP blood pressure BPM beats per minute cm centimetre CNS central nervous system CRF corticotrophin releasing factor DAMGO [D-Ala2,N-Me-Phea,Gty-o151-nnkephalin 5, DPDPE [D-Pen2' ] -Enkephalin DSLET [D- Sef,Leut,Thrul -Enkephalin g gr¿ìms HP hotplate ip intraperitonealy icv intracerebroventri cul ar sc subcutaneously hrs hours LMA locomotor activity m metre M moles/litre mg milligram min minute ml millilitre mM millimoles/litre mmHg millimetres of mercury NGF nerve growth factor NP alcohol non-preferring rat P alcohol preferring rat POMC pro-opioiomelanocortin RAS renin-angiotensin system SAMS sympathoadrenalmedullary sYstem SEM standard error of the mean xll
SHR spontaneously hypertensive rat WKY Wistar Kyoto rat WK-HA hyperactive normotensive rat WK-HT hypertensive normally active rat pg microgram CHAPTER 1
INTRODUCTION
1.1 Hypertension
High blood pressure is one of the most prevalent diseases in western countries. In Australia
9000 16.70/0 of men and l2.7Yo of women were found to be hypertensive out of a gfoup of
(National Heart Foundation, 1989). Hypertension is classified as either primary, where the cause is unknowrL or secondary, where the cause is known, eg phaeochromocytoma or
primary renovascular disease. The majority of hypertensive people in Western society have hypertension.
The cause of primary hypertension most likely involves a combination of a polygenetic
predisposition (Ganten,l993;Williams et al., 1990) and enviromental factors. These factors
include dietary habits such as salt and alcohol consumption patterns. Salt intake (\ileinberger
et al., lg82) and heavy alcohol consumption (Shaper et al', l98l; Shaper et al', 1988) have
both been shown to be associated with increased blood pressure in Western European
populations. Fat consumption and increased body mass have also been shown to correlate
with the levels of blood pressure in these populations (Shaper et al., l98l). In addition to
these dietary factors, social factors have been implicated in the development of hypertension.
An increased cardiovascular risk was shown to be associated with lower socio-economic
manual status in the British Cardiovascular Risk Study, but the increased risk demonstrated in
workers was probably a result of dietary habits such as the increased alcohol consumption 2 which was shown in this group (Shaper el al., l98l). High blood pressure and cultural factors
have been investigated and a cross cultural study demonstrated that blood pressures were
higher in developed Western type societies compared to simple hunter-gatherer societies
(Waldron et al., lg82). These workers concluded that level of psychosocial stress, which
varied with the cultural setting, was probably an important factor in determining population
levels of blood pressure. They considered that the differences in blood pressure could not be
ascribed to diet.
The interaction between multiple genes and multiple enviromental factors such as those
discussed above indicate that a single mechanism in the initiation and development of
hypertension is unlikely. Each of these factors may have quite a subtle influence on blood
pressure control and produce a gradual resetting of blood pressure at a higher level over
time.
This complexity is reflected in the range of drug therapies for high blood pressure in humans.
They include a number of classes of drugs targeted at a number of mechanisms involved in the
control of blood pressure. The varied therapies indicate that a range of approaches to the
control of this disease are successful. As discussed below, drugs which are effective in
lowering blood pressure a¡e varied in their chemistry and in their mechanisms of action. Beta
blockers are aimed at inhibiting the stimulation of cardiac output via the sympathetic nervous
system. Inhibitors of angiotensin converting enzyme (ACE) reduce the production of the
potent vasoconstrictor angiotensin II (AII). Calcium antagonists cause vasodilation via a
direct effect on vascular smooth muscle and diuretics are effective by reducing the plasma
volume and, as a result, the cardiac output. J
tilithout control of high blood pressure by these and other therapies, hypertension is likely to lead to a number of life threatening pathologies if allowed to proceed. Hypertensive people may suffer from a variety of undesirable cardiovascular events such as stroke, heart failure and renal failure (Hobbs et al., L992;Zanchetti & Mancia, 1987; Samuelsson et al., 1987).
A large body of useful information on hypertension has been obtained from human studies and this has contributed to improvements in dosage regimes and therapy. Research on humans however, suffers due to the limitations placed on the level of experimental manipulation that can be undertaken without breaching ethical considerations. Animal models of hypertension were developed to allow appropriate experimental manipulation of the many factors and systems thought to be important in control of blood pressure. These models have revealed many of the mechanisms involved in blood pressure control and allowed the development of new drugs and therapies.
1.2 Animal models of hypertension
1.2.1Renal
Renal models of hypertension in the rabbit, dog and rat have been produced by disrupting renal function with surgical intervention. Clipping of renal arteries to reduce blood flow, removal of a kidney and wrapping the kidney in cellophane have all been employed (Page et al., 1955 Goldblatt, 1960). These interventions are sometimes accompanied by treatment with deoxycorticosterone acetate (DOCA) andlor salt loading (Seyle, 1942). Such methods 4
have been used for many decades and have provided important information on hypertension.
However, models which involve the disruption of normal kidney function are most relevent to the study of secondary renal hypertension. They have less relevence for primary hypertension and methodologicatly, they are time consuming. Furthermore, due to the fairly severe disruption of the animal's physiology in these models, the results can be difficult to interpret.
1.2.2 Ctenetic
A number of genetic animal models of hypertension have been described, including Milan,
Sabra, New Zealand and Okomoto strains of rat. The first two of these have a significant renal component to the development of hypertension and in the case of the Sabra do not develop hypertension without further intervention (eg salt loading) (Yamamori, l98l). All of these strains do, however, reduce the problems associated with surgical procedures.
1.2.3 Spontaneousþ hypertensive rat (SHR)
This is the last of the strains mentioned above and was developed by Okomoto and co- workers in Japan in the early 1960s. The strain is the result of selective inbreeding of the offspring of a single pair of Wistar rats which displayed elevated systolic blood pressure.
Twenty generations later l}OYo of these animals proceeded to develop high blood pressure in adulthood. The SHR is normotensive at birth and up until about 4 weeks of age. Thereafrer blood pressure progressively rises to reach a plateau at a¡ound l8 weeks of age. The blood pressure remains high for the rest of the animal's life. The genetic control for this animal is the
Wistar-Kyoto rat (WKÐ which is the normotensive strain of rat from which the spontaneously hypertensive rat (SfR) was developed (Okamoto & Aoki, 1963). This is the 5 most popula¡ animal model of primary hypertension and has been studied extensively. Surgery
and drug treatment are not required to produce hypertension in this strain.
Dietary and environmental factors influence the development of the disease in SHRs as they
do in humans. Sensitivity of blood pressure levels to salt intake has been demonstrated in
these animals (Bachmanov, 1989; Bertino & Beauchamp, 1988) Increasing environmental
stress has also been shown to be effective in raising blood pressure in SHRs (Kvetnansþ øf
al., 1979; Knardahl & Hendley, 1990; McCarty et al., 1978). Effective drug therapy for
hypertension in these animals is also similar to that which is effective in treating high blood
pressure in humans (Yamori, 1991).
Interestingl¡ the opioid antagonist naloxone is also capable of lowering blood pressure in
these animals @elbarre et al., 1982), which suggests a role for endogenous opioids in the
hypertensive characteristic of this strain.
1.2.4 Sub-strains of SHR
The behaviour of SHRS has been compared to WKYs and a number of differences have been
reported (Sagvolden et al., 1992; Danysz et al., 1983). The most distinctive of these is an
increase in locomotor activity (LMA) (Sutterer et al., l9E4; Danysz et al., 1983; Whitehorn
el al., 1983; Knardaht & Sagvolden, 1979). The increased locomotor activity may involve
dopamine, as blockade with the selective dopamine D-2 antagonist sulpiride has less inhibitory
effect on LMA in the SHRS compared to WKYs (van den Buuse et al., 1992- van den Buuse
& de Jong, 1989). Selective recombinant inbreeding has separated the hyperactive and 6 hypertensive traits into two separate and distinct strains derived from the SHR (Sagvolden ef al., 1992) and Chapter 3 of this thesis reports some experiments in these animals.
1.3 Blood pressure mechanisms
A number of physiological systems involved in the control of blood pressure have been identified. These mechanisms have largely been uncovered from studies on animal models of hypertension and brief descriptions of some of these mechanisms a¡e contained in this section.
L3.1 Baroreceptor reflex arc
This is a major component in the control of blood pressure. High pressure mechanoreceptors located in the carotid sinuses, aortic arct¡ and carotid arteries respond to increases in blood pressure by increasing afferent activity. These afferents terminate in the nucleus tractus sola¡is of the medulla. From here, neurons project to the caudal ventromedulla where they synapse with neurons which project to the rostral ventro lateral medulla and connect with bulbospinal neurons (Chalmers et al., 1992). Low pressure mechanoreceptors situated in the heart and in the lung perform similar functions and this is referred to as the cardiopulmonary receptor reflex. The activation of these receptors results in an increase in vagal activity and a decrease in sympathetic tone (Randich & Maixner, 1984).
Baroreceptor and cardiopulmonary reflexes have been examined in the SHR by pharmacologcally manipulating blood pressure and thus stimulating the reflexes. An impairment of both the baroreceptor and cardiopulmonary reflexes has been reported in the
SHR compared to the WKY (Widdop et al., 1990). A reduced vagally mediated baroreceptor 7 response to the injection of the vasoconstrictor, phenylephrine or the vasodilator, nitroprusside a¡d a reduced cardiopulmonary (Bezold-Jarisch) response to phenyldiguanide was demonstrated in conscious SHRs. The reduced baroreceptor response in the SHR can be
corrected with chronic but not acute treatment with the angiotensin type one (ATl) receptor
antagonist, E)(P 3174 (Bartholomeusz & Widdop, 1995). Thus, it would seem that the SHR
may have a reduced capacity to control blood pressure via these reflexes and that angiotensin
may contribute to this inadequacy.
1.3.2 Sympathetic nervous system
The sympathetic nerves play an important role in the control of blood pressure. The release of
catecholamines from sympathetic nerve endings is a major mechanism for the control
peripheral resistance and ca¡diac output.
The sympathetic nervous system also plays a role in the development of hypertension in
genetically hypertensive animals. This has been demonstrated by chemical sympathectomy
with 6-hydrory dopamine (Yamori et al., 1972), or by nerve grou/th factor (NGF) antiserum
and guanethidine (Læ et al., l99lb). These treatments, which destroy sympathetic nerve
terminals, attenuate the development of hypertension in SHRs.
A number of str¡dies have shown that the SHR has an overdeveloped sympathetic neryous
system as indicated by increased content of noradrenaline in blood vessels (Head, 1989; Head
et al., 1985).There is also an increase in the number of nerve bundles and thei¡ densities as
indicated by electron microscopy and histofluorescence (Cassis et al., 1985). Sympathetic
nerves in the blood vessels of SHRs take up significantly higher amounts of 3H- 8 norepinephrine than vessels from WKYs (Webb et al., l98l). Increased nerve traffic in sympathetic nerves of SHRs (Judy et al., 1976), and increased overflow of 3H-norepinephrine from blood vessels have also been reported (Keeton & Biediger, 1988; Zsoter et al., 1982).
Thus, from these and other studies it is clear that SHRs have an increased number and activity of noradrenergic sympathetic nerve terminals in blood vessels.
The increased innervation of these tissues may well be the result of an increased expression of nerve growth factor (NGF) This is a protein considered essential for the development and maintenance of sympathetic nerves (Levi-Montalcini & Hamburger, l95l). Elevated levels of
NGF have been reported in the blood vessels of the young SHR (Donohue ef al., 1989)-
Increased expression of NGF as measured by mRNA levels for this protein has also been
reported in the young SHR (Falckh et al., 1992b;Falckh et al., 1992a). Neonat¿l treatment of
normotensive animals with NGF results in vascular changes similar to those seen in
hypertensive animals (Zettler et al.,l99l). Therefore, the increased expression and content of
NGF seen in the blood vessels of hypertensive animals may be the reason for increased
sympathetic innervation in these animals.
The undecapeptide, substance P, is found in the dorsal root ganglia of the spinal cord and is
released from sensory nerve endings. The level of this peptide is increased by NGF, and
reduced by antibody to NGF, indicating that sensory nerves are also dependent on this protein
in a similar manner to noradrenergic sympathetic nerves (Otten et al., 1980). The level of
mRNA for substance P in adult sensory neurons has also been shown to be increased by NGF
(Lindsay & Harmar, 1989). Hence, the possibility of a common link between hypertension and
pain responses in these animals exists at the level of the s€nsory nerves' 9
1.3,3 The Renin-Angiotensin System
The Renin-Angiotensin System (RAS) has an important role in blood pressure control through the production of the potent circulating vasoconstrictor, angiotensin II (AII). An outline of the system is shown in Figure l.l. Renin produced in the kidney acts on angiotensinogen from the liver to form angiotensin I (AI). Angiotensin I in the blood is a substrate for angiotensin converting enzyme (ACE), primarily located in pulmonary blood vessel endothelium, and the product of this reaction is the active peptide, AII (Lee et al., l99lb). AII is the major active peptide of the system, although the importance of other fragments such as angiotensin III is becoming apparent (Radhakrishnan & SinL l99a; Harding et al., 1987).
Two types of angiotensin receptors have been demonstrated thus far and they exist in a variety of tissues. The ATI t1rye which mediates the pressor effect of AII, is inhibited by the highly selective non peptide antagonist losartan (Timmermans et al., 1993). The reducing agent, dithiothreitol, also diminishes the binding capabilities of this receptor type, probably by interacting with the cysteine residues on the receptor which are essential for receptor function
(Timmermans ef al., 1993). The second receptor is the AT2 type which is inhibited by another non peptide antagonist termed PDl23l77, but this receptor does not have a clearly defined physiological role (Pucell et al., t99l). The AT2 receptor is found in high concentrations in foetal tissues and is thought to to be important in development (Tsutsumi & Saavedr4 l99l;
Tsutsumi et al., 1993; Cook et al., l99l). t0
Systcmíc
Liver
Angiotensinogen Tissue renln + Renal nnln renal renin Angiotensin f other protea3es Lung ACE e.g.r Anglotensln Chymase Prolease Protease fnactfw -Actlve" fragmentr lngmentc Ang lII' Ang II/' Ang 1.7 Anglotensln II I ANGIOTENSN E¡¡tracellular RECEPTORS Gene regutatlon' lntemallza tlon-') anglotenslnogen lnacttvatlon renln Cellular rosponso ACE contractlon anglotensln secretlon fêoeptor
Fig l.l The renin-angiotensin system. Modified from Timmeflnans et al', 1993 n
Losartan is a higfly selective antagonist of the ATI receptor compared to the AT2 with a ratio of 30,000 to I (Chiu et al., 1990) This drug has an oral bioavailability in rats of 33o/o
(Wong et al., 1990) and a half life of 5.7 hours in the same species (Christ et al., 1994). It is oxidised by the liver to form an active metabolite E)G3174, which is a non-competitive antagonist of the ATI receptor and has a longer duration of action (Wong et al., 1990). The contribution of EXP3174 to the antihypertensive action of losartan in long term treatment is not clear.
The majority of ATI receptors are linked via G-protein to phospholipase C and increase
intracellular calcium levels via inositol trisphosphate when acted upon by AII. Some
receptors are linked to phospholipase A or D and increase arachadonate and its products
(Timmerm ans et al., 1993).
The receptor- cellular response coupling for the AT2 receptor is not as well defined as that
for the ATI receptor and it may be that it is not coupled to G-protein (Mukoyama et al.,
lee3).
Both of these receptors have been cloned. The ATI receptor has been isolated from rwo
sources and cloned: one from rat vascula¡ tissue (Murphy et al., 1991), and the other from the
bovine adrenal gland (Sasaki et al., l99l). Continuing molecular studies are identifing
subtypes of the ATI receptor (Mukoyanaet al., 1993), but the physiological relevance of
these subtypes is not clear at present (Timmermans et al., 1993). t2
The cloning of the AT2 receptor (Nakajima et al., 1993) has led to the production of mice without a functional gene for the AT2 receptor (Ichiki et al., 1995; Hein et al., 1995). Two gfoups have recently reported on studies in these animals. One group reported no change in blood pressure (Hein et al., 1995) and the other increased systolic and diastolic blood pressures and increased sensitivity to AII (Ichiki et al., 1995). Both groups reported decreased ambulatory activity. Hypoalgesia was reported by one group (Ichiki et al., 1995), but it was not clear as to the method employed to establish this. These preliminary data suggest that the ca¡diovascula¡ and pain physiology of the genetically manipulated animals is different from normal mice, but this needs further investigation. From these experiments it can be concluded that the high AT2 receptor concentrations that are normally found in foetal mice are not essential for development. Despite the absence of these receptors the animals described in these reports developed normally to adulthood.
Studies which involve the disruption of genes, such as those described above, have not been performed in rats due to technical problems in introducing the targeting vector into the embryonic cells of this species. Similar experiments in rats, and especially the SI{R, which disrupted the ATI tlpe receptor gene would be of great benefit as $'e have the most information on blood pressure control for this species. Unfortunately, the measurement of blood pressure in the conscious mouse also presents technical difrculties. It is probable that the animals are subjected to a considerable degree of stress from the surgery and restraint th¿t are required and this would probably influence their blood pressure. Thus, the blood pressure measurements following disruption of the AT2 receptor gene may well have been influenced by these factors. l3
ACE inhibitors such as captopril, when administered to young SHRS during the development phase of hypertension, prevent the characteristic rise in blood pressure seen in untreated animals. Furthermore, when the drug is withdrawn, the blood pressure in these animals remains low compared to untreated SHRs (Harrap et al., 1994; Harrap et al., 1990). The mechanism of this sustained effect of the drug is unknown and the subject of intensive research, but it does suggest an action of this drug other than an acute effect on circulating
AII levels via inhibition of peripheral angiotensin converting enzyme. Similar effects have been reported for other ACE inhibitors such as perindopril (Christensen et al., 1989). The non-peptide ATI antagonist losa¡tan also displays a persistent lowering effect on the blood pressure of the SHR after it has been withdrawn (Mortonet al., 1992).
The RAS is important in blood pressure control and the development of specific non-peptide antagonists and molecular biolological receptor studies promise to lead to a new understanding of the role of tissue RAS in disease. In addition , it is now clear that aside from the well known RAS described above, which has an endocrine functior¡ there are also local
tissue RASs (Dzau, 1987) The brain is one tissue in which a RAS has been described
(Steckelings et al., 1992a; Phillips, 1987; Ganten et al., 1975) and this will be discussed
below in 1.3.5.
1.3.4 Opioids
In this section the role of opioids in blood pressure will be discussed. The opioid system
(Figl.2) and its role in pain is described in more detail in 1.5-4. 'ua¡s,{s qtr '166I 'Árane¡¿ u¡og PeSPo¡4¡ p¡o¡do eql Z't
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u;qtlropuc.rtcg auourroq ¡n¡clrt ¡qo¡ ôu!¡¡lnrut¡¡ t¡^toutloul'a1.8 .¡tlp.r¡rre¡ul ¡ï¡.ulqtlo¡¡ocl¡ro3 auour¡oq Out¡rlnu¡!¡¡ ¡¡Acoull¡r¡¡'sqdlv
þt l5
Binding studies using the non-selective opioid receptor ligand 3H-naltrexone (Rahmattt et al.,
l99l;Martucci & Hahn, 1979) have shown that the number of opioid receptors in SHR brain is increased when compared to the WKY. The latter study (Rahmani et al., 1991), where a number of brain regions were examined, showed that this increase was confined to the hypothalamus (Rahmani et al., l99l). 3H'[D-Ala',N-Me-Phea,Gly-ol5¡-Enkephalin
(DAMGO), a selective mu ligand, has been shown to bind to receptors located only in
discrete brain regions in these strains. Binding of this ligand in the hypothalamus and mid-
brain was higher in SHR animals compared to WKYs (Gulati et al., 1990). Other opioid
receptor types have also been studied. Ligand binding to membranes prepared from different
brain regions revealed an increase in kappa binding sites in the hlpothalamus and cortex of
SHR5, but no difference for mu or delta receptor sites in any of the areas sh¡died (Bhargava &
Das, 1986). An increase in delta binding sites has been reported in the amygdala of the SHR
@hargava & nanman¡, 1993) and decreased dynorphin binding in the hippocampus
(McConnaughey et al., 1992).
Autoradiographic studies investigating the binding of 3H-DAMGO, and 3H-[D-Pen2r,]-
Enkephalin (DPDPE), a selective delta ligand, in SHR and Sprague Dawley rats, revealed
similar distributions of binding sites in a number of brain regions, There was an increased
density of binding of DAMGO in a number of regions involved in blood pressure control and
locomotion for SHRs (Kujirai et al., l99l). Unfortunately, these experiments did not include the WKY strain.
It would appear that there are different distributions of opioid receptors in the \ryKY and SHR
and that they are associated with areas of the brain thought to be involved in the control of l6 blood pressure, zuch as the hypothalamus. With the examination of more discrete regions of the brain and the continued development of more specific ligands and improvements in
sensitivity ofthe methods a clearer picture may emerge.
Differences between the strains in their response to opioids have been demonstrated at the
level of the neuroeffector junction in the periphery. In perfirsed mesenteric artery preparations
from SHR and WKY animals, responses to electrical stimulation of noradrenergic sympathetic
nerves have been studied. Enkephalins inhibit the release of noradrenaline by electrical
stimulation in these preparations via presynaptic opioid receptors and this effect is more
pronounced in SHRs (Tsuda & Masuyam4 1989).
The acute cardiovascular effects of opioid agonists in these strains have been investigated in
anaesttretised animals. A comparison of icv and iv routes of administration for the mu agonist
morphiceptin, delta agonist [D-Sel,Leut,Thrul-Enkephalin (DSLET), and kappa agonist U-
50, 488H revealed some differences in immediate cardiovascula¡ responses. When
administered iv, the blood pressure responses were similar in WKY, SHR and Renal
hypertensive rats. Morphiceptin and U-50, 488H decreased blood pressure and DSLET
increased blood pressure. \Uhen these drugs were administered icv the same results were
obøined for the WKY animals, whereas the two hypertensive strains showed blood pressure
responses in an opposite direction to those seen with iv administration (Widy Tyszkiewicz &
Czlonkowski, l99l). These results indicate that the hypertensive animals have a dif[erent
response to central administration of these agonists which is not evident in the normotensive
strain and may indicate a difference in central opioid receptor function. t7
Chronic treatment with the non-selective opioid antagonist, naloxone, inhibited the age- dependant rise in blood pressure normally seen in SHRs (Quock et al., 1984; Szilagyi, 1988).
The effect of chronic blockade of specific opioid receptor types has also been tested. Chronic treatment with the kappa antagonists l\tR2266 or MRl452 from 6-10 weeks of age in SHRs attenuated the rise in blood pressure normally seen (Kraft et al., 1991a). No changes in beta
endorphin, enkephalin or catecholamines was observed in the hypothalamus, brainstem or
midbrain with this treatment. A simila¡ experiment with a delta antagonist (ICII54 129) also
attenuated the rise in blood pressure in young SHRs (Kraft et al., l99lb). Catecholamines,
enkephalin and beta endorphin in the brain regions were again measured with this treatment.
An increase in catecholamine and beta endorphin levels was observed in the hypothalamus of
the SHR.
In these experiments with chronic treatments, although the rise in blood pressure was reduced
or delayed by opioid antagonists, the animals still had blood Pressures which were
significantly higher than the WKY control animals. On withdrawal of treatment the blood
pressures quickly rose to the levels seen in untreated SHRs.
It would appear from the studies above that mu, delta and kappa opioid receptors are
involved in the control of blood pressure in hypertensive rats. However, to what extent this is
a direct influence on blood pressure control mechanisms or an indirect effect via other systems
in which opioids play a role is unclear. l8
1.3. 5 Central mechanisms
The brain is involved in the control and integration of all of the blood pressure control systems mentioned above. Some reference to this has already been made in the individual sections. The nucleus tractus solitarus (NTS) of the dorsomedial medulla, the rostral ventrolateral medulla (RVM) and the caudal ventrolateral medulla (CWM) are the centres involved in the baroreceptor reflex . The excitatory transmitters found in the neurons which
et al', descend from the RVM include epinephrine, amino acids and zubstance P (Chalmers
lgg2). Enkephatin containing neurons have also been shown to project from the NTS to the
RVM (Morilak et al., 1989) and the microinjection of opioids in these areas can produce
the either pressor or depressor changes in blood pressure depending on the precise location of
injection (punnen & Sapru, 1936). An inhibitory gamma amino butyric acid containing
pathway between the CVLM and the RVM has also been descriH (Li et al., l99l; Blessing
in &,Li,l9S9). Components of the RAS are found in central structures as referred to above
section 1.3.3. AII is found in high concentrations in structures identified as important in the
control of blood pressure. The NTS of the medulla" the paraventricular nucleus and other
structures in the hypothalamus have cells which contain AII and appropriate receptors
(Steckelings et al., lggz).Injection of angiotensin II into the lateral ventricle gives rise to an
increase in blood pressure and this is enhanced in the case of the SHR (Wright et al-, l9E7;
Hoftn¿n et al., lg77). These responses can be blocked by prior injection of the non-selective
peptide AII antagonist, saralasin (Phillips et al., 1977).
.C There a¡e a number of parallel and compfment_ary systems involved in the control of blood
pressure both within and outside the CNS. The precise nature of these systems is still under l9 investigation, but in view of the many and varied active substances involved it is likely that the integrated response will be complex.
1.4 Pain mechanisms
1.4.1 Definition of pain
Pain is a very subjective experience and involves a complex response, usually as the result of injury. Attempts to define pain in an objective sense have proved to be difficult. There is no clea¡ correlation between the severity of injuries and the level of reported pain by victims of traumatic accidents (Melzack et al., 1982). Thus, to define pain in terms of the level of stimulus is not useful. Similarly, to define pain in terms of the physiological response is also problematic as people with injuries and complaining of pain often have, for example, normal respiration and heart rates (Chapmaî et al., 1985). The International Association for the
Study of Pain has defined pain thus: '?ain is an unpleasant sensory and emotional experience
associated with actual or potential tissue damage." By the necessary inclusion of the word
"emotion" this definition limits the possibility of directly researching pain using animal models,
as \ile have great difficuþ in assessing emotion in animals. Because of these and ethical
considerations the great majority of in vivo animal studies investigate nociception rather than
pain as defined above. 20
1.4.2 Nociception
One of the most important roles of the nervous system is to provide warning to the organism of impending injury. Since the primary site of injury from the external environment is the skin, it is in this organ that a group of highly specialised nerve fibres have evolved. These have been termed nociceptors, because they respond to noxious stimuli. The most widely studied Foup of nociceptors are the C-fibre mechano-heat receptors. These receptors have a receptive field a¡ea of about l9mrn, a heat threshold of 43oC and a conduction velocþ of 1.2 m/s. They are thought to be responsible for burning second pain.
The second most widely studied group are the A-fibre mechano-heat afferents of two types.
The type I are fast conducting fibres (3ln/s) and are responsible for the primary hyperalgesia following injury. Type2 receptors are thought to be responsible for sharp, first pain and have conduction velocities of about l5m/s.
Nociceptors exist in other organs such as muscle and tooth pulp and prezumably they play a role in deep pain (Campbell et al., 1989).
1.4.3 Afferent pain pathways
The dorsal root ganglia pass the sensory information from the periphery on to the spinal cord.
The neurotransmitter released by these nociceptive afferents is probably substance P.
Substance P is an ll amino acid peptide @uler & GaddunU l93lþhich is found in large concentrations in the dorsal horn of the spinal cord. Presynaptic inhibition of substance P release by opioids and/or post synaptic inhibition of its action at these synapses appear to be the first sites of modulation of nociceptive input (Besson & ChaouctU 1987). 2l
The spinothalamic tract is the main ascending pathway for transmission of nociceptive inputs to the brain. The termination of this tract in the thalamus of humans, monkeys and rats is chiefly the ventral posterior lateral nucleus. Ascending pain pathways terminate in the ventrobasal area of the thalamus and it has been shown that increased electrical activity in these neurons is associated with noxious stimuli. Thalamic structures have dense connections with the somatosensory cortex where higher processing of nociceptive inputs may take place
(Willis, 1989).
1.4.4 Opioids
The first opioids were discovered by Hughes and Kosterlitzin 1975. They consisted of two tetrapeptides differing by the C-terminal amino acid and termed Met-enkephalin and Leu- enkephalin (Hughes et al., 1975). These are found throughout the CNS and in the gastrointestinal tract and the adrenal medulla. Met-enkephalin is the opioid found in the highest concentration in brain and is in particulady high concentrations in the hypothalamus and striatum (Giraud et al., 1983). It has a high affinity for the kappa opioid receptor, with lesser affinity for the mu receptor and almost no affinity for the delta type (Corbett et al., le82).
The dynorphins are found throughout the CNS and have a similar distribution to the enkephalins. Their affinity for kappa and mu receptors is similar to that of the enkephalins, but unlike the enkephalins, they display some affinity for the delta receptor (Corbett et al., 1982). 22
Pro-opiomelanocortin (POMC) is the prohormone for adrenocorticotrophin (ACTH) and melanocyte stimulating hormones as well as beta endorphin. Its major site of expression is in the pituitary, but it is also found in the hypothalamus (Lopez Calderon et al., 1991). The major active opioid product of POMC, beta-endorphin is found in these regions and it is also detectable in plasma and CSF (Antonio Martinez et al., 1990).
Figure 1.2 shows the opioids and their precursors. POMC, pro-dynorphin and pro-enkephalin are the primary gene products from which the active opioids beta endorphin, dynorphins and the enkephalins a¡e formed (Pleuvry, l99l).
Receptors for the endogenous opioids are found throughout the brain and spinal cord and are widely distributed in the periphery. The three t)¡pes: mu, delta and kappa a¡e linked via G proteins to either potassium channels, in the case of the mu and deltq or to calcium channels, in the case ofthe kappa receptor. All three receptors are coupled to adenylate cyclase, and generall¡ agonist occupation of the receptors results in inhibition of this enzyme (Childers, lee3).
The enkephalins have a short half life in vivo due to the rapid degradation of these peptides by neutral endopeptidase-24,11(NEP). The pharmacology of this enzyme has been extensively reviewed, @atel et al., 1993;Roques et al., 1993). It is a member of the Zn metallopeptidase g.oup of enzrymes which includes ACE. They are membrane bound and a¡e widely distributed throughout the CNS. Inhibition of these enz.ymes can lead to analgesia (Maldonado et al.,
1994; Roques et al., 1980). A large number of inhibitors of these enzymes have beerç and continue to be, developed in the hope that drugs which modtfy the opioid system in a 23 therapeutically useful way will be discovered. Cardiovascular disease and pain control a¡e two of the most likely a¡eas of use for such agents (Patel et al., 1993).
The opioid system with its many active peptides, multiplicity of receptors and processing enzymes is still a rich field for research and provides many possible sites of action for new drugs.
1.4.5 Endogenous pain control mechanisms
The relationship betweeen injury and pain as prwiously referred to in t.5.1 is not always
obvious. Severe injuries a¡e not always accompanied by pairL at least not until some time after
the accident (Wall, 1979;Melzack et al.,l9S2). The discovery of descending pathways which
could inhibit respons€s evoked in the spinal cord and subsequent resea¡ch revealed a
descending system which attenuated afferent inputs (Hagbarth & Kenr, 1954).
The best described system is opioid mediated and involves the periaqueductal grey (PAG)
matter of the mid braiq the rostral ventromedial medulla (RVM) and the dorsal horn of the
have the spinal cord @asbaum & Fields, 1978). These structures contain opioids and
appropriate receptors. The PAG has a high density of predominantly p receptor sites and
contains enkephalins and dynorphin (Goodman et al., 1980). Enkephalin containing neurons
sensitive to the microinjection of opioids are found in the RVM (Lleweþ et al., 1986;
Akaike et al., 1978). The superficial layers of the dorsal horn contain enkephalin and
dynorphin, have binding sites for all classes of opioid receptor and respond to the injection of
opioid with analgesia (Yaksh & Rudy, 1976). 24
1.5 Pain perception and hypertension
1.5.1 Animals
The response of SHRs to nociceptive stimulation is blunted compared to the WKY. SHRS
have longer latencies in the hotplate test (Sitsen & de Jong, 1984; Virus et al., 1983; Maixner
et al., lg82). The increased hotplate latencies are reduced by treatment with naloxone,
suggesting a role for opioids (Saavedra, l98l; Maixner et al., 1982). Experimental rat models
of hlryertension where the animals are made hypertensive by treatment with
deorycorticosterone and salt treatment or by renal clipping have also been reported to
produce hypoalgesic animals (Zanfu et al., l9S0). However, this was not confirmed by other
workers who observed no hlpoalgesia in the experimental models of hypertension (Sitsen &
de Jong 1984). Both of these studies used hot plate testing and the difference in results is
¡¡tzztrng. Raising blood pres$rre with alpha adrenergic agonists such as phørylephrine results
in an immediate hypoalgesiq but this has only been demonstrated with acute dosing (Randich
& Maixner, 1984).
Tail flick latencies have also been reported to be extended in the SHR (Saavedr4 l98l), but
the majority of studies have reported no difference between SHRs and WKYs (Virus et al.,
1983; Sitsen & de Jong, 1984). Tail flick response to the application of heat is retained even
when the spinal column is lesioned and is therefore thought to be mainly a spinal reflex
(Barton et al., 1980) This would suggest that the altered pain perception in SHRs is more
likely to be at a higher level of the nervous system than the spinal cord. 25
Sensitivity to the prototypic opioid agonist morphine is also altered in SHRS. Body temperature of SHRs, but not WKY or Wistar rats, is lowered by ip morphine (Sitsen et al',
1987). SHRs have a greater and longer analgesia after iv morphine and this cannot be
explained in terms of altered pharmacokinetics. Measurement of morphine in both strains
after iv dosing indicated that the drug has simila¡ plasma levels, volume of distribution and
similar elimination kinetics in the two groups of rats (Bhargava & Villar, 1992)'
1.5.2 Humans
Humans \ilith high blood pressure also have an altered perception of pain. Responses to tooth
pulp stimulation indicate that untreated hypertensive subjects have a reduced sensitivity to
pain when compared to normotensive subjects (Ghione et al., 1988; Zamir & Shuber, 1980).
In another study, simila¡ results were obtained when the response to painfirl cutaneous
electrical stimulation was shown to be inversely correlated with blood pressr¡re (Rosa et al.,
lgg4). When a group of normotensive people were tested using the finger pressure method,
again there was a correlation between pain response and blood pressure @ruehl et al-, 1992).
Thus, it would seem that, using a number of diflerent methods for testing acute pain
perception, high blood pressure is associated with a decreased response. Clinically, this may
be important. Sufferers from high blood pressure are predisposed to ca¡diac disease. Silent
cardiac ischemia is a condition which is associated with sudden death from ca¡diac arrest
(Miller et al., 1990; Sheps et al., 1988). These patients do not experience the normal pain of
angina associated with a compromised coronary blood flow and therefore do not seek medical
advice. It is therefore possible that the hypoalgesia in hypertensive patients is related to the
incidence of silent cardiac ischemia. 26
1.6 Adaption to stress
L6.1 Definition and occurrence.
Stress, like pain is difficult to define in an objective fashion. Physical stress induced by injury or an inappropriate physicat environment is easy to quantify by the level of stimulus, but stress is the response to the stimulus, and this is influenced by numerous factors. The integration of these factors may result in a discrepancy between perceived circumstances and expectations which may result in a stress reaction at low observed levels of stimulus. Stress reactions involve many homeostatic systems, including the hypothalamic-pituitary-adrenal, sympatho- adrenal and endogenous opioid systems (Emrich & Mi[ar\ 1982' Milla¡r, l98l; Millan &
EmrictU l98l; Goldstein, 1987). In humans and animals high levels of stress are believed to be involved in the development of disease. Suppression of immune fi¡nction mediated by opioids has been implicated in the development and progression of rnany pathologies including cancer(Licinio et al., 1995; Tejwani et al., l99l; Plotnikoff, l98E; Shavit et al., 1985).
Consumption of drugs with abuse potential is reported to increase in stressed populations
(Lindenberg et al., 1993; Rhodes & Jason, 1990).
I .6.2 Physiological mechanisms
The hypothalamic-pituitary-adrenal a¡ris is the best known system involved in stress. Release of corticotrophin-releasing-factor (CRJ) from the hypothalamus results in the release of
ACTH from the pituitary gland. In turrç ACTH stimulates the release of corticosterone from the adrenal cortex. The hypothalamus is also the main subcortical centre regulating sympathetic activity and the sympathoadrenomedullary system (SAMS) is the other important system involved in response to stress. Activation of this system stimulates the release of 27 epinephrine from the adrenal medulla. These systems are much more complicated than described here and involve the recruitment and integration of many neural and hormonal components (Goldstein, 1987). Among these a¡e the opioids.
A number of changes in these systems have been observed in hypertensive animals. A lower level of release of ACTH and smaller content of CRF in the median eminence and posterior pituitary were soen in young SHRs when compared with WKYs (Hashimoto et al., 1989).
Hypothalamic vasopressin is reduced in the young SI{R, while posterior pituitary levels a¡e elevated and these levels were not influenced when hlpertension was prevented by ACE inhibition or sympathetic denervation (Sladek et al., 1988). A decreased pituitary content and release of oxytocin has been reported in the adult Sfn with an increased content and release of vasopressin (Rosella Dampman et al., 1985). The release of vasopressiq but not oxytocin, was decreased by acute treatment with naloxone, indicating a possible role for the opioids in the modulation of pituitary hormone release.
The biorythesis of POMC, the precurser of p-endorphi4 in the intermediate lobe of the pituitary of the SHR has been shown to be reduced. This lower production of POMC can be reversed by antihypertensive treatment with propranalol, hydralazine or captopril (Felder &
Garland, 1989). A reduction in the expression of POMC as measured by mRNA levels has also beeri demonstrated in the anterior pituitary of the adult SHR. In these experiments a concurrent reduction in beta endorphin immunoreactivity and ACTH immunoreactive cells was demonstrated. This was investigated in the hypertensive and in the hyperactive substrains of the SHR and this pattern was confined to the hypertensive and not the hlperactive
¡,i n_L , substrain (Braas & Hendley, 19 4). 28
In view of their important role in physiological responses to stress, the adrenal glands have been studied in the SHR. This has been investigated by complete or partial adrenalectomy in combination with other manipulations. Bilateral adrenalectomy prevents the rise in blood pressure in the young SHR and reduces the blood pressure in adult SHRS (Ruch el al., 1984).
These workers suggested a role for glucocorticoids in maint¿ining hypertension in these
animals as betamethasone treatment restored blood pressure in adrenalectomised animals. In
another study, SHRs which had been adrenalectomised also showed a drop in blood pressue
which could be reversed with corticosterone replacernent (Hashimoto et al., 1989). Bilateral
demedullation attenuated, but did not prevent the rise in BP in young SHRS and had no effect
on blood pressure in the adult animal @orkowski & Quinq 1983). Collectively, these results
suggest that the adrenal cortex may be more important than the adrenal medulla in its
contribution to the increased blood pressrre in the SHR. This is srpported by more recent
data when adrenaline was shown to have no effect on blood presln¡re in normotensive SD and
\ryKY rats and hlpertensive SHR and stroke-prone SHR animals (Jablonskis & Howe, 1994).
In contrast to these reports, there is evidence that the adrenal medulla is more active in the
SHR. SHRs sympathectomised soon a.fter birth with a combination of antiserum to nerve
growth factor and guanethidine, still had blood pressures higber than age matched WKYs, but
this difference was abolished by adrenal demedullation (Lee et al., l99lb). Stimulation of the
entire spinal cord in pithed SHRs resulted in increased blood pressure and heart rate responses
compared to WKYs and in these experiments adrenalectomy abolished these differences oC¡ baween the strains @ucher & Stoclet, 1987). Cold stress (+ increased the urinary
excretion of adrenaline in juvenile (4 week) but not adult (l2week) SHRs suggesting an
increased response of the adrenal medulla to stimulation in prehypertensive SHRs (Yamori ef 29 a/., 1985). It would appear from these reports that both the cortex and medulla of the adrenal glands are probably important in the development of hypertension in SHRs but that they may have a lesser role to play once high blood pressure is established.
Overall it would appear that the pituitary-adrenal aris has a part to play in the development and maintenance of blood pressure in the SHR. A major role of this physiological system is in the response of the animal to stress and therefore it is possible that the changes observed in the SHR indicate a modified response to stress in this strain.
1.6.3 Behavioural factors
A number of studies have indicated that increased stress can raise blood pressure in rats
(Henry et al., 1993; Knardahl & Hendley, 1990; Kna¡dahl & Hendley, l99l). Increased stress can also caus€ changes in behaviour. For example, ethanol conzumption is increased in animals stressed by immobilisation or foot shock @ohorecþ, 1990).
The consumption patterns for a number of substances in hypertensive animals can also be diferent from those of normotensive animals. The SHR has been shown to have a preference for sodiunu calcium and potassium salt solutions (Ferrell & Dreith, 1986; Bertino &
Beauchamp, 1988; Bachmanov, 1989). This taste preference was investigated by recording electrical activity from the chorda t),pani nerve with and without the application of an epithelial sodium transport blocker in WKY and SHR animals (Formaker & Hill, 1990). The authors concluded that differences in taste receptor function could not explain the preference for salt in the SHR. 30
Thus, it would seem that the link between stress, hypertension and consummatory behaviour described in humans may well extend to animals. The study of the self administration of drugs with abuse potential such as ethanol by the SHR strain of rat may well provide further insights to these links.
1.7 Introduction summary
This introduction has been an attempt to review the literature relevent to the studies which will be described in the following chapters. Hopefully, it \¡/ilI have provided sufficient background for the reader to follow the subsequent chapters with ease.
Essential hypertension is a disease of multifactorial etiology which is widespread in the western world. Researoh on this disease is highly dependent on the use of animal models of which the spontaneousþ hlpertensive rat has emerged as the mo$ widely used. This is an appropriate model as it has many of the characteristics of the human disease in its dwelopment and outcomes. It can be treated beneficially with the same treatments as the human can and the disease can be exacerbated by similar factors.
The SHR displays other genetic traits which are not associated with hypertension, for example increased locomotor activity. The independence of this trait has been dernonstrated by breeding sub-strains of the SHR which express either the hypertensive or the hlperactive phenotlpe. 3l
Pain perception is reduced in the SHR as in the human hypertensive, and antihypertensive drugs modi$ this in animals and humans. The influence of cardiovascular drugs on pain perception may have important clinical implications in patients with cardiac disease.
The possibility of a commonality of mechanisms involved in hypertension, pain perception and behaviour has been recognised but the interactions have not been established.
Response to environmental and psychosocial stress is recognised as important in the
development of disease and the level of substance abuse in society and the SHR may provide
a useful tool in studying this.
I. 8 AIMS
The general aim of this thesis was to investigate the relationship between nociception and
hypertensiorL primarily in the SHR. The use of the SHR as a model for stress-induced
conzumption was also a secondary aim.
Previous studies had suffered from a number of inadequacies. Generally the acute, immediate
effects of manipulations or drug tre¿tments had been studied. However, disease and its
treatment are generally of long duration which allows some physiological adapations to take
place. For these reasons, drugs were generally given for a number of days in the experiments
presented here. 32
Due consideration had not always been given to the influence of manipulations on other aspects of the animal's physiology in interpreting specificity of effects. A number of parameters $/ere measured concurrently in the studies presented here in an attempt to identify non-selective effects of the drugs administered.
Since the primary aim of most previous studies was to research the cause of hypertension" the experiments concentrated on the development phase of hypertension. This has the problem of constantly changng physiology in the animal, making interpretion of the results difficult. For the experiments described in this thesis, we used adult animals in which hypertension was well established, held under constant conditions.
Specifically, the aims were:
l.To establish if the reported decreased response to pain in the SHR was a consêquence of hypertension.
2.To identifr the mediators of the reduced pain response
3.To identifu the site of action of the mediators
4. To improve the methodology in pain/stresVbehavioural measurement.
5.To investigate the consumption of alcohol in the SHR. 33
CHAPTER 2
GENERAL METHODS
2.1 Animals
2. l.l Source of strains and housing conditions
SHR and WKY rats,200-300g, were obtained from Animal Resource Centre, Western
Australia. Albino Wistars rats 200-3009, were bred and supplied by the University of
Adelaide Animal Services. Hlryeractive (WK- HA) and hypertensive (WK-HT) strains were
obtained from the CSIRO Department of Human Nutrition Animal Houso, OTlalloran Hill,
South Australia. The latter animals had been imported from Vermont, U.S.A some months
previously. All animals were housed in the cleatr or quarantine barrier facility of the Medical
School fuiimal House, Universþ of Adelaide, for at least 2 weeks prior to the
coÍtmencement of studies. The animals were housed 4 rats to a cage except where indicated.
They were maintained on a 12-12 hr lighVdark cycle at l9-23oc and h¿d free access to food
and water unless otherwise indicated.
prior to commencement of all studies, approval was obtained from the Animal Ethics
Committee of the University of Adelaide. 34
2. 1.2 Surgical procedures
The implantation of osmotic minipumps was undertaken using the methods recommended by the manufacturer, ALZA ( Palo Alto, California USA) as demonstrated on their videotape;
" Implantation of ALZET minipumps in rats and mice". The animals were anaesthetised with a
mixture of methohexitone sodium l0mg/ml and pentoba¡bitone sodium 60mg/ml, 9:l
administered intraperitonealy (ip) at a dose of 5ml/kg. The area of the neck and upper back
was shaved and swabbed with antiseptic. A lcm transverse incision was made in the skin
between the scapulae and a 4cm subcutaneous pocket was created by blunt dissection along
the midline in a cu¡dal direction. The minipump was inserted and the wound closed with
suture.The wound was dusted with topical antibiotic powder containing: neomycin sulphate
2.5mglg nitroflurazone 2mglg phenylmercuric nitrate 0.05mg/g and benzocaine 5mg/g. A
single sc injection ( O.lml) of an antibiotic mixture containing trimethoprim 8Omg/ml and
sulphadiazine 400m9/ml was administered immediately after the operation. The operation was
undert¿ken under aseptic conditions and the recovery was monitored by the staffveterina¡ian.
Testing of animals was not undertaken until l0 days post operatively when the animals
appeared to be compløely recovered. The wounds had healed by this time and their
loçomotor activities were simila¡ to those of animals which had not undergone surgery.
Surgical methods to enable intracerebroventricula¡ (iÐ infusions and telemetric measurernent
of blood pressure and heart rate will be described in the relevant chapters.
2.1 .3. Drug administration
Drugs for oral administration were dissolved in the drinking water at the appropriate concentrations and the volume consumed each day was measured. The drugs were made up 35
Delivery portal
Removable caP
Flange
Flow moderator
lmpermeable reservoir wall
Osmotic agont
Semipermeable membrane
<- Aqueoug Envlronment
Reservoir
Fig.2.l Schematic diag¡am of a Model 2OO2 AI'æT@ osmotic minipump' 36
fresh each day and a dark drinking bottle was employed to minimise the possibilþ of degradation catalysed by light. Drugs for parenteral administration were dissolved in sterile sodium chloride solution (l54mM). For norepinephrine, ascorbic acid (290¡rM) was added to the solution.
Osmotic minipumps were used for the administration of drugs in some experiments where longer term treatment was required, but the drugs were not suitable for addition to drinking water. Alzet model2002 pumps were used (Figure 2.1). These pumps a¡e 3cm long by 0.7cm
diameter and deliver at a rate of O.5¡rl/trour for up to 14 days. The subcutaneous (s.c.) route
was chosen in preference to intravenous (i.v.) administration as this was considered less
trar¡matic surgically and had been reported as equally effective (Criffin et al., l99l). Pumps
were primed by incubation at 3T C for four hours prior to insertion. This was important to
prevent access of tissue fluids to the drug reservoir which may have caused degradation of
drugs by enzymic action. The administration of drugs intracerebroventricularly was used in
some experiments and the precise methods are described in the appropriate chapters.
2.2 Blood pressure me¡surement
2.2.1. Indirect blood pressure measurements
Indirect blood pressure measurements were made by the tail-cuffmethod (Bunag,1973)'
Rats were restrained in a perspex tube l00mm in diameter and heated for l0 minutes with
warm air until the air temperature adjacent to the animals was approximately 32o C. A model 37
803 Doppler flowmeter (Parks Electronic Laboratory. Beaverton, Oregon) was used to detect blood flow in the caudal artery. The ultrasonic probe placed on the ventral surface of the tail was used to detect the blood flow, which could be occluded by inflation of a cuff connected to a anaeroid sphygmomanometer pressure Üt;*- A number of readings were taken until three consistent measurements were obtained. This device was checked against a mercury manometer on a regular monthly basis and after any routine maintenance
A comparison between tail-cuff and direct carotid artery methods of measuring blood pressure was undertaken in anaesthetised rats to verify the reliability of the tail-cuff
procedure. WKY rats were anaesthetised as described under surgical procedures above and
carotid a¡tery blood pressure recorded as described below. Tail-cuff blood pressure
measurements were undertaken at the same time as direct pressures were being recorded. The results showed that the methods gave similar estimates of systolic blood pressure (Figure 2.2).
2.2.2. Direct blood pressure measurements
Ca¡otid artery blood pressures were meas¡ured in rats, anaesthetised as described above. The
right carotid artery was exposed and tied off distal to the site of insertion for the catheter.
The proximal end of the exposed section of the artery was occluded with a ligature and an
incision made in the a¡tery. A plastic catheter 0.8mm internal diameter and l.2mm external
diameter, filled with heparinised saline, was inserted into the artery and tied in place. The
catheter was attached to a pressure transducer ( Statham PC23) which was connected to a
cha¡t recorder. The transducer and recorder were calibrated against a mercury manometer
prior to use. 38
150 o) ¡-. (t) (t) () l-r €iPO.,a, I 00 oEoÉr EE c)v o 50 (t) (t)h
0 TAIL CUFF CAROTID
Fig.2.2 Systolic blood pressure simultaneousþ measured by tail-cuffand direct carotid cannula in anaesthetised WKY rats. Meanstsem are shown, nd. 39
A Data Sciences telemetric data collection system was used tq directly monitor'iblood \ -"-_ pressure in some experiments. TAI IPA-C4O transmitters, RAl010 receivers and a Dataquest
IV aquisition system were employed. Full methodology is described in chapter 7.
2.3 Nociception tests
2.3.1 Hotplate test
The standa¡d method (Woolfe & MacDonald, lgß) modified by the use of an electronically
controlled heating block was used. The block had a smooth metal surface 250 by 250 mm
and was set to a temperature of 52fl.1oC. The plate was surrounded by a transparent
perspo( barrier 100 mm high. The animals were placed on the heated surface, and the end
point was the time when they climbed over the barrier or licked their feet. In the latter case,
the animals were rernoved from the hot plate immediately. The time benveen the animal being
placed on the hotplate and the end point was meazured in seconds. Accurate electronic
feedback temperature control was important as variations in temperature of lo C were found
to cause quite noticeable changes in latency. The end point of this test is achieved by the
animal demonstrating an organised behaviour and therefore involves supraspinal processing
(Chapman et al., l9S5). Repeated testing is not advisable with this procedure as learning will
influence the latencies.
The efFect of morphine on the hotplate latency of WKY rats Ìvas used to demonstrate that this
method could measure graded levels of analgesia. (Fig 2.3). Rats rilere injected
subcutaneously with morphine sulphate at doses in the range of l0 to 45 pmoVkg and placed 40
1
75 go o) crt X 50 CÚ
\oo\ 25
01020304050 Morphine dose (¡rmol/kg s.c.)
100
g() 75 (l) GI
ã 50 É \o o\ 25
0102030 Morphine dose (pmol/kg s.c.)
Figure 2.3 The effect of morphine on hotplate (upper panel) and tail-flick latencies (lower panel) in WKY rats. Means t sen! n:6. 4t
on the hotplate. Their latency was measured and expressed as Yo of mærimum. Maximum latency was set at one minute to avoid the possibility of tissue damage. A dose dependent increase in latency was observed'
2.3.2Tatl-flick test
The standard method @'Amore & SmittL l94l) was modified by the use of Peltier diodes as
used here a heat source. The original test used radiant energy focused on the tail. The device
was designed and built in the University of Adelaide at the request of the author. It utilised a
peltier effect heat pump ( lvfidland Ross, Canrbridge, MA) which is a device that generates or
absorbs heat when a current passes through a junction of two dissimilar metals (Fink &
Christiensen,lg3g). The current was supplied by an amplifier driving a MOSFET bridge' The
ventral surface of the animal's tail was laid upon the diodes and the po\üer supply activated.
This raised the temperature of the diodes to a maximum of 60oC. The animal was free to
remove its tail from the heat source at any time and the time taken for the animal to lift its tail
was recorded.
In both the hot-plate and tail-flick tests, inspection of the ani_mals feet and tail did not reveal
tissue damage.
The effect of morphine on the tail-flick latency of tilKY rats was used to demonstrate that this
method could measure graded levels of analgesia in a similar fashion to that described for the
hotplate (Fig 2.3). 42
2.4 Locomotor activity (LMA)
LMA was measured by placing the animal in a transparent perspex chamber 25 X 25cm square by l8 cm high with a wire mesh lid. A l0xl0 array of photocells 1.5 cm above the floor of the cage detected the movement of the animal. These photocells were linked electronically to an IBM compatible computer which analysed the signals from the detectors and determined the location of the animal every l00ms. The animals were monitored for
2gmins after a 2 minute familiarisation period. All locomotor measurements were made between 9 and l1 am. The actual distance moved per unit time could be measured by this apparatus which allowed the expression of the results as metres per second (m/s) for the length of the trial.
2.5 St¡tisticrl eneþsis
Data analysis was undertaken using the GraphPad Instat or Prism computer software
packages. fuialysis of va¡iance (ANOVA) followed by Tukey-Kramer, Dunnet's, Student t-
tests, paired or unpaired, was used where va¡iances of groups were not different. Where a
significant difference in group va¡iances existed, equivalent non-parametric tests were used. In
all studies p< 0.05 was taken as significant. Values are expressed as mean t sern for the
number of estimates shown. Lwels of significance at p< 0.05, p< 0.01 and p< 0.00lare
indicated by * (#), ** (lÉ#) *d +**(###) respectively. 43
CHAPTER 3
STIIDIES ON TWO STRAINS OF RAT DERIVED FROM THE SHR AI\[D THE
INFLTTENCE OF GENDER
3.1 Introduction
3.1.1 WK-HA and WK-HT
Two of the most distinctive behavioural characteristics of the SHR are an increased level of locomotor activþ (Sagvolden et al., 1992; Danysz el al., 1983) and a decreased response to nociceptive stimulation as provided by a hot plate (Sitsen & de Jong, 1984; Virus et al., 1983;
Maixner et al., lg82). Locomotor activity in the SHR is about double that seen in the WKY
(Sutterer et a1.,1984; Danysz et aL.,1983; Knardahl & Sagvoldera 1979). Lowering of blood pressure by treating SHRs with the directly acting vasodilator hydralazine was shown not to lower the LMA in these animals (Whitehorn et al., 1983). In the same study the blood pressure of the normotensive WKYs was increased by applying a renal artery clip and this procedure did not increase locomotor activity (Whitehorn et al., l9S3). The authors concluded th¿t increased LMA was an inherent property of the SHR and was independent of blood pressure.
Subsequent to these results, the same group of workers developed new substrains of the SHR and WKY by selective recombinant inbreeding (Hendley & Ohlsson, l99l). The Wistar-
Kyoto hyperactive (WK-HA) and the rilistar-Kyoto hypertensive (WK-HT) are two strains 44 of animals produced by this program. The WK-HA retains the trait of increased locomotor
activity which is seen in the SI{R" but it is not hypertensive. Conversely, the \ryK-HT
develops hypertension in the same way as the SI{& but is not hyperactive. Both of these
strains are derived from the SHR.
These strains have been used to study the increased cardiovascular reactivity to stress that has
a been reported in the SHR (Knardahl & Hendley, 1990). A low pressure jet of air directed to
rat's head for 5 minutes results in increased blood pressure. A greater increase in blood
pressure was observed in the SHR and WK-HA groups compared to the WKY and WK-HT
groups (Knardahl & Hendley, 1990). Foot shock stress has also been used to test the
reactivity of these strains (Hendley et al., 1988). The procedure involved a train of electrical
pulses through the grid floor of the cage with a stimulus of lmA for a duration of 0.6sec and
a frequency of one every 6 seconds for a minute. Direct blood pressure was monitored and
blood samples were taken for analysis of catecholamines. Foot shock elicited a greater heart
rate and blood pressure resporuye in the \ryK-HA and SHR than the WK-HT and WKY and
these correlated with increased levels of circulating catecholamines (Hendley et al., 1988)'
These experiments indicated that the increased cardiovascular reactivity to stress in the SHR
is also evident in the WK-HA but not in the WK-I{T strain.
The open field test, where an animal is placed in a novel open environment, has been used to
test the response of these animals to stressful situations. The sensitivity of the pituitary
adrenal axis in these animals has also been evaluated by challenge with iv CRF and subsequent
measurement of the release of stress hormones such as ACTH, aldosterone, corticosterone
et 1993). and various other indices of neuroendocrine response in these strains (Castanon al., 45
The results of these studies indicate differences in their neuroendocrine profile and cardiovascular response to stress which collectively suggest that it is the hyperactive rather than the hypertensive trait that is involved in heightened response to stress in these animals.
Although the reactivity of these strains when subjected to stress has been studied in some detail, there a¡e no reports on the sensitivity of these strains of animals to pain. Animals subjected to a variety of stress procedures display hypoalgesia in nociceptive tests @ieretti ef al., l99l;Millan & Emricb l98l). The hypoalgesia displayed in these situations is associated with the release of endogenous opioids (Vaswani et aI., l9S8). In view of this, it might be expected that the reduced response to nociception in the SHR would be a result of stress induced analgesia and therefore would be linked to the hyperactive rather than hypertensive trait. In this chapter \¡/e compared the two strains for their sensitivity to a nociceptive stimulus provided by a hot plate to establish if altered pain perception was linked to the hyperactive or the hlpertensive trait.
3.1.2 Gender
The influence of gender on the blood pressure, LMA and nociception in WKY and
SHR5 was also sh¡died. Gender differences have been reported in SHR animals for blood pressure (Vincent et al., 1994;Iams & Wexler, 1979; Cambotti et al., 1984; Bachmann et al',
l99l) and locomotor activity (Hendley et al., 1985).
Blood pressure in adult male SHRs has been reported to be 10-30 mm of Hg higher than females of the same age (Vincent et al., 1994; Cambotti el al., 1984). Neonatal castration of males reduces this difference and neonatal androgen treatment of females increases their blood pressure towa¡ds the levels seen in males (Cambotti et al., l9S4). Similar results have been 46 reported with these treatments being applied at 30 days of age (Iams & Wexler, 1979').
Recently, the density of alpha 2 adrenoceptors prepared from renal membranes has been compared in male and female SHRS (Gong et al., 1994). The study showed 60% fewer receptors in the female SI{R, compared to the male, and this correlated with blood pressure.
The receptor levels and blood pressure in the males were lowered by castration and androgen treatment of females increased the levels of both. Endothelium-dependent reloration of blood vessels has also been compa¡ed in female and male SHRs. Endothelium- dependent responses of the thoracic aorta mediated by nitric oxide were more pronounced in female SHRs (Kauser
& Rubanyi, 1995). The lower level of adrenoreceptors and the lesser degree of endothelial dysfirnction seen in female SHRs may account for the lower blood pressure levels in this sex.
The locomotor activity of female SHRS is greater than that of males and this is more evident in animals greater than 6 months of age (Hendley et al., 1985). This trait has also been shown in female rats of nonhypertensive albino strains (Beatty & Fessler, 1976a). Female and male animals of different ages were tested for a number of behaviours, including open field activity and foot shock threshold. Juvenile animals showed no sex difference in either of these parameters, but adult females showed more locomotor activity and a lower shock threshold than males.
There are no reports of a systematic study of the influence of gender on nociceptive behaviour in SHR animals, but a number of studies have been undertaken in other nonhypertensive strains of rats. As mentioned above, foot shock threshold has been measured in non-hypertensive albino rats @eatty & Fessler, 1976a) and shown to be lower in female animals. In a separate study, these authors investigated the influence of gon4dectomy and sex 47 hormone treatment and concluded that the differences in shock threshold were related to gonadal hormones (Beatty & Fessler, 1976b). The latency of male Sprague -Dawley rats in the hot plate and tail-flick analgesic tests is reduced by castration and this effect is partially reversed by testosterone administration, indicating a modulatory role for gonadal hormones in the response to nociception in this strain (Forman et al., 1989). In tail flick and foot shock tests female Sprague-Dawley rats display less morphine and swim stress induced analgesia than male animals @omero & Bodnar, 1986).
In most studies using the SHR as a model of hypertension, male rather than female rats are used, presumably for the usual reason that it avoids the problem of fluctuating sex hormones and associated physiological fluctuations. The male SHR is also considered a more appropriate model of hypertension than the female as hypertension is more prevalent in male humans @achmann et al., 1991). In view of these reports on gender differences in many of the parameters to be studied in this projeú't, locomotor activity and behaviour of male and female SHR and WKY animals to nociceptive stimulation were compared to identify gender differences prior to further experiments.
3.2 Methods
3.2.1 Animals
Male 2OO-250g WK-HA and WK-HT rats were originally imported from the laboratories of
Professor E. Hendley, Burlington Vermont by the Division of Human Nutrition of CSIRO.
During the experiments described here, they were housed in the quarantine banier facility of 48 the Medical School Animal House of the University of Adelaide and were on a l2l|2 light dark cycle with free access to food and water
\ryKY and SHR rats 200-2509 , of both sexes were obtained and housed as described in the general methods.
3.2.2Blood pressure, hotplate and LMA measurements
For the experiments on the influence of gender in the SHR and WKY, tail-cuff blood
pressure, nociception and LMA \¡rere measured as described in Chapter 2. In the experiments
on the hyperactive and hypertensive strains, tail cuff blood pressure and locomotor activity ,!,, i iI.,,r*,,-,, 1i,..,_ , data were provided by L. Jabonilskis of CSIRO using methods similar to those described in
the Chapter 2. The hot plate data were obtained as described in the general methods.
3.3 Rcsults
3.3.I \ryK-HA and WK-HT animals
Systolic blood pressures were significantly higher in the WK-HT strain than in the WK-HA
animals. The WK-HT animals had systolic blood pressures of 128+4 mmHg and the WK-HA
animals pressures of I l3+3 mmHg (Fig 3.1). Locomotor activity was also significantly
different bøween the two strains with a value of 188+27 counts/I5 min for the WK-IIT group
and 664+80 countll5 min for the WK-HA group of animals ( Fig 3.1). These values show
similar relative differences to previous reports (Whitehorn et al., 1983; Danysz et al., 1983; 49
150 500 6 ** rT{T É IHA EF ':t(t)c) ã loo 1000 LC) ø) rt) *¡*t3 'l' = >q lJ¡ øl pË Ë.oÞc) óo å50o o *** .o
0 BP HP LMA
Figure 3.1 Systolic blood pressures @P), hotplate latencies (If) and locomotor activities
(LMA) of hypertensive (WK-HT) and hyperactive (WK-HA) strains of rat. Means *sem, n:
9-l l. *'rp<0.01, ***p<0.001, t-test 50
Kna¡dahl & Sagvolden, 1979), but are not directly comparable to results in other chapters as
LMA was measured by line crossing in this section and by infrared grid in all other chapters.
The hypertensive (WK-I{'[) group of animals had a latency on the hot plate of 36.7+3'8 compared to 18.8+2.4 s. for the hyperactive (WK-HA) group (Fig 3.1). These latencies show a signiñcantly reduced response to the hotplate in the WK-HT animals compared to the tilK-
HA strain.
3.3.2 Gender
Blood pressures of male and female WKYs were 139+2 and 127*.4ñîflg respectively,
compared $/ith 207+8 and lg5+4 for male and female SHRs (Fig. 3.2). These were
significantly different between strains, but not between genders. Locomotor actMties of male
and female WKYs were 9.2* 2.0 and 13.911.3 X l0-3 m/s, respectively, as compared to
18.5+l.land 2l .5+ 2.4 Xl0-3 m/s for SHRs ( Fig 3.2) They show significantly higher values
for the hypertensive rats and sligbtly, but not statistically significantly, higher levels for the
females compared to the males of both strains. Hotplate latencies of male and female WKYs
were 28.5+4.8 and 28.2+5.2 s. respectively, comparedto 42.7+4.3 and 45.3 +3.4 s. for male
and female SHRS. These differences were statistically significant in the case of females but did
not quite reach significance in the case of males.
Overall, there were clea¡ differences between WKY and SHR rats of the same sex in blood
pressure, LMA and hot plate latency. Only in the latter case, and only for males, did these fail
to reach significance. Tail flick latencies were not significantly different between any of the
groups. There were no statistically significant differences between sexes of the sa¡ne strain for
any of the parameters measured. 5l
Blood pressure
300
*** *** TCD 200 E E o. o 100
0
I-r Male WlCl @ Femaþ WKf Male SHR I- Female SHR Locomotor activity 30 *
ao * È or20 o I Ë .È ro (t
0
Figure 3.2 Blood pressures and locomotor activity of male and female WKY and SHR rats.
Meanstsem, n: 6 afe shown. * p<0.05, 'r** p<0.001 compared to opposite gender same strain, Tukey-Kramer test. 52
Hotplate * 50
.^,40() o -30 (J c 9zo -5 1
æ Female WKY
Tail-flick I Femaþ SHR
5
o to (J c 2.5 o +,6 J
00
Figure 3.3 Hotplate and tail-flick latencies for male and female WKY and SHR rats.
Meanstsem, o: 6. + p<0.05 compared to opposite gender same strain, Tukey-Kramer test 53
3.4 Discussion
These results indicate that the altered response to nociception as indicated by increased hot
plate latencies in the SHR is also a characteristic of the hypertensive but normoactive WK-HT
strain derived from the SHR. It is not evident in the hyperactive but normotensive WK-HA
strain. The difFerence in latencies between the WK-HT and WK-HA animals is of the same
order as seen when SHRs and rWKYs are compared (Fig. 3.3), and this is consistent with
previous studies (Sitsen & de Jong, 1984; Virus et al., 1983; Maixner et al., 1982). The
experimental design was limited somewhat by quarantine restrictions on the animals imported
from the USA. Direct comparisons between the imported \ryK-HT and WK-HA strains and
the local SHR and WKY strains in the same experiment was not possible. However, from the
limited studies reported here, it appears that hypertension, but not increased locomotor
activity, is the trait more closely associated with altered pain responses in the SHR. Ahhough
the hyperactive and not the hypertensive trait has been associated with an increased
ca¡diovascular reactivity to stress as measured by blood pressure and blood flow changes
(Knardahl & Hendley, 1990), this may not extend to stress induced analgesia.
The two substrains of the SHR described here would have proved useful for some further
studies, but availability and quarantine restrictions prevented further use of these animals .
Therefore, the SHR strain was used for the remainder of this thesis, but with locomotor activity monitored in each experiment to indicate effects of drugs or manipulations which were either nonspecific or were associated with the hyperactive rather than the hypertensive and analgesic traits. 54
Although it did not reach significance in this study, there is a suggestion from the data that the female animals have higher LMd as has been reported (Hendley et al., 1985). The previous report only showed a clear gender difference in LMA when the animals were greater than 6 months old and the animals used here were only 3 to 4 months old at the time of testing. This may be an explanation for the discrepancy. The blood pressure data also a slightly higher blood pressure in the male animals of both strains, but not as great a difference as has been reported by other workers (Gong el al., 1994; Iams & Wexler, 1979). Perhaps with greater group numbers a difference would emerge, but this seems unlikely in view of the small
standard errors in the data.
In view of the similarity between sexes in both WKY and SI{R, in blood pressure, LMA and
nociceptive behaviour, male animals were used for the remainder of this thesis. This allowed
direct comparison between this work and the majority of previous work which had used male
animals.
Acknowledgments
I wish to thank Dr. P. Howe, Division of Human NutritiorU CSIRO, for donating the \ilK-HA , i¡ and WK-HT animals, and Dr. L. Jabloniskis of the same laboratory for providing blood
pressure and locomotor activity data on these animals. 55
CHAPTER 4
THE EFFECT OF ANTIHYPERTENSTVE DRUGS ON NOCICEPTION IN THE
SHR AND WI(Y RAT.
4.1 Introduction
'fhe relationship between blood pressure and responses to pain in rat models of hypertension is controversial. The reduced response to nociception in the SHR rat has been shown to be shared by other rat models of hypertension. In one study, one clip renal hypertensive rats and rats made hypertensive by DOCA-salt treatment displayed similar increased latencies on the hot plate (Zarrir et a1.,1980). However, other workers have shown that the one clip renal and
DOCA-salt models of hypertension do not result in increased latencies on the hotplate test
(Sitsen & de Jong, 1984). In order to examine if the hypoalgesia in the SHR was a consequence of increased blood pressure, the influence of antihypertensive treatment has also been studied. Oral administration of the antihypertensive drugs hydralazine and captopril to
SHRS and WKYs from 5 to l0 weeks of age did not alter nociceptive responses on the hotplate when tested at l0 weeks (Sitsen & de Jong, 1984).
The role of opioids in mediating the hypoalgesia seen in hypertensive animals was also examined in the studies mentioned above. Naloxone, which is capable of blocking central and peripheral opioid receptors, abolished the hypoalgesia seen in the hypertensive animals (ZamÍ et al., 1980). A quaternary salt of naloxone, N-methylnaloxone bromide, which cannot 56 penetrate central sites, did not reverse the hypoalgesia seen in the hypertensive animals (Sitsen
& de Jong, 1984). This suggested a role for central opioids in the reduced response to pain in the hypertensive animals. Combined central and peripheral blockade of opioid receptors with naloxone did not influence blood pressure in this study. Both papers concluded that central opioids were involved in the analgesia, but in one study they concluded that the analgesia was closely linked to blood pressure and in the other that it was not.
The alternative approach of raising the blood pressure of normotensive animals and measuring nociception, rather than lowering the blood pressure of hypertensive animals, as described above, has also been employed. Blood pressure has been raised with bolus iv injections of the alpha adrenoreceptor agonist phenylephrine and tail-flick latencies measured
(Randich & Maixner, 1984). The increase in tail-flick latencies observed when phenylephrine was inñ¡sed could be blocked by the alpha adrenoreceptor antagonist phentolamine, but was unaffected by naloxone. This latter study was limited to immediate effects of these manipulations on nociception. As the drugs were administered iv in a bolus, the changes in blood pressure were transient, and the authors only measured tail-flick responses for 3 minutes after the peak blood pressure response. It would seem from these studies and the results reported in Chapter one that a reduced response to nociception is observed in the
SI{R, but that the relationship between blood pressure and pain regulatory systems is not clear.
There are a number of active peptides and processing enzymes common to both blood pressure and pain regulatory mechanisms. These may provide an area where interactions between these systems operate and explain the interaction between blood pressure and 57 nociception. These include Substance P and bradykinin as well as the opioids. In addition ,
there is evidence that levels of all of these substances can be influenced by the RAS.
SHRs have been shown to have increased levels of NGF (Donohue et al., 1989), which is
thought to be responsible for the sympathetic noradrenergic hyperinnewation seen in the
peripheral tissues of these animals (Cassis et a1.,1985). Sensory nerves are also dependent on
NGF for their development and maintenance (Lindsay & Harmar, 1989). Substance P, a
peptide thought to be important in pain transmission, is found in high concentration in some
sensory nerves. It is also a substrate for angiotensin converting enzyme. Levels of substance P
can be increased in slices of rat spinal cord by treatment with the ACE inhibitor captopril
(Mauborgne et a1.,1987). Thus, drugs which influence the RAS may well also influence pain
mechanisms via an effect on Substance P.
ACE is also the primary enzyme responsible for the breakdown of bradykinin (Ishida et al-,
1989; Sheikh & Kaplan, 1936). Bradykinin is a potent vasodilator and is thought to be
involved in the antihypertensive action of ACE inhibitors such as captopril (van den Buuse &
Kerkhofl l99l; Bao et al., 1992\.It is also a peptide associated with nociception (Dray et
al., 1992; Chapman & Dickensorl 1992; Bauer et al., 1992).Intravenous bradykinin inhibits
the tail-flick nociceptive reflex in the rat and this can be prevented by neonatal destruction of
substance P containing sensory neurons @auer et al., 1992). Blockade of bradykinin
receptors in the dorsal horn sensory neurons of the rat with the bradykinin receptor antagonist
HOEI49 reduces the response of these neurons to nociceptive stimulation with formalin
(Chapman & Dickenson, 1992). 58
Enkephalinase (EC24,11), which is responsible for the degradation of enkaphalins, is a Zn
metallopeptidase. This group of enzymes includes ACE and inhibition of these enzymes can
lead to analgesia in mice and rats (Maldonado et al., 1994;Roques et al', 1980). Captopril is
capable of potentiating morphine analgesia in rodents (Ercan et al., 1980; Das ef al., 1982).
These findings, in combination with the development of more selective non peptide
antagonists for angiotensin receptors, provided an opportunity to re-examine the relationship
between nociception and pain in the SHR, The evidence described above suggests that the
RAS and, in particular, the level of ACE activity, may be influencing the levels of peptides
involved in pain processing. In order to investigate further the relationship between blood
pressure and pain, the effect of two hypertensive drugs which have mechanisms of action via
the RAS were examined: captopril, the ACE inhibitor, and losartan, the ATI receptor
antagonist. Two antihypertensive drugs which have mechanisms of action which do not
involve the RAS were also tested for comparison: hydralaane, the directly acting vasodilator
and verapamil, a calcium antagonist. The latter drug was also included as calcium is thought
to be important in the mechanism of pain perception and calcium antagonists have been
shown to influence the analgesic efFects of drugs such as morphine (Smith & Stevens, 1995;
Kuzmin et al., 1994; Carta et al., 1990). Animals with established hypertension as well as
normotensive controls were studied.
Initially, the efFects of acute treatment by ip injection of captopril and hydralazine were tested.
In subsequent experiments drugs were administered in the drinking water to eliminate the
stress involved with injections and in an attempt to deliver the drugs continuously to the site
of action. 59
Tail-flick was used as a measure of reflex response to pain and the hot plate test as a response which involved supraspinal sensory processing. Locomotor activity was measured as it is elevated in genetically hypertensive rats (Whitehorn el al., 1983; Knardahl & Saryolden,
lgTg'), and also as an index of any general psychomotor depressive effects of the drugs used.
4.2 Mcthods
4.2.lAnimals and drugs
Male SHRs and WKYs 3004009 were used. They were housed as previously described in
Chapter 2. Drugs for injection were dissolved in sterile l54mM sodium chloride at the
appropriate concentration and injected in a volume of 1.0 mÛkg. Captopril (O.7mglml),
hydralazine HCI (0.2mglml), losartan (0.2 mgln,l) and verapamil (0.1,0.3&l.0mg/ml) were
dissolved in distilled water and the resulting solutions were made available to the animals in
their drinking bottle for 3 days prior to testing. Dark drinking bottles were used as a
precaution against light catalysed degradation of the drugs. Drug solutions were replaced
daily and the volume consumed recorded.
4.2.2. Blood pressure and behavioural measurements
Tail-cuff blood pressure, locomotor activity, tail-flick and hotplate latencies were measured
as described in Chapter 2.
In the acute experiment, animals were tested for hotplate latencies 30 minutes after they were
injected with drugs 60
4.3 Results
4 3 I Effects of injected drugs
Prior to drug treatment, WKY hot plate latencies were of the order of l0 seconds and
significantly different from the SHRs which were 30 seconds. Injection with saline caused a
significant reduction in latency in the WKYs from 10.6+1.0 seconds to 6.710.7. However,
saline had no effect in the SHR. Captopril was without effect in the WKY, but reduced the
latency in the SHR from 25.7X4.9 seconds to 17.2!4.0 (Table 4.la). These data suggested an
effect of captopril in reducing latencies, but the significant effect of saline in the WKY made the method suspect. Prior to treatment with hydralazine or saline in a second experiment (
Table 4.lb), the WKY latency was l0.7tl.l, a value simila¡ to that above. The latency in the
SHR group was 22.3!1.7. Saline caused a significant decrease in latencies in WKY and SHR
animals. Hydralazine reduced the latencies in the SHR but not the WKY. Again, the effect of the drug in reducing latencies was difficult to interpret because of the saline effects.
4.3 .2 Drug doses consumed
The doses of drugs ingested by the animals through their drinking water were: captopril
95Ðmg/r.glday, hydralazine l9+lmg/kÚðzy,losartan l8+2 mg/kg/day and verapamil 7t0.3,
2OÐ. and 5815 mglkdday. 6l
Table 4.la
\ryKY SHR
No injection l0 6+1 0 29.6L3]ffi
Saline ip 6.7*0.7* 25.7+4.9
Captopril ip 95+1.2 t7.2+4.0*
Table 4.lb
WKY SHR
No injection 10.7+l.l 22.3L1.1ffi
Saline ip 5.0+0.3** 14.2+.1.7**
Hydralazine ip t5.2+4.2 14.3+1.4**
Table 4.1
The effects of (a) saline and captopril 5Omg/kg ip or (b) saline andhydralazine 2Omglkg in
WKY and SHR rats.
Hot plate latencies are shown as meansf SEM, n:6-12. * p<0.05,**p<0.01 compared to same strain ,no injection.llll# p<0.001 compared to WKY, no injection, t-test. 62
4.3.3 Effects of oral captopril
The untreated SHR when compared to \ryKY animals showed higher blood pressures, higher locomotor activity, no difference in tail flick latencies and higher hotplate latencies ( Fig.a.l ).
Captopril lowered the blood pressure in the SHR strain by 44 mmHg and in the WKY by 20
mmHg. ( Fig.a.I ). It had no efFect on locomotor activity or tail flick latency in either strain of
rat. In the hotplate test, captopril eliminated the latency difference between the strains.
4.3.4 F:trect of oral hydralazine
Hydralazine treatment caused a marked reduction in blood pressure in the SHR animals and a
smaller reduction in the WKY (Fig. a.2 ) Thus, the usual difference between the strains was
abolished by hydralazine treatment. Hydralazine did not significantly influence locomotor
activity or tail flick in either strain. Hot plate latencies were not altered sufficiently to
eliminate the normal difference between the strains. There was still a significant difFerence in
hot plate latency between the strains in the presence of hydralazine.
4.3.5 Effects of oral losartan
There was no significant lowering of blood pressure by losartan at the dose used.
Locomotor activity and tail flick latencies were unaltered. The difference in hotplate latencies
between the strains was markedly reduced by losartan treatment ( Fig. a.3). 63
EWKY r----'twKY ØSHR nw H ^sR ttt # :EÀÉ Þ
CAPTO CONT CAPTO coN[
I 75 25 ØSTIR f-f wKY ø-SFIR -IWKY o o É t t^o Ëa go o :99 66 o o d Fi
CONT CAPTO CONT CAPIO
Fig.4.l
Blood pressure, locomotor activity, hotplate and tail-flick latencies in the presence and * absence of captoprilgsmg/r¡glday p.o. Means + SEM, n:12 p<0.05 compared to WKY for the same treatment. # compared to same strain different treatment, t-test. 64
r-----'rwKY r,?SHR ØSÍTR f----wKY E2Ã 4#4 >Þ !Eb.Ë ### qo I .:t õ
cotIT HYDRAL CONT HYDRAL
t----1wKY ?7772SllR r---rwKY P-sI{R
o ço l5 oÉ o 6^ !ao!? ,Zû*8 Ëä a
.6
IIYDR,A.L CONT TTYDRAL
Fig.4.2
Blood pressure, locomotor activity, hotplate and tail-flick latencies in the presence and r absence of hydralazine lgmgkglday p.o. Means I SEM, r-l2 p<0.05 compared to WKY for the same treatment. # compared to same strain different treatment, t-test. 65
7V^SHR r-----twrcY :WKY vzTvÀslIR to 4d '5É Ë.8 €É €= ñ
LOSART CONT LOSART
r-rwKY vrmsl:IR r--lwKY 7V?STIR 0 v tr .2À åa 11fl lf¡çO 'é" ;st fxx 3-Ëä t
CONT I,OSART
Fig.4.3
Blood pressure, locomotor activity, hotplate and tail-flick latencies in the presence and absence of losartan lSmglk/day p.o. Means + SEM n:12 * p<0.05 compared to WKY for the same treatment. # compared to same strain different treatment, t-test. 66
Locomotor ætivity -TWKY Blood pressure vvTzslR
M tt* **a fl r*+
*t 6 E t 'fr
È ., Êa o
58 7 20 58 7 n
r----wKY Tail-flick u--sl-R Hoþlate # t++
o q) () q) Ø Ø ** o o É c) c) d -ì ¡C6
7205872058
Figure 4.4
The eflect of verapamil on blood pressure, locomotor activity, hotplate and tail-flick latencies in the WKY and SHR. Dose of verapamil is shown in mg/kg/day po for three days. Means *
SEM, * p<0.05 compared to WKY at the same dose. # compared to same strain at the lowest dose, t-test. 67
4..3.6 Effect of oral verapamil
Verapamil lowered BP slightly in both the WKYand SHR at the highest dose. It did not
abolish the difference in blood pressure between the two strains. (Fig.a.a).
The differences in locomotor activity between the two strains remained even at the highest
dose of verapamil. The difFerence in hotplate latencies between the strains was unaffected by
the lowest dose of verapamil, reduced at the intermediate dose and increased at the highest
dose (Fig.4.4). There were no effects of verapamil on tail-flick latencies at any dose used.
4.4 Discussion
The results of this study confirmed the findings in Chapter 3 and previous findings (Whitehorn
et al., 1983, Knardahl & Sagvolden, 1979; Zamir et al., 1980; Maixner et al., 1982),
suggesting that the SHR has a lower sensitivity to pain in the hotplate test and higher
locomotor activity. None of the treatments altered locomotor activity in these animals, except
for a slight stimulation at the higher doses of verapamil, indicating that the drug effects on
nociceptive behavior were not due to non-specific CNS depression. The lack of difference in
the tail flick test indicates that the difference in pain sensitivity in the two strains is probably
mediated at a supraspinal level and is in accordance with the results of other studies (Huang &
Shyu, 1987; Virus et al., t98l) showing no difference in tail flick latencies between these
strains. 68
A number of differences between the two strains emerged with drug treatment. Acute treatment of the animals with ip injections indicated that saline was as effective as captopril and hydralazine in reducing latencies in some, but not all cases. It was concluded from these results that the stress involved in handling the animals for injection combined with the lack of steady state drug concentrations at the time of testing made this method of drug administration unreliable for these studies. Acute administration of drugs was not used in the remainder of this thesis. Orally administered hydralazine was most effective in lowering the blood pressure of the SHRS to levels prevailing in the normotensive rats, but had little influence on the hot plate latencies. This finding is in agreement with other findings where the
animals were chronically treated from a young age (Sitsen & de Jong, 1984) and indicates that the nociceptive differences in the strains is not a consequence of the differences in blood pressure. Captopril at the dose used was less effective than hydralazine in lowering blood pressure in the SI{R, but eliminated the differences in hot plate latencies. This result is not in
accordance with the findings of Sitsen and de Jong, who also treated rats with this drug and
did not alter pain perception (Sitsen & de Jong, 1984). The animals in their study were treated long term between the ages of 5 and l0 weeks during the developing phase of hypertension
and at a lower dose than used in the studies described here. These differences in experimental design may be an explanation for the lack of effect of captopril in that report.
Losartan at, or below, the threshold dose for lowering blood pressure in the SHR markedly reduced the hotplate differences (Fig 4.4). This drug and its active metabolite E)G3174 are highly selective for the angiotensin ATI receptor (Timmerrnans et al., 1993; Wong et al.,
1990). The latter result adds weight to the captopril data presented here and implicates angiotensin in the modulation of nociception in these animals. 69
The relatively small effects of captopril and losartan on blood pressure could be explained in terms of the timing and duration of administration. When administered long term during the development phase of hypertension, ACE inhibitors and losartan have been shown to have a continuing effect in preventing hypertension in the SHR. (Lee et al., l99la; Hanap et al.,
1990a). In the present study the drugs were only administered for three days, and at a time when the hypertension was well established.
Collectively, the rezults highlight the interplay between renin- angiotensin mechanisms and
intrinsic analgesic mechanisms. The effect of captopril on pain perception seen here could
possibly be explained by altered levels of peptides such as substance P, bradykinin or the
opioids due to the inhibition of peptidases. However, the results with losartan, which is
highly selective for the ATI receptor and has no reported influence on peptide processing,
contradicts this hypothesis. The effect of captopril on hot plate latency is more likely to be
due to reduced AII levels, leading in turn to a reduction in the activation of ATI receptors.
Although a subtle influence of blocking ATI receptors on the levels of active peptides other
than angiotensin is possible, it seems much more likely that the simila¡ effects of captopril and
Iosartan on nociceptive responses in the SHR are mediated via the ATI receptor. It follows
that agents which reduce the activation of ATI receptors in the SHR also reduce the extended
hot plate latencies seen in this strain by a mechanism that is independent of blood pressure.
Consistent with this conclusion, it has been reported (Haulica et al., 1986) that icv AII
produced a large analgesic effect in rats and that this was blocked by the non selective
angiotensin antagonist saralasin and the opiate antagonist naloxone. 70
If indeed the hypoalgesia seen in the SHR is mediated via ATI receptors, then treatment of normotensive rats with AII should increase hot plate latencies. Furthermore, if this does occur and the effect is independent of angiotensin's role in raising blood pressure, raising the blood pressure by other means should not influence nociception. These ideas will be pursued in the next chapter. 71
CHAPTER 5
THE EIIFECTS OF PERIPIIERALLY ADMIMSTERf,D ANGIOTENSIN TI AND
NOREPINEPHRINE ON BLOOD PRESSURE AND NOCICEPTION IN lryISTAR
AND }VI(Y RATS.
5.1 Introduction
In the previous chapter captopril was shown to decrease latencies of SHRs in the hot plate test (Irvine et al., 1995). The ATI receptor antagonist losartan had similar effects in the SI{R, but hydralaÅne, an anti-hypertensive agent acting independently of the renin-angiotensin systenr, did not influence the increased latencies of the SHRs (Irvine et al., 1995).
One hypothesis to explain the action of ACE inhibitors zuch as captopril on pain sensitivity is that they influence endogenous opioid systems by virtue of their abilþ to inhibit enzymes such as neutral endopeptidase 24.1 1, the enzyme responsible for the breakdown of enkephalin
@oques & Beaumont, l99O; Erdos & Skidgel, 1989). However, the observation that losartar¡ a specific AT-l receptor antagonist, has a similar effect to captopril on nociception suggests that the efFect on pain described above is in some way mediated by AII acting at AT-
I receptors. In addition, Aff itself has been shown to have an analgesic effect when acutely administered intracerebroventricularly (icv) in rats and rabbits and this effect is blocked by the nonselective AII antagonist saralasin (Fedoseeva et al., 1990; Haulica et al., 1986). 72
In view of the possibility that increased levels of AII are responsible for the inc¡eased hot plate latencies in the SHR, the present studies investigated the effect of AII infusions in the normotensive Wistar-Kyoto (!VKÐ rat and in the outbred albino Wistar strain. If angiotensin plays a role in altered pain responses in the SHRs, then infusion of angiotensin in the normotensive rat strains should mimic the pattern of nociceptive responses seen in the hypertensive SHR strain.
As a control, a hypertensive agent which acts independently of the renin-angiotensin system, norepinephrine, was used to demonstrate that any change in nociceptive response was not secondary to a change in blood pressure. It would also provide an indication of whether increased sympathetic outflow of norepinephrine was responsible for the analgesia, as has been suggested (Siren et al., 1989).
Tail flick was used as a measure of reflex response to pain and the hot plate test as a response
which involved supraspinal sensory processing. In addition, several measures served as
indices of drug action. Locomotor activity provided an index of any general psychomotor
is depressive or stimulant effects of the drugs administered. An increase in water consumption
observed in rats when very low levels of AII are administered icv (Gronan & York, 1979),
and this was used throughout these experiments to provide a sensitive indication of the access
of angiotensin to central structures.
AII and norepinephrine cannot be administered orally, but must be administered parentally.
Osmotic minipumps were utilised for this purpose in order that chronic dosing could be
undertaken without inducing handling stress associated with repeated injections. 73
Administration by the sc route is reported to give venous concentrations equivalent to those produced by iv infusion in the rat (Grifñn et al., l99l)
5.2 Methods
5.2.1 Animals
Adult, male WKY and SHR rats and albino Wistars weighing 300-4009 were used. They were housed individually in a l}ll2light /dark cycle, with food and water ad libitum.
5 .2.2 Drug administration
Losartan and AII were dissolved in normal saline and L- norepinephrine HCI in ascorbic saline
(260pM). These solutions were inserted in osmotic minipumps (Alzet model 2002), which deliver at 0.5 ¡tLlhr. for 14 days. The pumps were primed by incubation in sterile saline for 4
o hours at37 C immediately prior to use.
5.2.3 Surgery
Animals were anaesthetised with a mixture of methohexitone lOmg/ml and pentobarbitone
60mg/ml 9:1, 5mVkg and the osmotic minipumps were inserted subcutaneously between the
scapulae under aseptic conditions as described in Chapter 2. The wounds were closed with
suture clips and the animals allowed to recover. Testing took place l0 days subsequently. 74
5.2.4Blood pressure, behavioural tests and water consumption
Tail-cuff blood pressure, locomotor activity, tail-flick and hot plate latencies were measured as described in the general methods. Daily water consumption \ryas measured from day 5 to day 10 of drug treatment.
5.3 Results
5.3.1 Angiotensin in WKY animals
The saline treated SHR when compared to saline treated WKY animals showed higher blood
pressures, higher locomotor activity, no difference in tail flick latencies and higher hotplate latencies ( Fig.5.l).
Subcutaneous AII infusions in the WKY had no effect on any of the parameters measured at
144 and288 pgkg/day (Fig.5.l). The highest dose of angiotensin in the WKY produced a pattern similar to the untreated SHR. Blood pressure increased to the same level
as the SI{R, and hotplate latency increased to a similar level. There was no effect on locomotor activity and a slight increase in tail flick latency.
5.3.2 Angiotensin in outbred Wistar rats
The results from Wistar rats exposed to identical treatment conditions as the WKYs are
shown in Fig. 5.2. tilistar rats showed increased blood pressures at 288 and 576 pdkdday. 75
200 **¡l ++t ô äo s.
I 00 + oa p.. t, Êq V)
0 Blood pressure Locomotor activtty
40 ** *¡td. I.ElWKYAII 144
30 :WKYAII 288 (t) () 57ó c) I}VKYAII (n o Ê G) GI t rl t0
0 Hoþlate Tail-flick
Fig. 5.1. Means and SEMs, for blood pressure, locomotor activity, hotplate and tailflick * latencies in WKY and SHR rats. Effect of angiotensin [I in Vglkg/day for l0 days s.c. p<
0.05,** p<0.01*** p< 0.001 compared to WKY saline, Dunnet's test n: 6-8. 76
***
t** c) äo t. E É I O t))I P.'. Êa (t)
0 Blood pressure Locomotor activlty
l-* lAII 144 30 :AII 288 * IAII 576 A20 cc) () É * I€ro
0 Hoþlate Tail-flick
Fig 5.2. Means and SEMs for blood pressure, locomotor activity, hotplate and tailflick * *** latencies in Wistar rats. Effect of angiotensin II in pÚkdday for l0 days s.c. p( 0.05, p<0 001 compared to Wistar saline, Dunnet's test, n :7'll. 77
300
** C) ** 6 zoo s. Ér É )-{ O (¡)I * roo * ov) o
0 Blood pressure Locomotor activity
I lsallne 20 NE 50 INE IOO (n q)o (t)
() l0 É o) d *l
Hotplate Tail-flick
Fig.5.3. Means and SEMs for blood pressure, locomotor activity, hotplate and tailflick * ** latencies in WKY rats. Effect of norepinephrine mgk{day for l0 days s.c. p( 0.05, p<0.01 compared to \ilKY saline, Dunnet's test, n:8. 78
300 t-] Saline AII576 o æq l-a !+ >, ãÌ,t< al 200 ø) ôì oi3ä{ bE ñl I
Wistar IWKY
Fig. 5.4. Means and SEMs for water consumption in WKY and Wistar rats. Effect of
angiotensin llin ¡tglk{day compared to saline, t-test, n: 7 79
the There was no effect at any dose for locomotor activity, an increase in hotplate latency at
The overall highest dose and a small increase in tailflick latency at a dose of 288 Vglkg/day. pattern of responses was very similar to that seen in Fig. 5.1 for the WKY strain of rat administered angiotensin.
5.3.3 Norepinephrine in WKY rats
Norepinephrine at 50 and 100 mglkglday sc raised blood pressure to levels equal to those
decrease in acheived with AII, but did not affect hotplate or tail-flick latencies. There was a
locomotor activity at the highest dose, which was-interifl'ted as an indication that the animals \) were adversely affected by this high dose ( Fig. 5.3 ).
5.3.4 Water consumPtion
Water consumption in rilistar rats \ilas increased somewhat by the highest dose of AII but the
difference was not statistically significant. Therefore the Wistar and WKY rats were
unaffected even at this dose (576 Pekglday ) ( Fig'5'a ).
I)iscussion
The results of this study show that peripheral infusions of angiotensin at 567 vgkglday to
Wistar and WKy rats increased both blood pressure and hot plate latencies to values
comparable to those achieved by untreated SHRs. A small, but significant increase in tail-flick 80 latency was also observed, but there \ ias no change in locomotor activity. The marked increase in hotplate latency in the \ryKY animals with subcutaneous angiotensin infusion suggests that increased angiotensin levels may be responsible for the increased latencies in the hypertensive animals. From these results it appears that the altered nociceptive response of
SHRs, but not their altered activity, may be due to elevated angiotensin.
The lack of change in any of the behavioural parameters when blood pressure was increased with peripheral norepinephrine infusions suggests that the changes observed with angiotensin treatment a¡e not simply a consequence of increased blood pressure. These data contrast \¡vith those of Randich and Maixner who were able to increase blood pressure in rats with phenylephrine and demonstrate an increased tail-flick latency @andich & Mabrner, 1984). It should be noted that their experiments involved administering a bolus dose of phenyleptrine iv. Responses to this treatment were only measured for 3 minutes after administration.
Furthermore, the rats were conscious and restrained with indwelling jugular vein and carotid
artery catheters. This would undoubtedly have resulted in some stress to the animals which may have had an influence on their pain and ca¡diovascular responses.
In the present study, norepinephrine's depressive effect on locomotor activity at the highest dose and an observed loss of weight and condition of the coat in these animals indicates that this dose was indeed very high. In view of this, increased sympathetic outflow of noradrenaline is an unlikely explanation for the increased hotplate latencies observed following angiotensin administration. 8l
The results presented here clearly indicate a role for angiotensin in the regulation of nociception in these animals. This is in accordance with the findings of other workers
(Fedoseeva et al., 1990; Haulica et al., 1985) who demonstrated that icv administration of angiotensin to adult male Wistar rats produced a large increase in nociceptive threshold as measured by the tail flick test and this was blocked by the angiotensin antagonist saralasin. In the same study, saralasin also blocked analgesia produced by immobilisation stress (Haulica et al., l9B5). In the second report, analgesia produced by electrostimulation in the rabbit was abolished by administration of saralasin (Fedoseeva et al., 1990).
Naloxone blocks the increased hot-plate latency observed in the SHR (Zamir & Maixner,
1986), indicating that endogenous opioid systems are involved in the altered nociceptive
resporu¡e described here. Therefore, it is possible that peripheral angiotensin increases the
release of endogenous opioids or in some other way faciliøtes the efFectiveness of
endogenous opioid systems.
The dipsogenic efFect of angiotensin, mediated by stimulation of the subfornical organ, is well
known (Gronan & Yorlq 1979). The lack of dipsogenic effect when angiotensin was
administered peripherally to the WKY in the present experiments indicates that it is unlikely
that the drug was entering the CNS from the periphery to a significant extent. The action of
angiotensin on blood pressure and hot plate latencies was therefore likely to be at a peripheral
site.
However, in view of the findings on the icv effects of angiotensin reported by other workers
(Fedoseeva et al., 1990; Haulica et al., 1985), and in spite of the lack of central effects of 82 angiotensin on water consumption, it is possible that the effects of peripherally administered angiotensin seen here were the result of transport into the CNS. This is also supported by the experiments referred to in the previous chapter where the administration of N-methyl- naloxone, which does not enter the CNS, could not block the increased hotplate latencies seen in the SI¡R, whereas naloxone could (Sitsen & de Jong, 1984). This latter study suggests that the site of interest is central.
The evidence presented in this chapter indicated that peripheral administration of angiotensin to normotensive animals will increase their blood pressure and hotplate latencies.
Furthermore, raising their blood pressure with norepinephrine does not alter hotplate latencies. Although it seemed likely that the hypoalgesia produced by angiotensin treatment was via a peripheral mechanism, the possibility of a central site of action had to be investigated. 83
CHAPTER 6
THE EFFECTS OF THE CENTRAL ADMII\üSTRATION OF ANGIOTENSIN AND
LOSARTAN ON BLOOD PRESSURE AND NOCICEPTION IN \ryKY AND SHR
RATS.
6.1 Introduction
In Chapter 5 evidence was presented indicating that peripheral infusion of AII increased hot plate latencies in WKY and Wistar rats to levels similar to those seen in untreated SHRs. In addition, AII itself has an analgesic effect when acutely administered intracerebroventricularly
(icv) in rats and rabbits and this is blocked by the nonselective AII antagonist saralasin
the @edoseeva et al., 1990; Haulica et a1.,1986). These findings suggest the hypothesis that subcutaneous administration of AII may lead to an increased central level of angiotensin and that the CNS is where the efus of AII on hotplate latencies are mediated.
In order to study this in a more direct manner, the effects of central angiotensin infusions on nociception were investigated in the normotensive Wistar-Kyoto (WKY) rat strain.
All components of the renin-angiotensin system have been found in the brain as well as in peripheral tissues @uiz et al., 1990). In view of the central analgesic actions of angiotensin described by others (Fedoseeva et al., 1990; Haulica et al., 1986), and the suggestion that the central renin-angiotensin system is the primary site for the cardiovascular actions of ACE 84
inhibitors and angiotensin antagonists in SHRs (Steckelings et al., 1992a; Unger et al', l98l),
pain central administration of angiotensin was evaluated to clarif the site of action for modulation.
The central effect of the angiotensin AT-1 receptor antagonist losartan in the hypertensive
SHR strain was also investigated, as this drug has been shown to have an effect on blood pressure and nociception in SHRs. The effect of losa¡tan is presumably mediated via a direct effect on AT-l receptors, but controversy exists as to whether or not losartan has central
purpose actions when administered peripherally (Song el aI., 1991; Bui et at., 1992). The of the present study was to confirm the earlier findings and to determine if icv infusion of losartan had an effect on blood pressure or nociception.
Tail flick, hot plate tests and locomotor activity were used for the reas¡ons stated in previous
Chapters. An increase in water consumption is observed in rats when very low levels of
angiotensin II are administered icv (Gronan & York, lg7g), and this was used as verification that the angiotensin was gaining access to the cerebral ventricles. Osmotic minipumps were
utilised in order that chronic dosing could be undertaken without inducing handling stress
levels associated with repeated injections, and in an attempt to achieve constant drug
throughout the CNS. 85
6.2 Methods
6.2.1 Animals
Adult male, WKY and SHR rats weighing 300-4009, were used. They were housed individually in a l2ll2light /dark cycle, with food and water ad libitum.
Losartan and AII were dissolved in normal saline. These solutions were inserted in osmotic minipumps (Alzet model 2OO2), which deliver at 0.5 pLlhr.for 14 days. The pumps were
o primed by incubation in sterile saline for 4 hours at37 C immediately prior to use.
6.2.2 Surgcal procedures
Animals were anaesthetised with methohexitoneþentobarbital 9:1, 5ml&g and the osmotic minipumps were inserted for delivery of icv drugs. The crown of the head was shaved, the skin swabbed with disinfectant and a midline incision 2.5qn long was made from a point midway between the eyes to just in front of the ea¡s. A subcutaneous tunnel was made by blunt dissection to a position between the scapulae. A brain infusion kit (Alza) was utilised to allow transport of the drug from the pump to the brain ( Fig 6.1). The minipump with the flexible extension tube attached was inserted through the aperture in the skin of the head and placed in the previously described position between the scapulae. The end of the tube was attached to the icv needle. The needle (25G) was positioned at a depth of 4mm from the skull surface by means of the plastic spacers, lmm lateral and 3 mm posterior relative to Bregma and fixed in place with dental cement. The positioning tab (FiS 6.1) was retnoved, the end of the tube and butt of the needle were covered in dental cement to provide a smooth surface 86
At¿ETo Pump Irdn Fbw lrodorelor lntt¡þn C¡md¡ /
\ Remomble Tab AI¿ET. Cathöter Osmt¡c Tr.ùe Purp
0 l0
cm
Fig 6.1 A diagram of a model 2002 lúzet@ osmotic mini pump with a brain hfusion kit attached. 87
and the wound closed with suture clips. Antibiotics were administered as described in Chapter
5. The animals were allowed to recover for l0 days prior to testing. At the conclusion of the experiments the animals were decapitated under anaesthesia and the heads fixed in formalin.
The position of the needle in the ventricles was confirmed by visual inspection after dissection ofthe fixed head.
6.2.3 Blood pressure and behavioural measurements
Tail-cuffblood pressure, locomotor activity, tail-flick and hot plate latencies were performed as described in Chapter 2.Daily water consumption was measured from day 5 to day l0 of drug treatment.
6.3 Results
6.3.1 Infusions of angiotensin in WKYs
When angiotensin was infused icv in WKY rats there \¡/as an increase in blood pressure only
at a dose of A Vglkg/day. Blood pressures following administration of angiotensin at 28 and
Aa pglkg/day were not different from saline @ig 6.2). There was a substantial fall in
locomotor activity at 14 pgk{day with no effect at lower or higher doses. Hot plate latencies
were not significantly modified by angiotensin except at a dose of 28 ¡Ælkglday where a
decrease was seen. No effect on tail flick latency was observed for any dose ofthe drug. 88
200 **
c)
ào ¡à.
100 O (¿)a Ê. Êa U) ***
0 Blood pressure Locomotor acttvtty
l-l saline 30 rAII 4 IAII 14 IAII 28 (t) o :AII 144 o) (r,
(J * (,)É Ë I '.l
0 Hotplate Tail-flick
Fig.6.2 Means and SEMs for blood pressure, locomotor activity, hotplate and tailflick * latencies in WKY rats. Efu of angiotensin II in pdkdday for l0 days i.c.v. p< 0.05,t* p<0.01, *** p<0.001 compared to \ilKY saline, Dunnet's test, n:6-8. 89
200
c) o0 É. tr I 00 ts O (¡)I Or Êa 3 v)
Blood pressure Locomotor activity
30 I losartan 10
oan c) TA
() (l)É id I -l
Hoþlate Tail-flick
Fig.6.3 Means and SEMs for blood pressure, locomotor activity, hotplate and tailflick latencies in SHR rats. Effect of losartan mglkg/day for l0 days icv, Dunnet's test, n:6-8. 90
1 000 *+* 750 EG' b.!4 (n 500 E 250
0 sal AII 4
150
ñt I b€ {rtt lt 50
0 sal los3 losl0 icv
Fig.6.4 Means and SEMs for water consumption in WKY rats top panel and SHR rats bottom panel. Effect of angiotensin tI in þg/xglday and of losa¡tan m/kglday for l0 days, 't*{3 p<0.01 compared to saline, t-test, n = 7-8. 9l
6.3.2 Infusions of losartan in SHRs
Icv infusions of losartan at doses of 3 and l0 mg/kg/day had no effect on blood pressure, locomotor activity, hot plate or tail flick in the SHR strain ( Fig ó.3).
6.3.3 Water consumption
Water consumption was increased 3-5 fold by icv infusions of angiotensin at all doses tested.
Only the data for the lowest dose is reported as the higher doses gave similar values (Fig 6.a).
A slight, nonsignificant increase in water consumption was observed for both doses of losartan in the SHRs.
6.4 Discussion
The only effect of the drugs on locomotor activity in these studies occurred following administration of angiotensin at A ¡tglkglday icv to the WKY, where a considerable decrease in activity was observed. This decrease was not seen at lower or higher doses of angiotørsin and did not influence the interpretation of the nociceptive data described below
Although blood pressure was raised by icv angiotensin at l4¡tglkglday, there was no observed increase in hotplate or tail-flick latencies at this or any dose. The only statistically significant change in latency \ilas on the hotplate at a dose of 28pglkg/day.This resulted in a reduced hot plate latency. Thus, chronic icv infusion of angiotensin at a range of doses in WKY rats cannot mimic the longer hot plate latencies seen in the SHR. 92
The dipsogenic effect of angiotensin was clearly evident from the water consumption data, indicating that the icv route of administration was effective. The lack of dipsogenic effect in the previous chapter, when angiotensin was administered peripherally in the WKY, indicates that it is unlikely that the drug was entering the CNS from the periphery to a significant extent, and this cannot be an explanation for the effects on nociception and blood pressure.
Losartan has been reported to be capable of entering the brain from the peripheral circulation to block central angiotensin receptors (Song et al., 1991), but no effect of the drug on blood pressure, nociception or water consumption rvas observed in the present study in spite of the
chronic dosing at high concentrations directly into the ventricles. Acute icv doses of lOpg/kg block the blood pressure effects of icv AII, and orally administered losartan at a dose of
lgmg/]rg reduces blood pressure in SHRs @ePasquale et al., 1992). In the experiments
presented here losartan was administered at l}mg/l.glday icv and yet this very high dose did
not afu blood pressure, nociception or water conzumption. The lack of effect on blood
pressure following chronic icv administration of losartan to the SHR is consistent with two
recent studies in which the drug was given acutely by the same route and had no effect @aull
et al., 1995; DePasquale et al., 1992). Together, these results do not support a role for
central ATI receptors in the hypotensive effects of losartan nor in its effect on nociception.
Although angiotørsin is clearly involved in regulation of nociception in these animals, the
rezults do not zupport a role for centrally located angiotensin. This is not in accordance with
the findings of other workers who have demonstrated analgesic effects of icv angiotensin in
the rat and rabbit (Fedoseeva et al., 1990; Haulica et al., 1986). This discrepancy may be
explained in terms of dosing. The experiments described here used chronic infusions over l0 93 days prior to testing. This would allow diffi;sion of the drug from the site of injection to more distant structures in the CNS. In addition, a constant concentration of drug in the animal for l0 days may result in some adaptation. The experiments mentioned above (Fedoseeva et al.,
1990; Haulica et al., 1986) measured acute effects of bolus injections of angiotensin.
Naloxone blocks the increased hot-plate latency observed in the SHR (Zamir & Maixner,
1986), indicating that endogenous opioid systems are involved in the altered nociceptive response described here. Therefore, it is possible that peripheral angiotensin increases the release of endogenous opioids or in some other way facilitates the eflectiveness of endogenous opioid systems. There is some evidence to support this. Peripherally administered angiotensin II has been shown to influence the release of p-endorphin and ACTH in Holzman I ri t-- rats (Spinedi & Negro Vilar, 1983). In addition, there have been reports @raas t1.,.' f , u.-i ,, r cR u" 1994; Felder & Garland, 1989) of a decreased expression of POMC in therintermediate lobe of the SHR compared to the WKY. Thus, although it is outside the scope of this thesis, it is possible that detailed investigation of the influence of peripheral angiotensin on hypothalamo- pituitary release of stress hormones may provide a clearer understanding of the mechanisms involved in the decreased pain sensitivity of SHRS. 94
CHAPTER 7
RADIOTtrLEMETRIC BLOOD PRESSTTRE MONMORING IN THE SHR
7.1 Introduction
The measurement of blood pressure in the conscious rat has been restricted to two methods: direct cannulation of a suitable artery such as the carotid and measurement with a pressure transducer or indirect tail-cuff measurement @unag, 1973). Direct measurement gives the most reliable results, but can only be used for relatively short periods of time without problems in keeping the catheter patent. It also involves some type of restraint or limitation on the animal's movement. Tail-cuff measurements are less demanding technically and are suitable for long term studies, but suffer from the disadvantage of being indirect, discontinuous and the need to both heat and restrain the animals to allow detection of blood flow in the caudal artery. The issues of restraint stress, heat stress and environmental influences on physiological parameters is particularly important in the SHR. This strain has been shown to be highly reactive to its environment (Whitehorn et al., 1983) and this is reflected in cardiovascular reactivity (Knardahl & Hendley, 1990).
The development of small implants capable of measuring blood pressure directly in the rat and relaying this information by radiowaves would appear to offer a technological leap forward in this area of measurement. The benefits include continuous long term recording without 95 animal handling, measurement of diastolic and systolic blood pressures and heart rate simultaneously (BrocLway el al., 1991). Long term continuous monitoring and automatic data storage and reduction via computer is also possible.
Recent studies have shown that resting heart rate and blood pressures are considerably lower
in telemetered animals indicating that they are probably less stressed (Bazil et al., 1993; Guiol
el al., 1992;Nan¡aez et al., 1993). The effect appears more pronounced for heart rate than
blood pressure (Guiol el al., lgg2). Sprague-Dawley rats showed a slightly increased blood
pressure when tethered for femoral artery catheter measurements, but hea¡t rate was 50 BPM
higher than telemetered non-tethered animals. In hypertensive animals heart rate and blood
pressure is increased. SHRs had systolic blood presstres 2O4O mmHg higher when assessed
by tail cuff or indwelling arterial catheters. Hea¡t rate in these animals was 100 BPM higher
than when these parameters were measured using telemetry @azil et al., 1993). These and
other studies indicate that the physiological status of these animals is altered by the
procedures involved in blood pressure recording. It is possible that some of the drug effects
seen in animals which were subjected to the stress of blood pressure recording by traditional
methods would be different in a less stressed animal. Evidence for this has been provided for
one drug. The acute antihypertensive effects of captopril in the SHR a5¡ measured by tail cuff
manometry and direct carotid cannula are not evident when the blood pressure is meazured by
of the telemetry @azil et a1.,1993). In that study, acute effe,cts of a single oral administration
drug were tested in separate groups of animals subjected to telemetry, tail cuff or direct
carotid cannulation for measurement of blood pressure. These results suggested that the
effects ofcaptopril at least, and perhaps losartan and hydralazine, on blood pressure reported
in earlier chapters of this thesis were the result of effects on stress reduction during the 96 restraint of tail-cuffmeasurements rather than a direct effect on blood pressure. In view of the e- advantages offered by the new technology, radiotelemetric blood pressure monitoring equipment was purchased and the method was evaluated to establish its suitability for the type of research reported in this thesis.
In the study presented here, telemetered blood pressures were measured in conscious animals over a 24hour period. The short term influence of mild handling stress on blood pressure and heart rate was examined. Telemetered values were measured while the animals were restrained for tail cuff estimates and unrestrained, in their home cage environment, A
comparison between tail-cufl telemetry and blood pressures obtained by direct carotid
cannulation was also performed in the anaesthetised SHR.
Following this assessment, tail-cuff blood pressures and telemetered blood pressures were
compared in untreated conscious SHRs and when treated with captopril, losartan and
hydralazine.
7.2 Methods
7.2.1 Animals and drug administration
Adult male SHR rats 300-4009, age-matched were used. They were housed individually in a
normal light /dark cycle with food and water ad libitum. Losartan, captopril and hydralazine 97
\ryere made up fresh each day and administered in the normal drinking water for 3 days prior to blood pressure measurements. The amount consumed was measured daily
7.2.2 Surgery for radiotelemetric implants
Implants (TAl IPA-C4O, Data Sciences International) were inserted as previously described
(Brockway et al., l99l). Briefly, the animals were anaesthetised with a mixture of
methohexitone sodium lOmg/ml and pentobarbitone sodium 60mg/ml, 9:1, administered at a
dose of 5ml/kg intraperitoneally and the telemetry devices were inserted in the peritoneal
cavity under aseptic conditions. A non-occluding catheter with a antithrombogenic tip (Fig
7.1) @rockway et al., 1991) was inserted in the abdominal aorta with the tip just below the
renal arteries and was fixed in place with tissue adhesive. The body of the implant was
immobilised by suturing to the ventral abdominal wall and the wounds closed with suture clips
(Fi9.7.2). Antibiotics were administered as described for minipump insertion in Chapter 2.
The clips were removed 5 days later and the animals allowed to recover for at least l0 days
prior to experiments.
7 .2.3 T r ansducer/transmitter
TA l 1PA-C4O implants (Fig 7.3) were used. These devices consist of an a¡terial catheter, a
pressure transducer and a radiotransmitter. They are 9gs in weight, 4.5cc in volume and
covered with a non allergenic plastic coating. A small ridge on one side of the cylinder is used
to sew the implant to the abdominal wall as descibed above. These devices are switched on
and off by means of a magnetic switch which is activated by briefly holding a small magnet
close to the animal's abdomen and have a battery life of 6 months continual use 98
Thin-watled tiP with anti- thrombogenic film aPPlied
ompatible gell NoncomPress able fluid
Gatheter tip of chronic blood pressure implant.'
Figure 7.1 The tip of the indwelling catheter used in the telemetric implants. 99
'lmplantable iadio-transmitter'
Recelver Computer
Figure 7.2The position of the implant and the catheter in the rat 100
out in the The implants are calibrated by the manufacturer, but a further check was carried
in a sealed laboratory prior to use.They were checked for accuracy and linearity by placing
means of a glass container which could be pressurised in a graded fashion and monitored by mercury manometer. No detectable errors were found'
7.2.4 Receivers
placed RAl0l0 receivers were used to pick up the signal emitted by the transmitters.They are range under the individual animal's cage and are capable of detecting signals up to a of
device and approximately 50 cm. A number of receivers can be connected to a multiplexing
7.4). this connects to a specialised data board in a 486 personal computer (Figure
7.2.5 Sampling parameters and data storage
The waveform sampling rate was s€t at 2l)ltzwith a l00Hz filter. Samples of ten seconds
24 hour duration were taken as often as \ilas required by the experimental protocol: hourly for
and mean data and every minute in most other cases. Heart rate as well as systolic, diastolic,
blood pressures were derived from the waveform and stored on disk'
7.2.6 Baseline blood Pressures in their Baseline telemetered blood pressures and heart rates were measured with the animals
from its cage home cage. The effect of brief handling was assessed by gently lifting the animal
and immediately replacing it in the s¿rme cage. llll il ro I a
Figure 7.3 The TA1lPA-C4O blood pressure ímplant' t02
Implanted rats
Receiver Receiver
Multiplo Power recording Figure 7.4 shows a schematic diagram ofthe Data Sciences@ radiotelemetric system 103 For direct comparison between tail cuff and telemetered blood pressures, animals were restrained in plastic tubes l00mm in diameter and heated in the normal manner for tail cuff blood pressure as described in the General Methods (Bunag, 1973). Telemetered blood pressure readings from these animals were recorded at the same time. 7.2.7 Carotid blood pressures Direct blood pressure readings in anaesthetised animals were made via a plastic catheter 0.8mm ID, l.2mm OD, inserted into the right carotid artery and connected to a PC23 Statham pressure transducer and a chart recorder. Full details ofthe surgery are described in Chapter 2. Telemetered BP readings were undertaken simultaneously. 7.3 Results 7 3.1 Twenty four hour readings Heart rate and systolic and diastolic blood pressures measured by radiotelemetry over a 24 hour period are shown in Fig 7.5. The mean heart rate in untreated, unrestrained, conscious SHRs was 319+7 beats per minute (BPM), ranging from 379 in the evening to 272 BPM in the afternoon (Fig.7.5). Systolic blood pressure was 179t2 mmHg, ranging from 160 to 193 mmHg over the day. Diastolic blood pressures followed a similar pattern to that seen for SBP, averaging 116_2 mmHg, with a range of 100 to 128 mmHg ( Fig 7.5). These readings are similar to those reported in the literature for SHRs using telemetry @azil et al., 1993; Kohno et al., 1994). 104 7.3.2Influence of brief handling The effect of brief handling on blood pressure and heart rate is shown in Fig. 7'6. Blood pressure had returned to normal after about 20 minutes, representing a drop in systolic blood pressure of 40 mmHg. ( 204+12 to 164+6 mmHg ). A smaller effect was observed for diastolic pressure. Heart rate was 431+ l4mmHg immediately after handling and dropped to 316+6 mmHg at l0 minutes. This type of handling is less intrusive than holding an animal for injection, for example, but produced a considerable cardiovascular response. 7.3.3 Unrestrained readings Systolic blood pressuÍes in untreated SHRs as measured by telemetry in unrestrained animals yielded values of 17816 mmHg. When animals were restrained SBP was significantly higher at 207+5 mmHg (Fig.7.7). This indicates that the restraint procedures for tail-cuff blood pressures is likely to cause an increase in pressure of the order of 30 mmHg in this strain. 7.3.4 Readings in anaesthetised animals Anaesthesia allowed a direct comparison of carotid, tail-cuff and telemetric blood pressure measurements in the same animal.Systolic blood pressures measured by telemetry and direct carotid cannula in anaesthetised SHRs were very similar. Mean BPs +SEM l8l+13 and 193+17 mmHg for telemetry and carotid catheter respectively. Simultaneous tail-cuff pressures in the same animals were lower, 120+8 mmHg. (Fig 7'8a). The lower value obsewed for the tail-cuffmethod is ,riþriring in view of the good correlation between tail-cuff -.-1- -- 105 heart rate v 500 systolic A Io) diastolic EC 400 Eo-L --oO- ko 300 II,',lrl,rrrl,,r,,llll.' 0)¡úE o_ot- t- 200 r,IIrtIII'I IITtTTIIIIIII o9o¡ o 100 ^.Ir^^Irr^r Ir¡-r^rII¡r¡r ct 0 06121824 Hours Figure 7 .5 Hleart rate (BPM) and systolic and diastolic blood pressures (mrnHg) recorded by telemetry in SHRs. Recordings were made hourly for 24 hours. Values are means + SEM, n:3. l0ó Blood pressure 300 +- syst öo + mean É + dias c) 200 * U) (t) G) A. € 100 o o Êq 0 0 l0 20 30 40 50 minutes 500 È Êa (.) *** *** *** *** GI 300 þ c) 200 100 01020304050 minutes Figure 7.6 The recovery from mild handling of blood pressure and heart rate in the SHR' *** Values are expressed as means +SEM n:3. *: p<0.05, p<0.001 compared to time 0, Dunnet's test. 107 300 *tr è0 É Êr (t)ca I 00 0 No restraint Restaint Figure 7.7 Systolic blood pressures (mmHg) in conscious restrained and unresttained SHRs measured by telemetry. Values are means+SEM, n:5' ** p<0.01, t-test. 108 300 ä0 200 ** À (t)Êa 100 Teløneûy Tail-cuff Carotid 300 äf) ,ß* rJi 200 ts E À m U) 100 0 Telemetry Tail-cuff Figure 7.8 A comparison between methods of meazuring blood pressure in SHRs. Upper panel, anaesthetised animals. Lower panel, conscious animals. Values are expressed as means t SEM, n:5. ** p<0.01, t-test. 109 Fig 3 Unrestrained 225 + captopril losartan 200 -¡- à0 --+-- hydralazine HÉ 175 È € l5o + 125 0 low high Restrained 225 200 Òf) * tì-- ti É t É 17s r** À É 150 ¡l** t25 0 low high Figure 7.9 Systolic blood pressures measured by telemetry in conscious SHRs with no treatment and with two doses of the drugs shown. Upper panel, unrestrained. Lower panel, restrained. Values are expressed as means t SEM n:5.* p<0.05, ** p<0.01, Dunnet's test. Low : 105,26 and 7 mglk{day Fúgh : 226,85 and 27 mglkglday for captopril, losartan and hydralazine respectively. r10 and carotid blood pressures shown for WKY animals in Chapter 2 and may be a characteristic of the SHR. 7.3.5 Telemetry vs tail-cuffin conscious animals ln order to,.lrrelfVrlo*p-!\a.lemetry and tail-cuffblood pressures in the normal situation where tail-cuffreadings would be used, simultaneous tail-cuffand telemetry blood pressures were measured in conscious animals. These readings indicated tail-cuff readings some 40 mmHg lower than the telemetered measurements. Blood pressure by telemetry was 207+5 mmHg and by tail-cufl 164+10 mmHg (Fig.7.8b). This result was in accordance with the measurements made in the anaesthetised animals. 7.3.6 Drug effects The influence of restraint on the effects of antihypertensive drugs on telernetered blood pressures is shown in Fig 7.9 The effects of low and high doses of the 3 drugs used indicate that in the unrestrained animal, only the high dose of captopril had a significant hypotensive effect. (Fig 7.9a). In the restrained situation, all drugs were hypotensive at the higher dose while only captopril and hydralazine \ilere at the lower dose (Fig 7.9b). These data indicate that the hypotensive potency of these drugs is influenced by restraint. The actual doses of drug consumed by individual animals was calculated after measuring the volume of drug solution drunk on each day of treatment. The drug doses consumed by the animals for the low and high concentration of each drug were: captopril 105+7, 226t12; losartan 26Ð, 8 5t4; hydralazine 7 + l, 27 t4 mgkgl day . lll 7.4 Discussion The need for a method of measuring BP which minimises stress to the animal prompted the studies described here. The preliminary evidence that pharmacological responses, and not only baseline levels of BP and heart rate, were influenced by the method employed made this investigation particularly important. It is vital in investigating hypertensive mechanisms in experimental animals that the many physiological systems that can influence blood pressure are not reset by the methodology. The baseline values for animals in their home cage reported here are generally well below the normal values quoted in the literature (Hein et al., 1995). The effect on BP and heart rate of minor handling was quite dramatic and unexpected and should certainly be considered in studies which involve extensive handling of animals even in the absence ofbehavioural evidence ofstress- Restraint significantly elevated blood pressure in conscious SHRs by 30mm Hg. This difference is simila¡ to that reported previously when tail-cuffand telemetry \¡/ere compared in SHRs (Bazil et a1.,1993). Telemetry and direct carotid cannulation give identical estimates of SBP in anaesthetised SHRs which supports previous work in normotensive rats where there was good correlation between these methods in alagstleiised animals @rockway et al., leel). Tail-cuff blood pressure monitoring in anaesthetised SHRs gave low values which contrasts with the comparisons made in Chapter 3 where a similar validation experiment with WKYs gave good correlation between carotid cannula and tail-cuff systolic blood pressure readings' tt2 This may be related to changes in blood flow or artery geometry specific to the hypertensive strain (van den Buuse et al., 198ó; Fouda et al., 1987). Alternatively, blood flow to the tail may be altered by the radiotelemetry implant and this results in lower blood pressure estimates by tail-cuff. The data in Chapter 3 were obtained from animals which did not have a telemetry implant. Comparison between telemetry and tail-cuff in conscious animals indicated that here, too, the tail-cuffgave an estimate some 40mmHg less than telemetry. Antihypertensive drug effects with the dosing regime used here are more pronounced under restraint conditions and this effect is greater for hydralazine than for the two drugs which interact with the renin-angiotensin system. This suggests that stress may play a role in the antihlpertensive action of hydralazine. However, if the greater antihypertensive effect of hydralazine under restraint conditions was due to an influence on stress mechanisms it might be expected to alter nociception. There rvas no influence of hydralazine on nociception in the earlier chapters. Telemetry, by eliminating the stress inherent in the alternative methods of monitoring blood pressure and allowing the continuous long term monitoring of cardiovascula¡ parameters should now be adopted wherever possible. Scientifically, it is the method of choice and has the added advantages of being time efficient and more likely to be accepted by animal welfare g¡oups and animal ethics committees. ll3 in The very extensive literature on the role of the adrenals and sympathetic nervous system the control of blood pressure in animal models of hypertension (Milanes et al., l99l; all studies Knardahl & Hendley, 1990; Henry et al., 1993) should now be reexamined In may have undertaken utilising tail-cuff or indwelling catheters, it seems likely that the data been compromised by the stress ofthe recording method' 114 CHAPTER 8 PAIN PERCEPTION IN IIUMAN NORIVTOTENSTVE AND HYPERTENSTVE SUBJECTS: EFFECT OF DRUG TREATMENT. 8.1 Introduction The decreased sensitivity to pain in hypertensive animals described in previous chapters is also observed in humans (Rosa et al., 1994; Singh et al., 1984; Zanir el al., 1980). However, unlike the SHR where antihypertensive treatment influenced the response to nociception, limited human studies with antihypertensive treatment have not demonstrated any effect on pain perception (Ghione et al., l98E). The influence of antihypertensive drugs on nociception in SHRs is dependent on the class of antihypertensive drug administered. When blood pressure was lowered to normotensive levels in spontaneously hypertensive rats with the directly acting vasodilator hydralazine, no change in pain sensitivity was observed. However, both the AII converting enzyme (ACE) inhibitor captopril and the AII antagonist losartan abolished the difference in pain perception between hypertensive and normotensive strains of rat (Irvine et al., 1995). In humans, untreated hypertensive patients have decreased sensitivity to acute pain as compared to normotensives and this is unchanged when their blood pressure is controlled with beta blockers or diuretics (Ghione et al., 1988). This is not inconsistent with the animal results presented here in that neither of these drug classes have a mechanism of action which ll5 includes the RAS. The animal data presented here suggest that blocking the action of AII at a peripheral site is required to return pain perception to normal in the SHR. To date , no study in humans has examined the eflects on pain perception of drugs with this mechanism of action. There are now a number of ACE inhibitors available for hypertensive therapy with varying tissue specificities (Fabris et al., 1990; Ranadive et al., 1992). The concept that ACE inhibitors have varying abilities to enter and inhibit ACE in tissues is being explored to optimise the clinical efFectiveness of these drugs (Nash, 1992). One area of particular relevence to the work presented in this chapter is the role of tissue RAS in cardiac hypertrophy. ACE inhibition has been shown to sufress hypertension induced cardiac hypertrophy in Sprague-Dawley rats made typ"rtrnrJt*n high sodium diet (Freeman et " al., 1987). Similar results have been obtained when SHR animals are treated with ACg inhibitors (Clozel et al., 1992). In addition to the ACE inhibitors, orally active AII antagonists such as losartan a¡e now in clinical trials and they are likely to be in clinical use soon. These agents and their active metabolites have varying physiochemical properties and have the potential to affect central and peripheral RASs differentially (Song et al., l99l). In the following experiments we tested the hypothesis that the lowered sensitivity to pain in hypertensive patients is abolished by ACE inhibitors, but not by anti-hypertensive drugs which do not influence the RAS. ll6 8.2 Methods 8.2. I Experimental subjects Treated hypertensive subjects were recruited from the Hypertension Clinic at the Royal Adelaide Hospital and untreated normotensive subjects were recruited from the staff of the University of Adelaide. Four aged matched groups were studied: untreated normotensives (n:19), and hypertensives treated with an ACE inhibitor (n:18), a beta blocker (n:10) or a calcium antagonist (n:l 3). The study had the approval of the Research Ethics Committee of the Royal Adelaide Hospital prior to commencement. All subjects were fully informed both verbally and in writing and provided written informed consent prior to the study. 8.2.2 Blood pressure and heart rate Subjects rested in the supine position in a quiet roonL while beat-to-beat blood pressure was measured non-invasively from the finger using the technique of dynamic vascular unloading by volume clamping (Finapres, Ohmeda). This device consists of a photoelectric plethysmograph mounted in a cuffwhich is attached to a finger. The operation is controlled electronically and is capable of continously measuring blood pressure in a non-stressful fashion (Molhoek et al., l9B4). Heart rate was measured by standard ECG electrodes attached to the chest of the subject. The resting period lasted for at least ten minutes, and longer if required to achieve a stable baseline. t17 8.2.3 Cold pressor test The cold pressor test has been shown to be a reliable pain model which yielded dose response data for codeine in a double blind cross over design (Woltr el ql., 196ó). In the present study the left foot was immersed to the ankle in water at 4"C for two minutes. Immediately following removal of the foot from the water, the subject was asked to rate the level of discomfort or pain experienced during the cold pressor test, using an ungraduated visual analogue scale (length l00mm). Subjects were allowed to remove their foot at any time if they found the test intolerable but this did not occur. Blood pressure and heart rate were measured continuously throughout the test. E.3 Results 8.3.1 Age and resting blood pressures There were no significant differences in age between the groups. Mean age was 48+5 years. Resting systolic blood pressures (mmHg) were: normotensive group (N), 127+4, ACE, inhibitor group (A),132t3, beta blocker group (B), 137+7 and the calcium antagonist group (C), 13ft4. Resting diastolic pressures (mmHg) were; goup N, ó7+2, goup þ\ 63+2' g.oup B,68+4 and group C,7lL3. There were no significant differences between the groups for these parameters (Fig 8.1). 118 Blood pressure 150 lïïÌ]N (n:19) @l A(n:18) mB (n:10) of) IC (n:13) É E 50 0 Systolic Diastolic Figure 8.I Resting systolic and diastolic blood pressures in the four experimental groups immediately prior to testing. Normotensives (N), ACE inhibitor treated (A),Beta blocker treated (B) and Calcium antagonist treated (C). Meanstsem are shown. Cold pressor test 30 [trnN @A ÚB 20 IC s l0 0 Systolic Diastolic Heart rate Figure 8.2 shows theYo change in blood pressures and heart rate during exposure to the cold pressor test in the four experimental groups. Abbreviations and group numbers as shown in Fig 8.1. ll9 Cold pressor 50 N @A c) ilB L. ¡t rf o 30 IC ttto 'ñl êr l0 N A B c to the cold Figure 8.3 shows the pain scores in the four experimental groups in response ** p<0'01 compared to pressor test. Abbreviations and group numbers as shown in Fig 8' 1' N, t-test t20 8.3.2 Cold pressor test All groups showed a rise in blood pressure in response to the cold pressor test, as expected. However, there were no significant difFerences between treatment groups in relation to systolic blood pressure, diastolic blood pressure and heart rate responses (Fig. 8.2). Pain scores (mm) in response to the cold pressor test, were 42+4 for group N, 39-t5 for group A\ 22+5 for group B and 3Gt6 for group C (Fig 8.3). While goup B had a significantly lower pain score than the normotensives, neither groups A nor C differed significantly from each other or from the normotensive group. These data show that hypertensives who have had their blood pressure corrected with beta blockers still have the reduced response to pain reported to be cha¡acteristic in untreated hypertensives. This contrasts with the other two groups and in particular the ACE inhibitor treated one. This latter group had the same pain responses as untreated normotensives. t.4 Discussion The results in this chapter strongly support our earlier work in the SHR and suggest that drugs which reduce AII levels restore pain perception to normal in human hypertensives as well as in rats. The three groups of hypertensive subjects studied here had similar blood pressures, indicating that the drug therapies were equally effective in controlling blood pressure. However, this was not the case for their response to pain. There was no difference in pain perception between normotensives and hypertensives who had their BP controlled by ACE inhibitors. This contrasted with the beta blocker group who remained hypoalgesic as 121 had been previously reported (Ghione et al., 1988). In that study treatment of hypertensives with diuretics or betablockers at doses which corrected their hypertension did not correct their diminished response to pain as measured by tooth pulp stimulation. The patients receiving calcium antagonists were intermediate in their pain scores and this is consistent with studies in which calcium channel blockers have been shown to potentiate the analgesic effects of morphine in humans (Carta et al., 1999) and in rats (Gurdal et al., 1992). It appears from these and other studies that calcium is important in pain mechanisms and that antagonists are capable of causing or potentiating analgesia. The cold pressor test is used as a standard tool in autonomic physiology to examine the normal cardiovascular sympathetic reflex responses to an unpleasant stimulus. The similarity in cardiovascular response of all groups to the cold pressor procedure (Fig. 8.2) indicates that these drugs did not interfere with the normal sympathetic response. All groups had a similar increase in both diastolic and systolic blood pressure and heart rate in response to the stimulus. This suggests that the effect of ACE inhibitors reported here is possibly via a direct action on pain mechanisms. This study would have been strengthened by the inclusion of an untreated hypertensive group. The inclusion of such a group would have allowed confirmation of the other worker's studies, mentioned above, showing a decrease in response to pain. However, difficulties in recruiting untreated hypertensives and ethical limitations which prwented the withdrawal of therapy from treated patients did not allow this. t22 Previous studies have shown that untreated hypertensive humans have reduced nociception and it is reasonable to assume that the normal pain scores observed in the ACE inhibitor treated group represented a treatment efFect. Further investigation of this issue should include a placebo controlled, crossover trial with sequential measurements to confirm this finding. The clinical significance of this work may be in the problem of "silent" cardiac ischemia. This is a relatively common clinical condition, where, in spite of a compromised blood supply to the heart which would normally result in angina or chest pain, no pain is evident (Thaulow el al., 1993). This could be due to the reduced sensitivity to pain described here as many patients with cardiac disease also have high blood pressure. ACE inhibitors, unlike some of the other antihypertensive therapies, may restore pain perception to normal in these people. Hypertension is a major risk factor for ischaemic heart disease and if some but not all antihypertensive drugs alter pain perception the recognition of the onset of life threatening heart disease may be compromised with serious consequences. Acknowledgements I wish to sincerely thank Mr.Tim Nunan and Dr. Anne Tonkin of the Department of Clinical and Experimental Pharmacology, University of Adelaide, who carried out the technical aspects of these experiments on my behalf and whose expertise in human cardiovascular recording was essential to the success of the experiment. 123 CHAPTER 9 ALCOHOL CONSUMPTION IN THE }VKY AIID SHR 9.1 Introduction The RAS appears to play an important role in the development of hypertension in the SIil' especially in the early phase when ACE inhibition diminishes the development of hypertension. The blood pressure of the treated animals remains lower throughout life, even after withdrawal of drug treatment (Lee et al., l99O). The studies reported in this thesis confirmed the hypoalgesia of the SHR. Other authors have demonstrated that this hypoalgesia can be reversed with naloxone and therefore involves the opioid system (Sitsen & de Jong, 1983; Zanir et al., 1980). Earlier chapters also indicated that treatment with the ACE inhibitor captopril and the ATI receptor antagonist losartan altered the response of the SHR to pain . Therefore, in the SHR and WKY strains it is possible that a link exists between the RAS and the opioid system. It has been proposed, primarily by Grupp and co-workers, that alcohol consumption in rat and man is influenced by the activity of the RAS and that it can be modified by drugs which affect this system (Grupp et al., l99l). Peripheral administration of either AII or an ACE inhibitor reduces alcohol consumption in rats (Robertson et al., 1994; Grupp et ql., 1991).In humans, 124 captopril reduces alcohol consumption in moderately dependent drinkers (Pallavicini & Guivernau, 1994) In addition, other links between the RAS and alcohol consumption have been made. These studies have been undertaken with strains of rats which have a genetically determined preference for alcohol (P) or non-preference (NP). In these studies, P rats have been shown to have a lower plasma renin activity than NP rats (Grupp el al., l99l). Animals made hypertensive by the two kidney, one clip method, a model of hypertension which causes an increase in RAS activity, drink small amounts of alcohol compared to sham operated rats. One kidney, one clip hypertensive rats, a model which does not result in an increased RAS activity, drink similar amounts of alcohol to sham operated animals (Grupp et al., l99l). A dose dependent decrease in alcohol consumption was observed when rats were treated with AII subcutaneously (Grupp el al., l99l; Sazb et al., 1994). This reduction appears to be mediated through the ATI receptor as blockade of these receptors with losartan prevented the reduction in alcohol consumption. PDl23319, the AT2 receptor antagonist, had no effect (Grupp & Harding, 1994). These findings indicate that increased activity of the RAS, or treatment with angiotensin II to mimic this situation, results in a lower consumption of alcohol. However the same group of workers report a paradoxical decrease in alcohol consumption when ACE inhibitors a¡e administered to animals in otherwise identical experiments (Grupp et al., l99l). This has been explained with the hypothesis that ACE inhibition results in an increased level of angiotensin I which increases the amount of substrate available to central t25 ACE. This results in higher central levels of angiotensin [I, as central ACE has not been blocked. It is suggested that central ACE may be protected from the full effects of peripherally administered inhibitors due to limited access of these drugs to central sites. Thus, the action of ACE inhibitors in paradoxically reducing alcohol consumption is by an increased activation of central angiotensin ATI receptors (Grupp & Harding, 1994). This hypothesis has been tested directly by Fitts and co-workers who administered AII and captopril centrally and peripherally. In these experiments icv angiotensin II and peripheral captopril increased alcohol consumption. Icv captopril decreased the consumption of alcohol (Fitts, 1993a). The overall outcome of these conflicting results seemed to indicate that different methods employed in measuring consumption gave different results and that the doses of inhibitors used may be important in the interpretation of the results (Fitts, 1993a). More recently, Grupp's group also tested their hypothesis directly by blocking central AII receptors and observing the effect of peripherat administration of an ACE inhibitor. The reduction of alcohol consumption as a result of peripheral enalapil administration was unaffected when central AII recæptors were blocked with Sarl-Thr8-angiotensin II. This excluded a role for central AII in the action of ACE inhibitors on alcohol consumption and they concluded that their initial hypothesis was incorrect (Robertson ef al., 1994). Opioids have been implicated as mediators in the mechanisms which influence the consumption of alcohol. It has been shown that Sprague-Dawley rats with implanted gastric tubes will self-administer morphine by bar pressing. When the morphine solution is changed to an alcohol solution they self administer alcohol to a similar degree (Smith et al-, 1981). Low levels of endogenous opioid peptides may underlie elevated alcohol consumption in alcohol- 126 preferring mice (George et al., l99l).In this study, alcohol preferring mice had lower pain thresholds than non-preferring mice. Treatment with an opioid agonist increased pain threshold and decreased alcohol consumption. The consumption of alcohol in the two bottle choice paradigm has been reported to be higher in the SHR compared to the WKY (Khanna et ql., 1990). Considering the longer hot plate latencies in the SI{R, it would be expected that they would have low alcohol consumption if the low opioid-high alcohol consumption hypothesis in the mice was transferable to these animals. In view of the proposed roles of the RAS and opioid systems in alcohol consumption and the conflicting results to date, it was decided to examine alcohol consumption in the WKY and SHR strains and the influence on this of drugs which alter BP and pain sensitivity. In particular, drugs which influence the RAS were tested in the present study. 9.2 Experiment l: The influence of antihypertensive drugs This experiment investigated the influence of captopril, losartan and hydralazine on the consumption of alcohol in SHR and WKY animals. These drugs were chosen for similar reasons to those given in previous chapters: captopril as an ACE inhibitor, losartan to identify if ATI receptors were involved and hydralazine as a drug which altered blood pressure but not by involvment of the RAS. It was hoped that the data collected from the nociceptive experiments using these drugs would also help in identi$ing any links between nociceptive mechanisms and alcohol consumption. 127 9.2.1. Methods 9.2.1.1 Animals Male WKY and SHR animals 200-45}gwere used. They were caged individually on a l2ll2 light dark cycle and with food ad libitum as described in the Chapter 2. 9.2.2Two bottle choice test On days I to 5 increasing concentrations of ethyl alcohol ( starting at 2%) were supplied in the drinking water such that a concentration of 60/o vlv was achieved by day 5. During this time the animals had no other source of fluid. On days 5 to l0 two identical fluid bottles were provided and their position randomised daily. One bottle contained 6Vo vlv ethyl alcohol in water and the other contained water. The volume drunk from each bottle was measured daily and the total fluid and alcohol consumption in mls per kg of body weight per day calculated. 9.2.3 Drugtreatment and blood pressures Drugs were dissolved in the drinking water and water/alcohol solutions. The volume drunk each day was measured and the concentrations adjusted to deliver the required dose. The doses were: captopril l00mg/kg/day, losartan 3Omg/kglday and hydralanne Z0mglkdday They were administered from day I to day l0 as previously described. Blood pressure was measured by the tail cuff method. 9.2.2 Results and Discussion Blood pressures as well as total fluid and alcohol consumption in both WKYs and SHRs are 128 f--rwKY r---twKY vTzsHR a+* 77vaSllR HHH ã0 *l¡* d H (Ua É> E 100 BStâ -< o çÒI) ) o>è.y o cl €= .o t¡r m 0 Control Captopril Control Captopril 75 ! É,-o f----'lwKY ã-àso rz'zrtsLR o ò¡) o{ ### z3 H4# o--o.a as (t Control Caf,opil fluid Figure 9.1 shows the effect of captopril l0Omg/kg/day on blood pressure (mmHg), total rats. Results are expressd and ;yovlv alcohol consumption (mls/kgldÐ ir WKY and SHR * same treatment and as mean and sEM, n:6. indicates significance compared to wKY in the # indicates significance compared to untreated WKY, t-test' t29 W7V shr r__ uÀf r---lwKY +tt *** o d) #HH * ø7'SHR H#H Þ. !¡> øâ o od !>ol¿ o o. ã 50 €o o F-o ca control hydralazine control hyd,ralazine 75 r---wky É 7"-asbr o *** EiÞ.^ >r ÉÒb〠o>ol¿ EE ãv o control hydralazine Figure 9.2 shows the effect of hydralazine2}mglkglday, on blood pressure (mmHg), total fluid and 6o/ovlv alcohol consumption (mlVkg/dÐ in WKY and SHR rats. Results are * expressed as mean and SEM, n:6. indicates significance compard to WKY in the same treatment and # indicates significance compafed to untreated WKY, t-test' 130 presented inFig.9.l Captopril lowered blood pressure inthe SHR by 35mmHg but did not alter BP in WKY rats. In the untreated condition, total fluid consumption ( water/alcohol solution) for the WKY was 104.6+6.2 compared to 135.5+5.7 mls/kglday for the SHRs when the animals were untreated. In the animals treated with captopril the WKYs consumed 1 l5+8.6 and the SHRs 128+7 9 rnlslkg/day. Alcohol consumption in the untreated WKY group was 44.7+6.4 mls/kg/day compared to the untreated SHR group which drank 19.4+5.1 mlVkg/day. Captopril treated WKY rats drank 18.2+3.8 mls/kg/day compared to the treated SHR group which drank 24.7+mlslkdday. These results indicated a much higher consumption of alcohol in the normotensive animals and this effect was abolished with captopril. The lower consumption of alcohol by the SHR compared to the WKY was not expected in view of previous findings where the reverse was shown (Khanna et al., 1990). Similarly, the effect of captopril was surprising in lowering alcohol consumption in the WKY and not influencing consumption in the SHR. The effect of hydralazine on alcohol consumption is shown inFig.9.2. Blood pressures were not greatly different between untreated WKY and SHR rats and hydralazine treatment lowered the BP slightly in both the WKY and SHR. Total fluid consumption was similar in all groups exept the untreated SI{R" where the consumption was significantly higher by 2U/o. ln contrast to the results above, alcohol consumption in the untreated WKY was 23.&2.7 mllkg/day and 48.3+4 .2 rnls/kglday for untreated SHRs. Hydralazine reduced the SHRS consumption to that of the WKY Consumption levels were 24.&2.E mlykg/day and 19,9+2.2 mls/kglday respectively ( Fig 9.2). Thus , the difference in alcohol consumption was abolished by a drug which does not act via the RAS. l3l The opposite patterns of alcohol consumption for the untreated SHR and WKYs between the first (Fig 9.1) and second parts of this experiment (Fig 9.2) required that this discrepancy be investigated as a matter of priority before further experiments could be pursued. Close examination of the methods revealed the possibility of age being a factor in the variability of patterns of consumption and this was examined below. 9.3 Experiment 2: The influence of age Investigation of the ages of the animals used in the experiments above suggested that this fastor might explain the contrasting consumption patterns in the untreated animals. There appear to be no direct studies of aging and alcohol preference in animals or man. Epidemiological data suggest that age is a va¡iable associated with consumption of alcohol, such that decreases occur in later years of life, but no mechanisms have been established (Whalley, l9S0). Rats are reported to metabolise alcohol at a diminished rate as they age and this may influence their preference for alcohol. Twelve month old male rats have a decreased abilþ to eliminate parentally administered alcohol and this correlates with a decreased hepatic microsomal oxidising capacity when compared with two month old rats of the same sex and strain (Fernandez el al., 1988; Seitz et al., 1989). Thus, it is possible that age is an important variable to consider in the design and interpretation of animal experiments studying alcohol consumption. 132 The last experiment in this section was an attempt to test the influence of age on consumption of alcohol in the WKY and SHR. Hydralazine was excluded from the design and the ATI selective angiotensin antagonist losartan was included as this drug was considered more useful in identifying the mechanisms involved. 9.3.1Methods Male WKY and SHR animals 200-4509 were used. Animals described below as young were 3-6 months of age and those described as old, 9-10 months of age. They were caged individually. All other procedures were as described above with the exception that blood pressure was not measured in these animals. 9.3.2 Results Total fluid consumption in young \ryKY and SHR animals was similar in all groups and averaged a little over 120 mls/kglday. No significant influence of captopril or losartan was observed (Fig.9.3). The consumption of alcohol by the same animals showed the SHRs consumed 28311.9 mlVkg/day compared to 38+2.4 mlykg/day for WKYs when they were untreated. WKYs treated with losartan had a similar consumption to untreated animals, whereas SHRs slightly increased their consumption on the same drug. This change was not statistically significant. Captopril treatment reduced consumption of alcohol in both WKY and SHR animals significantly (Fig. 9.3). In the older animals total fluid consumption was higher in the SHR compared to WKY 120.2*2.8 vs 99.?t4.3 mlslkg/day. This trend was also seen for losartan treated groups, but was eliminated by captopril treatment (Fig 9.a). 133 YOUNG Total fluid consumption 200 l--l wKY vv?Àsl]Ã( (ü 'ùE & ú) E 0 CONT LOS CAP Theatment Ethanol consumption r-rwKY rcsHR 40 :| r; >, -ù€30 ### & ### J420 E CONT LOS CAP Treatment Figure 9.3 shows the amount of total fluid and 60/o vlv alcohol consumed by SHR and WKY rats 3-6 months old. The amounts are the daily consumption in mlVkg/day over a 5 day period. Means *SE are shown n: 6-12 in each goup. **p<0.0lcompared to WKY in the same treatment.llll# p<0.00lcompared to untreated \ryKY, t-test. CONT: no treatment, LOS : losartan and CAP: captopril. 134 oLt) Total fluid corsumption 200 V7VZSÍTF¿ r----WKY H## +t **+ .8 òo {Ø 0 COl.rI' LOS CAP Treatment Ethanol consumption 75 r---lwKY fæSHR * *** ** 5 ñt -ù€ {U) A 25 0 CONT LOS CAP Tieatment \üKY Figure 9.4 shows the amount of total fluid and 60/o vlv alcohol consumed by SHR and rats 9-10 months old. The amounts are the daily consumption in mlVkg/day over a 5 day \WKY period. Means *SE are shown n: 6-12.*p<0.05,**p<0.01,**p<0.001compared to in the same treatment. ### p<0.00lcompared to untreated WKY, t-test. CONT: no treatment, LOS : losartan and CAP: captopril. 135 Alcohol consumption was 34.5+4.0 in untreated WKYs and 49.1+3.4 in the untreated SHRs This pattern was not changed by treatment with losartan or captopril (Fig 9.a). 9.4 Discussion There is considerable evidence that genetic factors influence alcohol consumption in humans (Gelernter, 1995) and rodents (Crabbe et al., 1994). Research into the underlfng mechanisms involved has centred on the influence of alcohol on the CNS. Serotonergic, dopaminergic, GABA and opioid systems have all been shown to modulate alcohol consumption in animals (Litten & Allen, l99l). Alcohol prefening mice have a lower pain threshold and increased enkephalinase activity (George el al., 1991). Modulation of opioid systems has a profound effect on alcohol consumption (Sinclair, 1990; Litten & Allen, l99l). Naltrexone, the orally active opioid antagonist, is now used clinically in the treatment of alcoholism (Volpic,elli, 1995;Fern et a/., 1988). It has also been proposed that alcohol consumption in rat and man is controlled by the RAS and that alcohol consumption can be modified by drugs which affect this system (Grupp et al., l99l). Captopril has been shown to be effective in reducing alcohol consumpton in man @allavicini & Guivernau, 1994; Grupp el al., l99l). This has been challenged by other workers who have evidence in rodents that captopril increases alcohol consumption (Fitts, 1993a; Fitts, 1993b). Another ACE inhibitor, enalapril, at a dose which reduced blood pressure, was ineffective in reducing alcohol consumption in 42 normotensive alcoholics (Naranjo et al., l99l). 136 The experiments presented here initially indicated a higher consumption of alcohol by normotensive/algesic animals which was consistent with the results on mice reported above (George el al., l99l). In that report, increased alcohol consumption was seen in the mouse strain which had the shortest hotplate latencies. However, our results were not consistent with the previous findings in these rat strains where the SHR had a higher consumption of alcohol than the WKY (Khanna et al., 1990). In the first experiment reported here, treatment with captopril lowered alcohol consumption in WKYs, which agreed with the hypothesis of Grupp that peripheral ACE inhibition decreased alcohol consumption in animals and man (Grupp e/ al., l99l). The reversal of the consumption pattern for alcohol in untreated animals in the second experiment was suprising, but careful scrutiny of the methods indicated that the only apparent explanation was that the animals in the second study were considerably older. The pattern of increased alcohol consumption of SHRs when the animals were older is in accord with other findings (Khanna et aI., 1990). The effect of hydralazine in lowering alcohol consumption in the SHR was difficult to explain, especially as the dose of hydralazine used had little effect on BP. There was, however, a modest decrease in total fluid consumption by SHRs with this treatment. Hydralazine has the potential to interact with alcohol in ways not associated with its hypotensive actions. In vitro, it is capable of reacting strongly with acetaldehyde, the major product of alcohol metabolism. Adducts are formed in a direct chemical reaction (Nunez Vergara el al., l99l). It has been shown to inhibit gastric mucous secretion and thus increase the production of gastric ulcers produced by stress and alcohol in the rat (BhandaÍe et al., 1992; at Bekairi et al., 1994). These interactions may possibly be involved in modification of alcohol consumption by hydralazine seen in the present experiments. t37 An attempt to shed some light on these discrepancies was made by investigating the pattern of alcohol consumption in two age groups of WKYs, and SHRs Young SHR rats drank less alcohol than WKYs and this difference was abolished with captopril treatment (Fig.9.3). Losartan did not have an efFect at the dose used, which suggests that captopril did not exert its effect by influencing the concentration of AII at ATI receptors. Conversely, old SHR animals drank more alcohol than WKYs and this was not affected by treatment with captopril or losartan at the doses used. The age difference seen here may explain the variable results reported by different laboratories and is worthy of further study. However, the long time course ofthe experiments required prohibited pursuit ofthis line of research within the time constraints of this projec't. The data in this chapter, although somewhat confusing, do support a role for the RAS in the control of alcohol consumption. Alcohol consumption was influenced by captopril treatment. The relationship baween pain perception and alcohol consumption is still difficult to interpret as alcohol consumption did not always correlate with pain sensitivity in the strains of rat studied here. A further investigation specifically aimed at opioid mechanisms would be appropriate. 138 CHAPTER 10 DISCUSSION In this section an attempt will be made to tie together some of the results and outcomes of the previous chapters. Comparison will be made with more recent findings in the literature and future directions will be identified and discussed. The previous work a decade or so ago on pain perception and blood pressure had led to the belief that high blood pressure resulted in an increase in baroreceptor activation which, in turn, via increased activation of descending spinal tracts, inhibited nociceptive responses (Randich & Maixner, 1984). Opioids were believed to be involved as naloxone was effective in reversing the hypoalgesia associated with hypertension in rats. However, there was also contradictory evidence: prevention of the blood pressure increase in young SHRs did not return nociceptive responses to normal and some models of hypertension did not seem to have hypoalgesia (Sitsen & de Jong, 1983). Sino-aortic denervation in normotensive animals increases their blood pressure and heart rate, but has no influence in hot plate latencies (Sitsen et al., 1983). The experiments in Chapter 4 were based on two findings of the mid to late eighties. The first was that SHRs had increased sympathetic innervation as a result of increased expression of NGF (Donohue et al., 1989) and the second that ACE inhibitors had a sustained effect on blood pressure after withdrawal of treatment. The latter effect was not seen with other antihypertensive drugs (Christensen et al., l9E8; Harrap et al., 1986). These two findings, in 139 concert with the information that Substance P containing sympathetic sensory nerves were also dependant on NGF (Lindsay & Harmar, 1989), and that ACE inhibitors like captopril could increase levels of other peptides such as substance P or enkephalin (Hand a et al., 1991 ; Skidgel & Erdos, 1987; Orloff et al., 1986), led to an initial working hypothesis for this thesis. Increased NGF caused an abnormal development of peptide containing sensory nerves and ACE inhibitors by their action on peptidases may influence nociception in these animals. The results of Chapter 3 indicated that captopril could alter nociception in the SHR and that this was not dependent on blood pressure changes as reduction of blood pressure with hydralazine did not affect nociception. However, losartan, a drug which became available for research in the late eighties, had similar effects on nociception as captopril. These results, in combination with the information that other more selective ACE inhibitors (Harrap et al', 1990) and losartan (Morton et al., 1992) were capable of the same sustained hypotensive effect on SHRs as captopril, required a change of hypothesis. The increased latencies in the SHR could be returned to normal by treatment with a highly selective non peptide ATI receptor antagonist. Therefore, it appeared as if occupation of the ATI receptor by angiotensin II was required for the hypoalgesia in the SHR. The idea that peptidase inhibition was important was discarded. Experiments in Chapter 5 attempted to veriff the hypothesis that AII was the important element in the SHR's hypoalgesia by infusing norrnotensive WKYs with the peptide in an attempt to mimic the nociceptive pattern of responses seen in the hypertensive animal. This was successfully achieved. Infusion of norepinephrine at doses which increased blood pressure did not influence nociception, indicating that the effects of angiotensin II were not a result of 140 facilitation of release of norepinephrine from sympathetic nerves or simply a result of increased blood pressure. At this stage blood pressure had been manipulated up and down in the two strains with a variety of drugs whose mechanisms of action were varied, and the only drugs which had an effect on nociception were those which could alter the activation of AT1 receptors by angiotensin II. Local RASs, in particular the one found in brain, have been proposed as important in hypertension (Gohlke et al., 1989; Unger & Gohlke, 1992). Administration of an angiotensin antagonist blocks the acute effects of angiotensin II on blood pressure when both are administered icv @ePasquale et al., 1992). The two reports of an analgesic effect of angiotensin in the rat (Haulica et al., 1986; Haulica et al., l9g5) which had appeared prior to this project had involved icv administration of angiotensin II and the nonselective ATI and AT2 receptor antagonist saralasin. These results led to the assumption that the angiotensin effects on nociception were most probably via a central mechanism of action, although there was contradictory evidence. If, in the studies reported in Chapter 5, the peripherally administered angiotensin II had entered the CNS to any extent, an increase in drinking would have been observed. This is because the sub fornical organ is exquisitely sensitive to the application of angiotensin II (Thunhorst et al-, 1989), resulting in increased drinking. This was not observed in these experiments. t4t The results of the experiments involving icv administration of angiotensin and losartan in Chapter 6 were quite surprising. Angiotensin given to WKYs did not increase their latencies in the nociceptive tests, although it did increase blood pressure at some doses and had a huge dipsogenic effect, demonstrating that there was good access to the CNS. Secondly, losartan did not affect blood pressure, nociception or locomotor activity at doses shown to be well in excess of those required to block central AII receptors. These data indicate that the site of action of angiotensin II in the modification of nociceptive responses in these strains is not within the CNS and that the hypotensive action of losartan is also not within the CNS. Both of these findings challenge currently held views on the role of AII in blood pressure control and nociception. It should be noted that most experiments cited in the literature (Baum et al., 1983; Georgiev et al., lÇry¡2; DePasquale et al., 1992; Haulica et al., 1986; Haulica et al-, 1985) have used acute administration of drugs and observed the acute cardiovascular or analgesic response. This is especially true for studies involving icv administration. In this thesis the animals were generally treated for a number of days to allow the drug to equilibrate in the tissues and presumably this also resulted in some adaptive responses in the form of regulation of receptors or transduction mechanisms. The disadvantage of these dosing regimes is that the initial site of action is difficult to identify. On the other hand, there are a number of additional advantages in chronic treatment. The animals are less stressed when they do not have to be injected daily , which is important when cardiovascular and nociceptive responses are being measured. Steady state levels of drugs are achieved, which reduces the critical time element in deciding when to do the testing or measurements. Longer term treatments also more closely mimic the time scales of the pathology and therapy of disease' 142 The small human study reported in Chapter 8 suggests that the influence of ACE inhibitors on nociception in rats could also be demonstrated in humans. The lack of effect of other antihypertensive treatments in this study and by others (Ghione et al., 1988) suggests that, as in the rat, this effect may require the participation of ATI receptors. Pain is an important symptom in the diagnosis and clinical assessment of disease. Drugs which may unwittingly alter pain perception could confound these assessments. With losartan and other ATI selective antihypertensives in clinical trials at the time of writing and the vast array of ACE and mixed function peptidase inhibitors being developed for a variety of conditions, it would seem prudent to establish the precise role of ATI receptors in pain perception. In the belief that it was important to limit the amount of stress inflicted on the animals by the experimental procedures, chronic dosing regimes using minipumps were adopted. However, the measurement of blood pressure by tail-cuff remained as a procedure which is recognised as inducing stress (Bazil et al., 1993; Chiueh & Kopin, 1978). It was possible that the blood pressure responses reported in the earlier chapters for hydralazine, captopril and losartan were simply a result of an anti-stress action of the drugs during manipulation of the animal for blood pressure readings. Administration of these drugs with blood pressure monitored by telemetry indicated that their hypertensive action may in part involve an action on stress mechanisms as restraint increased their hypotensive effects. The introduction of telemetered cardiovascular recording described in Chapter 7 is an important step forward in cardiovascular and stress research. The use of telemetry for the measurment of blood pressure and other parameters will undoubtedly become widely adopted' 143 The studies on alcohol consumption described in Chapter 9 were started in parallel with the pain studies reported in the other chapters. They were undertaken because of the debate in the literature, at that time, as to the role of the RAS in alcohol consumption. The results of these experiments \¡/ere difficult to interpret and are indicative of the literature in this area which has been contradictory and confusing. It would appear from a report which appeared subsequent to the experiments described here that the argument for the involvement of central RAS in the control of alcohol consumption is not as strong as before @obertson et al., 1994). Direct icv application of ACE inhibitors or angiotensin receptor antagonists did not block the rise in alcohol consumption seen with the sc administration of an ACE inhibitor (Robertson et al., 1994). There remains the possibility that a RAS at a peripheral site is involved. These data support the concept which has developed throughout this thesis that peripheral rather than central actions of AII are important in a number of systems. It would be appropriate to pursue this line of rese¿rch in the SHR and WKY as they may well provide a useful model for the study of alcohol consumption. If, as the data here suggest, these strains change their alcohol preference with age, a number of intervention studies could be envisaged. The combination of a genetic disposition which changes with age may well provide a model for human alcohol abuse where genetic and time or age factors have been identified (Whalley, 1 980). In view of their aberrant alcohol consumption, their altered pain pathways and response to stress, the investigation of consummatory behaviours for other drugs with abuse potential may be fn¡itful in these animals. t44 APPENDIX The chemicals used in the experiments described in this thesis are listed below Ascorbic acid Ajax Chemicals, Australia. Captopril Donated by The Squibb Institute, Princeton, N.J Ethyl alcohol Ajax Chemicals Australia. Hydralazine HCI Sigma Chemical Co. Losa¡tan Donated by Du Pont Merck. Methohexitone sodium (Breital) Lily Morphine HCI McFarlane Smith,Edinburgh Pentobarbitone sodium (Nembutal) Boehringer Ingelheim. Sodium chloride Ajar Chemicals, Australia. Verapamil Sigma Chemical Co. t45 Bibliography AKAIKE, A., SHIBATA T., SATOH, M. AND TAKAGI, H. 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