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The cardiovascular effects of CGS 21680, an adenosine A2A receptor agonist and 17b-estradiol in rats… Nekooeian, Ali Akbar 1999

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THE CARDIOVASCULAR EFFECTS OF CGS 21680, AN ADENOSINE A RECEPTOR AGONIST AND 17B-ESTRADIOL IN RATS WITH IMPAIRED 2 A  CARDIAC FUNCTION  BY  ALI AKBAR NEKOOEIAN D . V . M . , Shiraz University, 1987 M.Sc., University of Toronto, 1993 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENT FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES Department of Pharmacology & Therapeutics Faculty of Medicine  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  © Ali Akbar Nekooeian, 1999  In presenting this  thesis  in partial  degree at the University of  fulfilment  of  the  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of this thesis for department  or  by  his  or  scholarly purposes may be granted by the head of her  representatives.  It  is  understood  that  copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  /  DE-6 (2/88)  II ABSTRACT  The first part of this thesis examined the acute cardiovascular effects of CGS 21680, a selective adenosine A 2 A receptor agonist, relative to those of sodium nitroprusside (SNP) and vehicle (normal saline) in male Sprague-Dawley rats with or without acute occlusion or chronic ligation of the left main coronary artery. Rats with chronic coronary ligation were studied 8 weeks after the operation. The second part examined the chronic effects of 17(3-estradiol, the most abundant naturally-occurring estrogen, relative to those of vehicle on in vivo cardiovascular function and response to vasoactive drugs in ovariectomised rats at 7 weeks after ligation of the left coronary artery.  It also; examined the effects of 17(3-estradiol on ex vivo contractile and  relaxant responses of aortic rings, pulmonary artery rings and portal vein strips to vasoactive drugs. The ovariectomised rats were implanted with 60 days sustained-release pellets containing 17(3-estradiol (1.5 mg) or vehicle one week prior to coronary ligation. The vasoactive drugs studied include noradrenaline, phenylephrine, N -intro-L-arginine methyl ester (L-NAME), acetylcholine and SNP.. In both parts of the studies, sham-operated  rats with normal  cardiovascular function were also prepared to facilitate the evaluation of changes elicited by CGS 21680 or 17(3-estradiol. In vivo studies were performed under sodium pentobarbitone or thiobutabarbital (Inactin) anaesthesia.  Cardiovascular assessments included measurements of mean arterial pressure  (MAP), central venous pressure (CVP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), cardiac output (CO), coronary arterial flow and conductance, mean circulatory filling pressure (MCFP), arterial resistance (RA) and venous resistance (Ry). M A P , C V P and L V E D P were measured with catheters inserted into the femoral artery, inferior vena cava and left ventricle, respectively. HR was counted from the upstroke of the arterial pulse pressure or a tachograph.  C O . a n d coronary arterial flow were measured using radioactively-labelled  Ill microspheres. M C F P was measured by transiently stopping the circulation via the inflation of a saline-filled balloon placed in the right atrium, and was calculated as V P P + (FAP - VPP)/60, where F A P and V P P represent the final arterial pressure and venous plateau pressure, respectively, at 5-7 sec following circulatory arrest, and 60 is the ratio of venous to arterial compliance.  R  A  and Ry were calculated as follows: RA = M A P / C O and Rv = (MCFP -  CVP)/CO. Coronary arterial conductance was calculated as coronary arterial flow/MAP. In rats without coronary artery occlusion or ligation, CGS 21680 (0.1, 0.3 and 1.0 ug/kg.min, i.v. infusion) decreased M A P and R A , and increased CO, HR as well as coronary arterial flow and conductance. Ganglionic blockade with hexamethonium (200 pg/kg.min, i.v. infusion) decreased M A P , CO and HR, but did not alter coronary arterial flow or conductance. In the presence of hexamethonium,. CGS 21680 decreased M A P and R , increased coronary A  arterial flow and conductance, but did not change CO or HR. The effects of CGS 21680 on CO and HR were, therefore, mediated via the alteration of baroreflex activity. Phenylephrine (7 p.g/kg.min, i.v. infusion) increased M A P and R A , reduced CO, HR and coronary arterial conductance, but did not alter coronary arterial flow. In the presence of phenylephrine, CGS 21680 reduced M A P , R , and increased CO, HR, as well as coronary arterial flow and :  conductance.  ,.: ,  A  •  Relative to sham-operation, acute occlusion as well as chronic ligation of the coronary artery reduced M A P , CO and rate of rise of left ventricular pressure (+dP/dt), and increased L V E D P , M C F P and R at 90 min and 8 weeks after the operation, respectively. CGS 21680 at v  1.0 p.g/kg.min in rats with acutely-occluded coronary arteries, and at 0.3 and 1.0 u.g/kg/min in rats with chronically-ligated coronary arteries, increased CO and HR, and reduced M A P , R , A  MCFP, R and L V E D P . v  SNP at a dose (4 ug/kg.min), which reduced M A P similar to CGS  IV 21680 (0.3 u.g/kg.min), caused similar reductions in R , L V E D P or R , and a similar increase in A  v  CO. However, SNP caused a greater decrease in M C F P but less tachycardia than CGS 21680. Ovariectomised rats with chronic ligation of the coronary artery had lower M A P , CO and left ventricular +dP/dt, but higher R , L V E D P , M C F P and R relative to sham-operated rats at 7 A  v  weeks post-ligation. Chronic ligation of the coronary artery reduced MAP-responses to i.v. bolus injections of L - N A M E , acetylcholine and SNP, but not to i.v. bolus or i.v. infusion of noradrenaline. Chronic coronary ligation also attenuated the relaxation response to acetylcholine in the portal vein, but. not aorta or pulmonary artery, in ex vivo studies. Moreover, it reduced contractile responses. t o X - N A M E , which reached statistical significance in the aorta and portal vein, but not pulmonary artery.  However, it did not alter the contractile responses to  phenylephrine or relaxation responses to SNP in any of these vessels. 17p-Estradiol increased CO, and augmented the M A P , R , M C F P and R responses to A  v  i.v. infusion of noradrenaline as well as the M A P response to i.v. bolus of L - N A M E . It also decreased R , Rv as.,well as the. M A P response to i.v. bolus of acetylcholine, but did not alter the A  M A P responses to i.v. bolus of SNP or noradrenaline. In ex vivo studies, 17p-estradiol increased contractile responses to phenylephrine in the aorta, decreased the response in the portal vein, and did not change it in the pulmonary artery. 17p-Estradiol also reduced the maximal relaxation responses ( E  max  ) to acetylcholine in the aorta, increased the response in the portal vein, and did  not change it in-the pulmonary artery. As well, it enhanced the contractile responses to L - N A M E in all types of vessels, but did not alter the relaxation responses to SNP in any of the vessels. These results indicate that rats with acute occlusion as well as chronic ligation of the coronary artery have signs of impaired cardiac function, namely reduced CO and left ventricular +dP/dt as well as increased L V E D P , Rv and M C F P suggesting the development of acute and chronic heart failure.,, Acute.administration of CGS 21680 increased CO and HR, dilated arterial and venous resistance vessels, as well as reduced L V E D P and M C F P in rats with acute or  V chronic heart failure. Chronic treatment of 17p-estradiol in ovariectomized rats with chronic heart failure also increased CO, elicited dilatation of arterial and venous resistance vessels, and reduced L V E D P as well as MCFP. The reductions of arterial resistance and L V E D P by CGS 21680 or 17P-estradiol reflect the reductions of cardiac afterload and preload, respectively. Chronic 17p-estradiol also increased in vivo pressor and ex vivo contractile responses of blood vessels to L - N A M E indicating restoration of the vasodilator activity of basal nitric oxide in rats with chronic heart failure. The findings of the present thesis suggest that CGS 21680 and 17pestradiol, by" virtue of their vasodilator properties, improved cardiac function in rat model of acute or chronic heart failure.  VI TABLE OF CONTENTS  page ABSTRACT  I  TABLE OF CONTENTS  VI  LIST OF TABLES  X  LIST OF FIGURES LIST OF ABBREVIATIONS  -  XII XV  ACKNOWLEDGEMENTS  XVII  DEDICATIONS  XVIII  1. INTRODUCTION  1  1.1. The purinoceptors  1  1.1.1. Subtypes of adenosine receptors 1.1.1.1. Adenosine A i receptors 1.1.1.2. Adenosine A2 receptors 1.1.1.3. Adenosine A 3 receptors  2 3 3 4  1.2. Cardiovascular pharmacology of adenosine 1.2.1. /« vitro pharmacology 1.2.2. In vivo pharmacology  5 6 7  1.3. Pharmacology of CGS 21680 1.3.1. Selectivity of CGS 21680 1.3.2. Cardiovascular pharmacology 1.3.2.1. In vitro pharmacology 1.3.2.2. In vivo pharmacology  8 8 9 9 10  1.4. Estrogens 1.4.1. Mechanisms, of cardioprotection by estrogens 1.4.2. Estrogen receptors 1.4.3. Cardiovascular pharmacology of estrogens 1.4.3.1. In vitro studies 1.4.3.2. Ex vivo studies 1.4.3.3. Human and animal in situ and in vivo studies 1.4.3.3.1. In situ studies 1.4.3.3.2. Acute studies . 1.4.3.3.3. Chronic studies  11 13 13 15 15 18 20 20 21 23  1.5. Heart failure 1.5.1. Acute heart failure 1.5.2. Chronic heart failure 1.5.3. Neurohormonal responses in heart failure 1.5.4. Assessment of infarct size 1.5.5. Factors influencing infarct size  27 28 29 31 32 34  1.6 Venous system 1.6.1. Venous terminology 1.6.1.1 Mean circulatory filling pressure 1.6.1.2. Venous capacitance 1.6.1.3. Stressed and unstressed blood volume 1.6.1.4. Venous.compliance 1.6.1.5. Venous resistance 1.6.2. Methods for studying venous function in vivo 1.6.2.1. Mean circulatory filling pressure technique 1.6.3. Measurement of mean circulatory filling pressure in rats 16.4. Limitations of the M C F P technique  34 47 38 38 38 38 39 40 40 41 41  1.7. Objective 1.7.1. Effects of CGS 21680 in fats with impaired cardiac function 1.7.2. Effects of 17p-estradiol in rats with impaired cardiac function  42 43 43  2. MATERIALS AND METHODS  45  2.1 Surgical preparation 2.1.1. Effects of CGS 2.1680 on haemodynamics in rats with and without impaired cardiac function 2.1.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function 2.1.1.2. Effects of CGS 21680 on haemodynamics in rats with acute occlusion of the coronary artery 2.1.1.3. Effects of CGS 21680 on haemodynamics in rats with chronic ligation of the coronary artery 2.1.2. Effects of 17p-estradiol in rats with impaired cardiac function  45 45 45 45 46 47  2.2. Instrumentation  48  2.3. Experimental design and protocols 2.3.1. Effects of CGS 21680 on haemodynamics in rats with and without impaired cardiac function 2.3.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function 2.3.1.2. Effects of CGS 21680 on haemodynamics in rats with acute occlusion of the coronary artery 2.3.1.3. Effects of CGS 21680 on haemodynamics in rats with chronic ligation of the coronary artery 2.3.2. Effects of 17p-estradiol in rats with impaired cardiac function  49 49 49 50 50 51  VIII 2.3.2.1 Effects of 17(3-estradiol on the activity of basal nitric oxide in rats with chronic ligation of the coronary artery 2.3.2.2. Effects of. 17p-estradiol on the activity of basal nitric oxide in blood vessels from rats with chronic ligation of the coronary artery 2.3.2.3. Effects of 17p-estradiol on mean circulatory filling pressure and venous resistance in rats with chronic ligation of the coronary artery  52  2.4. Measurement of cardiac output  52  2.5. Measurement of mean circulatory filling pressure  53  2.6. Measurement of total blood volume  54  2.7. Assessment of occluded zone  54  2.8. Assessment of surface area of infarct  54  2.9. Measurement of serum 17p-estradiol  55  2.10. Chemicals  55  2.11. Calculations and statistical analysis  56  3. RESULTS  58  51  51  3.1. Effects of CGS 21680 on haemodyamics in rats with and without impaired cardiac function 3.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function 3.1.1.1. Selectivity of CGS 21680 3.1.1.2. Effects of CGS 21680 on haemodynamics 3.1.2. Effects of CGS.21680 on haemodynamics in rats with acute occlusion of the coronary artery 3.1.3. Effects of CGS 21680 on haemodynamics in rats with chronic ligation of the coronary artery 3.2. Effects of 17p-estradiol in rats with impaired cardiac function 3.2.1. Effects of 17p-estradiol on the activity of basal nitric oxide in rats with chronic ligation of the coronary artery 3.2.2. Effects of 17P-estradiol on the activity of basal nitric oxide ' in.blood vessels from rat with chronic ligation of the coronary artery 3.2.3. Effects of 17p-estradiol on mean circulatory filling pressure and venous resistance in rats with chronic ligation of the coronary artery  58 58 58 58 70 77  88 88  93  98  IX 4. DISCUSSION 4.1 Effects of CGS 21680 on haemodyamics in rats with and without impaired cardiac function 4.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function 4.1.2. Effects of CGS 21680 on haemodynamics in rats with impaired cardiac function 4.1.2.1. Effects of acute occlusion of the coronary artery on haemodynamics 4.1.2.2. Effects of chronic ligation of the coronary artery on haemodynamics 4.1.2.3. Effects of CGS 21680 on haemodynamics  105  105 105 107 107 107 108  4.2. Effects of 17P-estradiol in rats with impaired cardiac function 4.2.1 Effects of 17p-estradiol on the activity of basal nitric oxide in rats with chronic ligation of the coronary artery 4.2.2. Effects of 17p-estradiol on the activity of basal nitric oxide in blood vessels from, rats with chronic ligation of the coronary artery 4.2.3. Effects of 17P-estradiol on mean circulatory filling pressure and venous resistance in rats with chronic ligation of the coronary artery  119  5. SUMMARY AND CONCLUSION  124  5.1. Effects of CGS 21680 on haemodynamics in rats with and without impaired cardiac function  124  5.2. Effects of 17p-estradiol in rats with impaired cardiac function  125  6. REFERENCES  127  112 112  116  LIST OF TABLES  Table 1. Baseline haemodynamics of rats without impaired cardiac function treated with saline, CGS 21680, hexamethonium, phenylephrine, and CGS 21680 in the presence of hexamethonium or phenylephrine. Table 2. Baseline haemodynamics of rats with acute sham operation treated with saline, and rats with acute occlusion of the coronary artery treated with saline or CGS 21680. Table 3. Haemodynamics of rats with acute sham operation treated with saline, and rats with acute occlusion of the coronary artery treated with saline or CGS 21680 at 90 min after coronary occlusion. Table 4. Body, ventricular, lung weights and infarct areas of rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline, CGS 21680 or SNP Table 5. Baseline haemodynamics of.rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline, CGS 21680 or SNP. Table 6. Infarct areas, and ventricular, lung and body weights of rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17(3-estradiol. Table 7. Baseline haemodynamics of rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17P-estradiol. Table 8. Contractile responses to phenylephrine in thoracic aortae, pulmonary arteries and portal veins from rats with chronic sham operation rats treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17p-estradiol. Table 9. Maximal response ( E ) and EC50 to A C h and SNP in thoracic aortae, pulmonary arteries and portal vein from rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17p-estradiol. max  Table 10. Infarct areas, ventricular, lung and body weights, haematocrit, total plasma volume and total blood volume of rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17p-estradiol.  Table 11. Baseline haemodynamics of rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary treated with vehicle or 17p-estradiol.  LIST OF FIGURES  Figure 1. Changes from the baseline M A P and R A in rats without impaired cardiac function treated with saline or CGS 21680. Figure 2. Changes from the baseline CO and H R in rats without impaired cardiac function treated with saline or CGS 21680. Figure 3. Changes from the baseline coronary arterial flow and conductance in rats without impaired cardiac function treated with saline or CGS 21680. Figure 4. Changes from the baseline M A P and R A in rats without impaired cardiac function treated with saline, CGS 21680, hexamethonium and CGS 21680 in the presence of hexamethonium. Figure 5. Changes, from the baseline CO and H R in rats without impaired cardiac function treated with saline, CGS 21680, hexamethonium and CGS 21680 in the presence of hexamethonium. Figure 6.- Changes from the baseline coronary arterial flow and conductance in rats without impaired cardiac function treated with saline,CGS 21680, hexamethonium and CGS 21680 in the presence of hexamethonium. Figure 7. Changes from the baseline M A P and R A in rats without impaired cardiac function treated, with saline, CGS 21680, phenylephrine and CGS 21680 in the presence of phenylephrine. Figure 8. Changes from the baseline CO and H R in rats without impaired cardiac function treated with saline, CGS 21680, phenylephrine and CGS 21680 in the presence of phenylephrine. Figure 9. Changes from the baseline coronary arterial flow and conductance in rats without impaired cardiac function treated with saline, CGS 21680, phenylephrine and CGS 21680 in the presence of phenylephrine. Figure 10. Changes from the pre-treatment values of M A P and R A in rats with acute sham operation treated with saline, and rats with acute occlusion of the coronary artery treated with saline or CGS 21680. . Figure 11. Changes from the pre-treatment values of CO and H R in rats with acute sham operation treated with saline, and rats with acute occlusion of the coronary artery treated with saline CGS 21680.  XIII Figure 12. Changes from the pre-treatment values of L V E D P and left ventricular +dP/dt in rats with acute sham operation treated with saline, and rats with acute occlusion of the coronary artery treated with saline or CGS 21680.  75  Figure 13. Changes from the pre-treatment values of MCFP and Rv in rats with acute sham operation treated with saline, and rats with acute occlusion of the coronary artery treated with saline or CGS 21680.  76  Figure 14. Changes from the baseline M A P and R A in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline or CGS 21680.  80  Figure 15. Changes from the baseline CO and HR in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline or CGS 21680.  81  Figure 16. Changes from the baseline L V E D P and left ventricular +dP/dt in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline or CGS 21680.  82  Figure 17. Changes from the baseline MCFP and Rv in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline or CGS 21680.  83  Figure 18. Changes from the baseline M A P and R A in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline, CGS 21680 or SNP.  84  Figure 19. Changes from the baseline CO and HR in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline, CGS 21680 or SNP.  85  Figure 20. Changes from the baseline L V E D P and left ventricular +dP/dt in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline, CGS 21680 or SNP. ,  86  Figure 21 Changes from the baseline MCFP and Ry in rats with chronic sham operation treated with saline, and rats with chronic ligation of the coronary artery treated with saline, CGS 21680 or SNP.  87  Figure 22. Dose-MAP,responses to N A and L - N A M E in rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17p-estradiol.  91  Figure 23. Dose-MAP responses to A C h and SNP in rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17p-estradiol. Figure 24. Contractile responses to L - N A M E in aortae, pulmonary arteries and portal veins from rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artery treated with vehicle or 17P-estradiol. Figure 25. Effects of saline and N A on M A P and R in rats with chronic sham operation treated with vehicle, and rat with chronic ligation of the coronary artery treated with vehicle or 17p-estradiol. A  Figure 26. Effects of saline and N A on CO and HR in rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary artrery treated with vehicle or 17p-estradiol. Figure 27. Effects of saline and N A on L V E D P and left ventricular +dP/dt in rats with chronic sham operation treated with vehicle, and rats with chronic ligation of the coronary treated with vehicle or 17P-estradiol. . Figure 28. Effects of saline and N A on M C F P and Rv in rats with chronic sham operation treated with vehicle, and rat with chronic ligation of the coronary artery treated with vehicle or 17P-estradiol.  LIST OF ABBREVIATIONS  ACh  acetylcholine  ADP  adenosine diphosphate  AMP  adenosine monophosphate  ANOVA  analysis of variance  ATP  adenosine triphosphate  cAMP  cyclic adenosine monophosphate  cGMP  cyclic guanosine monophosphate  CGS 21680  2-[p-(2-carboxyethyl)-phenethylamino]-5'-Nethylcarboxamidoadenosine  CO  cardiac output  CVP  central venous pressure  DMSO  dimethyl sulfoxide  DPCPX  1,3-dipropy-8-cyclopentyllxanthine  +dP/dt  rate of rise of pressure  E-CL  coronary-ligated rats treated with 17(3-estradiol  EDHF  endothelium-derived hyperpolarising factor  FAP  final  arterial pressure  HR  heart rate  i.p.  intraperitoneal  i.v.  intravenous  L-NAME  N -intro-L -arginine methyl ester  LVEDP  left ventricular end-diastolic pressure  MAP  mean arterial pressure  MCFP  mean circulatory filling pressure  NA  noradrenaline  NO  nitric oxide  NS  normal saline  R  arterial resistance  A  u  RCV  red cell volume  R  venous resistance  v  XVI s.c.  subcutaneous  SNP  sodium nitroprusside  TBV  total blood volume  TPR  total peripheral resistance  TPV  total plasma volume  V-CL  coronary-ligated rats treated with vehicle  VPP  venous plateau pressure  V-S  sham-operated rats treated with vehicle  XVII  ACKNOWLEDGEMENTS  I would like to express my sincere appreciation to my supervisors Drs. Catherine C. Y . Pang and Reza Tabrizchi for their careful guidance, excellent supervision, invaluable scientific insight and wise advice. I am especially grateful to Dr. Pang for her unmatched encouragement, meticulous constructive criticism, unique commitment to her students, and for sparing some time for whatever query I had.  I would like to thank the members of my advisory committee: Dr. Morley C. Sutter, Dr. Michael J. A . Walker and Dr. Simon W. Rabkin for the time and effort spent in evaluating my work and their insightful suggestions.  I would like to express my gratitude to Ms. Su Lin Lim for her assistance and making the lab a most pleasant place to work.  I am "grateful to staff of the Department of Pharmacology & Therapeutics: Mr. Christian Caritey, Mr. George Chua, Ms. Maureen Murphy, Ms. Wynne Leung, Ms. Janelle Stewart, and Ms. Bick Lu for their help during the course of my studies.  I am enormously indebted to the Ministry of Health of Islamic Republic of Iran and Heart & Stroke Foundation of British Columbia & Yukon for their financial support.  Most of all I am appreciative to my wife for unmatchable sacrifices, support and understanding, and our children, Parya and Mohammad.  XVIII  In the Name ofAllah, the merciful the compassionate  Dedications  This thesis is dedicated to:  My parents, My wife and our children  Parya and Mohammad  1. INTRODUCTION 1.1. The purinoceptors Interest in the physiology and pharmacology of purine nucleotides and nucleosides started almost 70 years ago, when Drury and Szent-Gyorgyi (1929) described the effects of adenosine and adenosine monophosphate (AMP) on the heart and circulation of several mammalian species. Afterwards, there were attempts to further elucidate the physiology and pharmacology of these compounds. A quarter of a century later, adenosine triphosphate (ATP) was shown to be. released upon antidromic stimulation of sensory nerves. This prompted Holton and Holton (1954) to suggest a vasodilating role for ATP. Subsequently, antidromic stimulation of the great auricular nerve was found to cause vasodilatation of rabbit ear vessels together with the release of ATP (Holton, 1959). A couple of years later, Gaarder and colleagues (1961) identified adenosine diphosphate (ADP) as an active component in the extract of red blood cells that caused the aggregation of platelets. In 1963, Berne further suggested that adenosine might be the physiological regulator of coronary arterial flow. The existence of receptors for purine nucleotides and nucleosides was first indicated through the observation that effects of adenosine in the heart could be blocked by caffeine (De Gubareff & Sleator, 1965). In 1978, Burnstock proposed a sub-classification of purine receptors into P| and P2 purinergic receptors on the basis of the following characteristics: 1) differing rank order of potencies of ATP, ADP, A M P and adenosine for the activation of purinergic receptors, 2) differing selectivity of blockade by antagonists, in particular the methylxanthines, 3) modulation of adenylyl cyclase activity by adenosine, but not A T P , and 4) induction of prostaglandin synthesis by ATP, but not adenosine. According to this classification, purinergic receptor agonists had the following order of potency for Pi-purinergic receptors: adenosine > A M P > A D P > A T P . The Pi-purinergic receptor agonists mediated their actions via adenylyl cyclase system, and their actions were  competitively antagonised by methylxanthines such as theophylline, aminophylline and caffeine. The P2-purinergic receptor agonists, on the other hand, had the reverse order of potency: ATP > A D P > A M P > adenosine, and did not act through adenylyl cyclase. Moreover, their actions were not inhibited by methylxanthines. purinoceptors (Fredholm et al., 1994).  The purinergic receptors were later renamed as The P|-purinoceptors are also known as adenosine  receptors, of which 4 subtypes have been identified, and will be discussed later.  The P 2 -  purinoceptors were further divided into P 2 , P2y, P2t, P211 and P27. subtypes (Dalziel & Westfall, X  1994; Bumstock,. 1997; Fredholm et al., 1997). As P2-purinoceptors are not the subject of investigation in the present study, they will not be discussed further.  1.1.1. Subtypes of adenosine receptors In  1979 van Calker and colleagues demonstrated that adenosine could alter the  concentration of cyclic adenosine monophosphate (cAMP) in cultured brain cells. The term A i was used to denote the receptors that mediated the inhibition of c A M P formation, and A2 to denote the receptors that activated the c A M P formation. Independently, Londos and colleagues (1980) termed adenosine receptors as R receptors due to their requirement of an intact ribose moiety for activation. They further subdivided adenosine receptors as Rj and R , corresponding a  to A] and A2 receptors respectively, because of their inhibitory (via Rj receptor) and activating (via R receptor) effects on adenylyl cyclase. The A1/A2 nomenclature is, however, widely a  accepted. Recent pharmacological and molecular biological studies have confirmed the existence of at least four subtypes of adenosine receptors namely, A i , A ( A A , A B ) and A3 (Fredholm et al., 2  2  2  1994). These receptors have the general chemical structures of rhodopsin-like superfamily of Gprotein-coupled receptors.  2  1.1.1.1. Adenosine A i receptors Adenosine A i receptors have been cloned from rat (Mahan et al., 1991), bovine (Tucker et al., 1992) and human (Libert at al., 1992) brains. These receptors have the following potency profile for adenosine analogues in inhibiting c A M P production in a variety of tissues: N - R - l 6  phenyl-2-propyl adenosine •= N -cyclo-hexyladenosine > N-ethylcarboxamidoadenosine = 26  chloroadenosine > adenosine > N -S-l-phenyl-2-propyladenosine (Dalziel & Westfall, 1994). 6  The A i receptors are found in brain, heart and kidney (Shryock & Belardinelli, 1997). The primary Ai-mediated cardiovascular effect is negative chronotropy.  1.1.1.2. Adenosine. A2 receptors Adenosine A2 receptors were shown to exist in a variety of vascular tissues. In humans, they were found in saphenous vein as well as coronary and internal mammary arteries (Makujina et al., 1992). They were also found in rat aorta (Lewis et al., 1994; Prentice & Hourani, 1996), pulmonary vasculature (Haynes et al., 1995), femoral artery and vein (Abiru et al., 1995), mesenteric artery (Hiley et al.,-1995) as well as guinea pig (Schiele & Schwaba, 1994) and pig (Abebe et al., 1994) coronary arteries.  The affinity of adenosine for A2 receptors is  approximately three orders of magnitude less than that for A | receptors. In 1983 Daly and. colleagues suggested the existence of two subtypes of A receptors due 2  to the existence of. low and high affinity binding sites for adenosine in brain tissues. Subsequently, A2 receptors were further sub-divided according to the observation that a large number of compounds had differing binding affinities for the rat striatal versus human fibroblast adenosine A2 receptors, and the terminology of A2A (striatal) and A2B (fibroblast) were proposed (Bruns et al., 1986). This sub-classification was later verified in an extensive functional study using a wide range .of adenosine, analogues in a variety of tissues including rat adipocytes and atria, guinea pig ilea, aortae and atria, dog coronary arteries as well as human platelets and 3  neutrophils (Gurden et al., 1993). The rank order of potency of agonists for A 2 A receptors in dog coronary arteries, human neutrophils and platelets was shown to be 2-(phenylamino)adenosine 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine ethylcarboxamidoadenosine  >  N -R-l-phenyl-2-propyladenosine 6  >  (CGS 21680) > N metrifudil  >  N-  cyclopentyladenosine > N-[(1S, ^ra»5)-2-hydroxycyclopentyl]adenosine = N -S-l-phenyl-26  propyladenosine. By contrast, the rank order of potency of the same agonists for A 2 B receptors was as follows: N-ethylcarboxamidoadenosine > metrifudil > N -R-l-phenyl-2-propyladenosine 6  =  N-cyclopentyladenosine  >  2-(phenylamino)  adenosine  =  N-[(1S,  trans)-2-  hydroxycyclopentyl]adenosine > N -S-l-phenyl-2-propyladenosine = CGS 21680 in guinea pig 6  aorta (Gurden at al., 1993).. The existence of two subtypes of A2 receptors was confirmed following cloning of these receptors in human brain (Furlong et al., 1992; Pierce et al., 1992), dog thyroid cells, (Maenhaut et al., 1990) as well as rat striatum (Fink et al., 1992) and brain (Stehle et al., 1992). The vasodilator or vasorelaxant activity of adenosine is partly mediated via the activation of A2 receptors (Kusachi et al., 1983).  1.1.1.3. Adenosine A 3 receptors , In 1986, Riberio and Sebastiao proposed that a third subtype of adenosine receptors, distinct from A i and A 2 , mediated the electrophysiological effects of adenosine. This receptor subtype, which was not coupled to adenylyl cyclase, but to C a  2+  channels and was distributed in  heart and nerve endings, was termed adenosine A 3 receptors (Riberio & Sebastiao, 1986). In 1992, Zhou and colleagues, cloned an adenosine receptor from rat testes, which negatively modulated the activity of adenylyl cyclase. The varying rank order of binding affinities for a number of A | and A2-selective ligands led the authors to conclude that the new receptor subtype was different from A | and A 2 receptors, and they proposed to call it A 3 receptor.  Similar  4  receptors were later cloned from rat heart and kidney (Zhou et al., 1992), human heart and lung (Sajjadi & Firestein, 1993) and aorta (Salvatore et al., 1993). The existence of the A 3 adenosine receptor was also demonstrated by in vivo studies. Fozard and  Carruthers  (1993a)  showed that intravenous  (i.v.)  infusion  of N -2-(46  aminophenyl)efhyladenosine, which was shown to have a high affinity for A 3 receptors in binding studies (Zhou et al., 1992), caused hypotension in pithed rats that had blood pressure elevated by i.v. infusion of angiotensin II. The hypotensive effect of this compound was not blocked by high doses (20-40 mg) of 8-sulfophenyltheophylline, a relatively nonselective adenosine receptor antagonist.  Therefore, it was proposed that the depressor response was  mediated by a receptor distinct from A | or A , which was called adenosine A 3 receptor. 2  1.2. Pharmacology of adenosine Adenosine is, a .ubiquitous endogenous purine nucleoside that modulates a variety of physiological functions. It is produced extra- and intracellularly in the heart via two distinct metabolic pathways. The first pathway, which is the predominant one, involves the hydrolysis of A M P to adenosine by 5'-nucleotidase, and the second pathway involves the catabolism of Sadenosylhomocysteine by S-adenosylhomocysteine hydrolase (Sparks & Bardenheuer, 1986; Olsson & Pearson, 1990). The half-life of adenosine in the interstitial space is from one to a few sec (Moser et al., 1989). Some of the adenosine in the interstitial space crosses the capillary wall into plasma, where it has a half-life of about a sec (Camm & Garratt, 1991). A large fraction of adenosine in plasma is taken up by myocardial cells, rephosphorylated by adenosine kinase to A M P , and reincorporated as adenine nucleotide (Wiedmeier et al., 1972). A significant fraction of it is degraded by adenosine deaminase to inosine and hypoxanthine (Rubio et al., 1972; Sparks & Bardenheuer, 1986).  5  1.2.1. /« vitro pharmacology Adenosine has been shown to cause relaxation of a variety of human and animal vascular tissues.  It relaxed precontracted rings from human saphenous vein as well as coronary and  internal mammary arteries (Makujina et al., 1992). It also relaxed precontracted rings from rat aorta (Moritoki et al., 1990; Rose'Meyer & Hope, 1990), mesenteric artery (Vourinen et al., 1992), portal vein (Sjoberg & Wahlstrom, 1975) and perfused mesenteric arterial bed (Hiley et al., 1995; Rubino et al., 1995; Tabrizchi & Lupichuk, 1995). Moreover, it relaxed guinea pig aorta (Headrick & Berne, 1990), pig coronary artery (Makujina et al., 1994), dog saphenous vein (Verhaeghe, 1977) as well as cerebral, coronary and mesenteric arteries (Toda et al., 1982), and bovine, coronary artery (Gushing et al., 1991).  Adenosine also relaxed isolated perfused  hindlimb arterial and venous beds of dog (Cotterrell & Karim, 1982) and perfused hepatic arterial bed of rabbit (Mathie et al., 1991). It caused coronary artery vasodilatation (Daut et al., 1990; von Beckerath et al., 1991; Vials & Burnstock, 1993), and increased coronary flow (Lewis & Hourani, 1997) in.isolated.perfused hearts. A number of mechanisms have been proposed to account for the adenosine-induced vascular relaxation. This relaxation appears to be partly endothelium-dependent, since it was partly inhibited by removal of the vascular endothelium (Rubanyi & Vanhoutte, 1985; Headrick & Berne, 1990; Vourinen et al., 1992; Hiley et a l , 1995) or treatment with methylene blue and haemoglobin (Moritoki et al., 1990; Rose'Meyer & Hope, 1990). Adenosine was shown to enhance the production of nitric oxide (NO) by vascular endothelial cells via a receptor-mediated mechanism (Li et al., 1995). On the other hand, adenosine-induced relaxation was shown to be endothelium-dependent,  but not mediated by NO.  This raises the possibility that other  endothelial factors, such as endothelium-derived hyperpolarising factor (EDHF) might be involved (Headrick & Berne, 1990). Adenosine might also elicit relaxation through inhibition of Ca  2+  influx (Ramagopal & Mustafa, 1988), augmentation of c A M P production (Cushing et al., 6  1991), hyperpolarisation of cell membrane (Sabouni et al., 1989), opening of ATP-sensitive K  +  channel (Kuo & Chancellor, 1995), and inhibition of phospholipase C activity (Long & Stone, 1987). However, A T P sensitive K channels were shown not to be involved in adenosine+  induced relaxation of porcine coronary arterial rings (Makujina et al., 1994) and rat mesenteric arterial bed (Tabrizchi & Lupichuk, 1995). How adenosine dilated the coronary arterial bed in isolated perfused hearts is as yet unsettled.  ,' Adenosine-induced coronary vasodilatation was shown to be partly due to :  endothelium-derived N O (Vials & Burnstock, 1993) as well as activation of large-conductance Ca -activated (Cabell et al., 1994) and ATP-sensitive K (Daut et al., 1990; von Beckerath et al., 2+  +  1991) channels.  However, N O was recently shown not to be involved in adenosine-induced  increases in coronary flow (Lewis & Hourani, 1997).  1.2.2. In vivo pharmacology Lv. infusion of adenosine in humans decreased total peripheral resistance (TPR), increased cardiac output (CO), but did not alter mean arterial pressure (MAP) (Bush et al., 1989; Edlund et al., 1990). It also decreased (DiMarco et al., 1983) or increased (Edlund et al., 1990) heart rate (HR). I.v. bolus adenosine decreased M A P and H R in dogs (Yoneyama et al., 1992) as well as normotensive (Jonzon et al., 1986; Stella et al., 1993) and hypertensive (Keddie et al., 1996) rats. I.v. infusion of adenosine caused coronary vasodilatation in dogs (Belloni & Hintze, 1991; Akatsuka et al., 1994), and hypotension in dogs (Belloni & Hintze, 1991) and rats (Glick et al., 1992; Hernandez et al., 1995). It also decreased TPR (Hernandez et al., 1995; Tabrizchi, 1997), but did not alter CO (Hernandez et al., 1995; Tabrizchi, 1997) in rats. Adenosine also reduced (Tabrizchi, 1997),, or did not change (Ohnishi et al., 1986) H R in rats.  It reduced mean  7  circulatory filling pressure (MCFP) (Glick et al., 1992; Tabrizchi, 1997) in rats, and dilated dorsal hand vein in humans (Ford et al., 1992). Intrarenal infusion of adenosine caused pressor responses in dogs (Katohli et al., 1983) and rats (Katohli et al., 1985), which were attributed to activation of renal sympathetic afferent nerves. It also constricted the hamster cheek pouch arterioles in vivo (Shepherd et al., 1996). The constriction was proposed to be due to A3 receptor-mediated degranulation of mast cells and subsequent release of vasoconstricting mediators. Adenosine was also shown to constrict the pulmonary vascular bed in sheep (Biaggioni et al., 1989), and to constrict as well as dilate it in cats (Neely jet al.,. 19.91; Neely & Matot, 1996). The effect of adenosine on pulmonary vascular bed was dependent on the prevailing tone; at basal tone it caused vasoconstriction, but at elevated tone it caused vasodilatation (Neely et al., 1991; Neely & Matot, 1996). The possible role of N O or ATP-sensitive K channels in the vasodilatation effect of +  adenosine is unclear. For example, N O partly mediated adenosine-induced decrease in TPR, but not the decrease in M A P (Hernandez et al., 1995). On the other hand, N O was not involved in adenosine-induced increases in forearm (Costa & Biaggionoi, 1998) and coronary (Shiode et al., 1996) arterial blood flows in human. The ATP-sensitive K channels have been reported to +  partly mediate the adenosine-induced hypotension (Belloni & Hintze, 1991) in rats (Hernandez et al., 1995) and coronary vasodilatation in dogs (Belloni & Hintze, 1991; Akatsuka et al., 1994). However,, these channels were not involved in adenosine-mediated hypotension in rats (Fozard & Carruthers, 1993b) and spinally-anaesthetised dogs (Yoneyama et al., 1992).  1.3. Pharmacology of CGS 21680 1.3.1. Selectivity of CGS 21680 CGS 21680 is a selective A 2 A receptor agonist, which elicits a saturable, reversible and high affinity (Kj = 15.5 nM) binding to rat striatal membrane. Binding studies showed that CGS 8  21680 was 170-fold more selective for adenosine A than A | receptors (Jarvis et al., 1989). CGS 2  21680 is poorly absorbed orally, and is largely degraded by hepatic first-pass metabolism. The half-life of CGS 21680 is 19 ± 4 min.after i.v. bolus injection in rats (Chovan et al., 1992).  1.3.2 Cardiovascular pharmacology 1.3.2.1. In.vitrp pharmacology  ......  CGS 21680 has been shown to relax a variety of human and animal vascular tissues. It relaxed precontracted rings from human saphenous vein as well as internal mammary and coronary arteries (Makujina et al., 1992). It also relaxed precontracted rings from rat aorta (Conti et al., 1993: Lewis et al., 1994), femoral artery and vein (Abiru et al., 1995) as well as rings from dog (Gurden et al., 1993), pig (Abebe et al., 1994; 1995) and cow (Conti et al., 1993) coronary arteries. CGS 21680 vasodilated perfused rat mesenteric arterial (Hiley et al., 1995) and rabbit hepatic arterial (Mathie et al., 1991) beds, and caused coronary vasodilatation in isolated perfused hearts from rat (Hutchison et al., 1989; Lewis & Hourani, 1997) and guinea pigs (Vials & Burnstock, 1993; Felsch et a l , 1994). CGS 21680-induced coronary vasodilatation might be partly mediated by endotheliumderived NO. N -intro-L-arginine methyl ester (L-NAME), a N O synthase inhibitor, attenuated G  CGS 21680-induced coronary, vasodilatation in isolated heart from guinea pig (Vials & Burnstock, 1993). Moreover, relaxation induced by CGS 21680 in porcine coronary artery was attenuated by N -monomefhyl-L-arginine, a N O synthase inhibitor (Abebe et al., 1995) or removal of vascular endothelium (Abebe et al., 1994). It is yet uncertain whether CGS 21680 has direct cardiac actions. CGS 21680 reduced the contractility of isolated perfused rabbit heart (Cano & Malik, 1992) and increased the amplitude of contraction of rat ventricular myocytes (Xu et al., 1996; Dobson & Fenton, 1997). By contrast, CGS 21680 did not affect the rate of contraction of isolated perfused rat heart 9  (Hutchison et al., 1989; Conti et al., 1993) and isolated rat right ventricle (Hernandez et al., 1994).  Moreover, binding studies revealed no specific binding sites for CGS 21680 in  membranes from right ventricle of rats (Hernandez et al., 1994).  1.3.2.2. In vivo pharmacology I.v. administrations of CGS 21680 caused hypotension in normotensive (Hutchison et al., 1989; Jackson et al., 1993; Patel et al., 1994; Harmon et a l , 1995), spontaneously hypertensive (Webb et al.,' 1991) and pithed (Fozard & Carruthers, 1993b) rats. It also increased (Hutchison et al., 1989) or did not affect (Patel et al., 1994; Hannon et al., 1995) HR in rats. Tachycardic effect of CGS-21680 was attenuated by pretreatment with metoprolol, a (31-adrenoceptor antagonist (Webb et al., 1991). Moreover, CGS 21680 did not cause tachycardia in pithed rats (Fozard & Carruthers, 1993b).  These observations suggest that tachycardic effects of CGS  21680 were likely caused by hypotension-induced reflex increase in sympathetic activity. CGS 21680, administered by i.v. infusion, increased CO, HR as well as plasma renin in spontaneously hypertensive rats (Webb et al., 1991). The increase in plasma renin activity was likely due to hypotension-induced increase in sympathetic activity, since intrarenal administration of CGS 21680 failed to increase the plasma renin activity (Levens et al., 1991). There is indication that.tolerance may develop following continual exposure to CGS 21680.  Chronic release of CGS. 21680 from a subcutaneous (s.c.) osmotic minipump, was  associated with reduced hypotensive response, which was attributed to the down-regulation of A2A receptors (Webb et al., 1993). By contrast twice daily i.p. administration of CGS 21680 for 21 days did not attenuate the hypotensive response. Therefore, existence of tolerance to CGS 21680 in the former study might be due to the continual presence of the drug (Casati et al., 1993).  .  10  1.4. Estrogens Estrogens are a family of sex hormones that are synthesised and released from the ovaries through concerted functions of theca and granulosa cells, and luteinizing and follicle stimulating hormones. Luteinizing hormone stimulates the theca cells to produce androgens. As androgens cross the basal membrane, they are converted to estrogens in response to stimulation of granulosa cells by follicle stimulating hormone. The most potent estrogens in human are 17(3estradiol followed- by estrone and estriol. Estrogens elicit a variety of sex-related activities including the development of secondary sexual characteristics, and positive feedback action on the hypothalamic-pituitary unit to synchronise the preovulatory release of gonadotropins (Shoham & Schachter, 1996). Epidemiological evidence indicates that estrogens have cardioprotective actions. Premenopausal women are 2-8 and 2-3 times at lower risk of coronary artery disease than men of the same age and postmenopausal women, respectively (Ryan, 1976; Gordon et al., 1978; Johansson et al., 1983; Lerner & Kannel, 1986; Bruckert & Turpin, 1995). The risk increases steeply after. menopause, and coronary artery disease becomes the leading cause of death in postmenopausal women (Gorodeski.. & Utian, 1994).  Estrogen replacement therapy in  postmenopausal women reduced the risk of morbidity and/or mortality from coronary artery disease (Bush et al., 1987; Gruchow et al., 1988; Henderson et a l , 1988; McFarland et al., 1989; Wolf et al., 1991; Grodstein et al., 1996).  Some studies, however, reported that estrogen  replacement did not have, beneficial effects (Pfeffer et al., 1978; Hernandez Avila, 1990; Rosenberg et al., 1993) or even had harmful effects (Jick et al., 1978; Wilson et al., 1985). Inconsistencies among epidemiological findings might be due to variations in sample sizes, duration of follow-ups, end-point of studies (death, chest pain or angiographic evidence) as well as dose of estrogen and duration of replacement therapy. Inconsistent findings might also be due to differences in health and environmental conditions including smoking, age, plasma 11  concentrations of lipoproteins, blood pressure, diabetes, weight, personal or parental history of coronary artery disease, atherosclerosis, social economic status, nutrition and access to health care.  Quantitative analysis of published epidemiological studies indicated that following  adjusting of cardiac risk factors, estrogen replacement was associated with a 50% reduction in the risk of coronary heart disease in postmenopausal women (Bush, 1990; Barrett-Connor & Bush, 1991; Stampfer & Colditz, 1991). Clinical trials. performed on the cardioprotective effects of estrogen have been small, short-term, and not designed to examine the outcome of coronary heart disease. A n analysis of published clinical .trials from 1973 and 1995 revealed that estrogen therapy of postmenopausal women offered no protection against cardiovascular events (Hemminki & McPherson, 1997). The Postmenopausal Estrogen-Progestin Intervention Trial (The Writing Group, 1995) compared the effects of estrogen alone, or in combination with progestin in postmenopausal women ranging from 45 to 64 years in a 12 months,period. This trial demonstrated that estrogen alone, or in combination with progestins, increased plasma high-density lipoproteins, and decreased plasma low-density lipoproteins. However, it did not monitor the morbidity or mortality of coronary heart disease.  The most recent randomised clinical trial (Hulley et al., 1998) examined the  effects of oral administration of conjugated equine estrogen (0.625 mg) plus medroxyprogestrone acetate (2.5 mg) in postmenopausal women aging from 49 to 77 years during a four-year period. It demonstrated that estrogen plus progestrone reduced plasma low density lipoproteins, increased plasma high-density lipoproteins as well as triglyceride, but failed to change mortality, or incidence or severity of nonfatal myocardial infarction (Hulley et al., 1998). It is, however, unclear whether the possible beneficial effects of estrogen were not masked by simultaneous use of progestins in this trial.  Progestins down-regulate estrogen  receptors, and may oppose estrogen effects (Sarrel, 1995). Medroxyprogestrone acetate was shown to attenuate the beneficial effects of estrogen on endothelium-dependent vasodilatation 12  (Williams et al.,'1994a) and progression of atherosclerosis (Adams et al., 1997) in coronary arteries of monkeys. Large, randomised and double-blind clinical trials using estrogen alone are essential to elucidate the usefulness of estrogen. However, such a trial could result in exposure of patients to harmful effects of estrogen such as increased incidence of endometrial cancer.  1.4.1. Mechanism of cardioprotection by estrogens Even though evidence is lacking, it is widely believed that estrogen replacement offers cardiac protection. Many mechanisms have been proposed to account for the possible protective effects of estrogens. Estrogens lowered the plasma concentrations of low-density lipoprotein (Barrett-Connor et ah, 1989; Nabulsi et al., 1993), and increased that of high-density lipoprotein (Barrett-Connor et a l , 1989; Hong et al., 1992; Nabulsi et al., 1993). The beneficial effects of estrogen on lipid profile is believed to account for 25%-50% of its cardioprotective effect (Ottosson et al., 1985; Bush et al., 1987).  Other actions of estrogens include antioxidation  (Shwaery et al., 1997) and inhibition of cell proliferation (Subbiah, 1998).  1.4.2. Estrogen receptors The estrogen receptors are a member of intracellular steroid/thyroid/retinoid receptor gene superfamily (Tsai & O'Malley, 1994). These receptors are latent transcription factors that are activated upon interaction with estrogen, which diffuses into cells through plasma membrane. The ligand-actiVated estrogen receptors interact with estrogen receptor response elements at the target D N A , resulting in the regulation of gene transcription as well as protein synthesis. The cellular localisation of these receptors is complex and controversial. Early studies suggested that estrogen receptor was located in cytoplasm, and that after binding to its ligand, translocated into the nucleus (Jensen & DeSombre, 1972). Later works demonstrated that estrogen receptors were also present in the nucleus (King & Greene, 1984; Welshons et al., 1984). However, more recent 13  work has demonstrated that estrogen receptors exist in cytoplasm as well as the nucleus (Freay et al., 1997). Pharmacological and molecular biological evidence have shown that estrogen receptors are present in heart and blood vessels from human and a variety of animals. These receptors were shown to exist in human aorta (Venkov et al., 1996) as well as uterine (Perrot-Applanat et al., 1988), mammary (Karas et al., 1994, 1995) and coronary (Losordo et al., 1994; Kim-Schulze et al., 1996) arteries. They were also found in human saphenous (Karas et al., 1994, 1995) and umbilical (Mikkola et al., .1995; Kim-Schulze et al., 1996; Venkov et al., 1996) veins. These receptors were also demonstrated to exist in baboon aorta (McGill & Sheridan, 1981; Lin et al., 1986), coronary artery (McGill & Sheridan, 1981) and myocardium (Lin & Shain, 1985), dog aorta and inferior vena cava (Horwitz & Horwitz, 1982), bovine aorta (Venkov et al., 1996), rabbit aorta (Colburn & Buonassisi, 1978) and uterine artery (Perrot-Applanat et al., 1988) as well as guinea pig uterine artery (Leiberman et al., 1990). Moreover, they were shown to exist in rat aorta (Nakao et al., 1981; Lin & Shain, 1985; Orimo et al., 1993; Bayard et al., 1995; Knauthe et al., 1996), portal vein (Knauthe et al., 1996), vena cava (Knauthe et al., 1996) as well as cardiac myocyte (Grohe et al., 1997; Stumpf et al., 1977) and mouse aorta (Freay et al., 1997; Rubanyi et al., 1997). Since the cloning of estrogen receptor in the 1980's (Green et al., 1986; Greene et al., 1986), there was a general acceptance that only one estrogen receptor (ERa) existed. More recently, a second subtype of estrogen receptor, ERp, was cloned from human thymus, spleen, ovary and testis (Mbsselman et al., 1996) as well as rat prostate, ovary, brain and pituitary (Kuiper et al., 1996, 1997). The ERp receptor was later shown to exist in rat oviduct, uterus, lung, bladder, testis, seminal vesicle and heart (Saunders et al., 1997). Some tissues such as kidney contain exclusively ERa, whereas others such as uterus, pituitary and epidydymis show a  14  predominance of ERa. Tissues such as ovary and prostate have either equal or greater levels of ERp (Kuiper et al., 1997). The ERp and ER« receptors were shown to have similar affinities for 17p-estradiol. The functions of subtypes of estrogen receptors, and the cellular mechanisms that translate ligand-receptor interactions into responses remain to be defined.  1.4.3. Cardiovascular pharmacology of estrogens Cardiovascular pharmacology of estrogen is influenced by the type of estrogen, duration of treatment (acute versus chronic), gender, species of animal, organ or tissue. 17|3-Estradiol, the most abundant estrogen in humans and animals, has been widely used to investigate the biological effects of estrogens. The following sections will focus mainly on studies involving 17|3-estradiol on cardiovascular tissues, organs or system.  1.4.3.1. In vitro studies 17(3-Estradiol has been shown to cause relaxation of vascular tissues from humans and a variety of animals. It relaxed precontracted rings of coronary (Miigge et al., 1993) and omental (Belfort et al., 1996) arteries from women. It also relaxed precontracted rings of aortae from female rabbits (Ma et al., 1997), coronary arteries from male (Jiang et al., 1991) and female (Jiang et al.,' 1991; Collins et a l , 1994) rabbits, and coronary arteries from male (White et al., 1995), castrated male (Bell et al., 1995; Han et al., 1995) and female (Bell et al., 1995; Han et al., 1995; White.et al., 1995) pigs. Moreover, it relaxed precontracted rings of aortae from male mice (Freay et al., 1997), and male (Ravi et al., 1994; Freay et a l , 1997; Tran et al., 1997) and female (Freay et al.,. 1997; Tran et al., 1997) rats as well as tail arteries from male (Shan et al., 1994) and,female (Lydrup & Nilsson, 1996) rats. 17(3-Estradiol also increased coronary arterial flow in isolated perfused hearts from male (Raddino et al., 1986; Eckstein et al., 1994) and 15  female (Raddino et al., 1986) rats, and decreased perfusion pressure in isolated perfused hearts from female rabbits (Ma et al., 1997). The mechanism of 17p-estradiol-induced vasorelaxation has not been fully investigated. It has been shown that vasorelaxation elicited by 17|3-estradiol is endothelium-dependent (Tran et al., 1997), endothelium-independent (Jiang et al., 1991; M a et al., 1997; Mugge et al., 1993) or 2_j_  due to Ca  antagonistic properties of the drug (Jiang et al., 1991). 17(3-Estradiol caused greater  relaxation in aortic rings from female than those from male rats, indicating a gender-dependency of action (Tran et al., 1997). Application of 17p-estradiol to vascular preparations for a period ranging from 20 min to 24 h can enhance vascular responses to relaxing agents.  17p-Estradiol potentiated the  endothelium-dependent relaxation to carbachol (Paredes-Carbajal et al., 1995), A-23187 (Bell et al., 1995) and acetylcholine (ACh) (Biriko & Majewski, 1998), but did not alter the endotheliumindependent relaxation to nitroglycerin (Bell et al., 1995). The influence of 17p-estradiol on A 23187-induced relaxation was agonist-specific and time-dependent, since it was not elicited by 17a-estradiol, and occurred after 18 h, but not 2 h, of incubation with 17p-estradiol (Bell et al., 1995). Application of 17p-estradiol to vascular preparations for a period ranging from 20 min to 24 h also modifies responses to contractile agents. It reduced maximal responses ( E  max  ) of aortic  rings to noradrenaline (NA) (Tran et al., 1997), phenylephrine (PE) (Ravi et a l , 1994; Thomas et al., 1995; Binko & Majewski, 1998), angiotensin II (Ravi et a l , 1994) and K (Thomas et al., +  1995). It also reduced the E  m a x  to endothelin in coronary arteries from female rabbits (Jiang et  al., 1992), and to adrenaline in perfused tail arteries from male rats (McNeill et al., 1996). The 17P-estradiol-induced attenuation of contractile response to N A might be gender-dependent, since it was more pronounced in aortae from female than those from male rats (Tran et al., 1997), 16  and occurred in perfused tail arteries from female, but not male, rats (McNeill et al., 1996). 17(3Estradiol also reduced vasoconstrictor responses to adrenergic nerve stimulation in tail arteries from both male and female rats,.although the reduction was greater in females (Garcia-Villalon et al., 1996).  . . . . . .  In contrast to these findings, 17p-estradiol potentiated the vasoconstrictor responses to N A , U-46619 (a thromboxane agonist) and K , but did not alter vasoconstriction to nerve +  stimulation in isolated perfused rat mesenteric arterial bed (Vargas et al., 1995). Moreover, incubation of rings'from female rat tail arteries with 17p-estradiol for 3-7 days increased the sensitivity to N A (Lydrup & Nilsson, 1996). 17P-Estradiol also modulates the release of vasoactive substances, and alters the activity of ion channels in vascular preparations. 17p-Estradiol increased the production of constitutive NO synthase in endothelial cells from bovine aorta (Hayashi et al., 1995, 1997) as well as human umbilical vein and aorta (Hishikawa et al., 1995). 17p-Estradiol also increased the synthesis of inducible N O synthase in endothelium-denuded rings from rat aorta (Binko & Majewski, 1998). Moreover, it stimulated prostacyclin (PGI2) release in rat aortic smooth muscle (Chang et al., 1980; Farhat et al., 1996b) and human umbilical vein endothelial (Mikkola et al., 1995) cells. 17p-Estradiol also increased the release of c A M P from rat pulmonary vascular smooth muscle cells (Farhat et al., 1996a), and reduced the release of endothelin (Best et al., 1998), an effect that was shared by estradiol valerate (Akishita et al., 1996) and ethinyl estradiol (Polderman et al., 1993).  17p-Estradiol was also shown to act as an opener of large-conductance Ca - and  voltage-activated K channels (White et al., 1995), and to be a C a +  2+  channel antagonist (Raddino  et al., 1986; Jiang et al., 1991, 1992; Nakajima et a l , 1994; Shan et al., 1994; Collins et al., 1996; Ogata et al., 1996)." •  .••  17  1.4.3.2. Ex vivo studies Ex vivo studies have been performed on vascular tissues from ovariectomised animals that had been treated with different doses of 17|3-estradiol for periods ranging from 3 days to 16 weeks. Ex vivo studies show that 17(3-estradiol may cause vasorelaxation, and modulate vascular responses to relaxing agents. 17p-Estradiol caused coronary vasodilatation in isolated perfused rabbit hearts (Gorodeski et al., 1995).  It restored endothelium-dependent relaxation in the  coronary arteries of hypercholesterolemic swine (Keaney et al., 1994).  It also augmented  endothelium-dependent relaxation in rings from rat aortae (Cheng et al., 1994; Vedernikov et al., 1997) as well as.rabbit femoral (Gisclard et al., 1988), dog coronary (Miller & Vanhoutte, 1991), pig coronary (Keaney et al., 1994) and sheep uterine (Veille et al., 1996) arteries.  However,  17p-estradiol did not alter the endothelium-dependent relaxation to A-23187 in rabbit femoral (Gisclard et al., 1988) and dog coronary (Miller & Vanhoutte, 1991) arteries as well as endothelium-dependent relaxation to A C h in rat aortae (Bolego et al., 1997; Vedernikov et al., 1997), and Msheep'renal, arteries. (Veille et al., 1996).  17p-Estradiol also did not alter  endothelium-independent relaxation in dog (Miller & Vanhoutte, 1991) and pig (Keaney et al., 1994) coronary arteries as well as rat aortae (Bolego et a l , 1997; Vedernikov et al., 1997). 17p-Estradiol was also shown to attenuate vascular responses to contractile agents ex vivo. It attenuated the contractile responses to angiotensin II (Cheng & Gruetter, 1992) and K  +  (Vedernikov et.;al., ,1,997) in rat aortae, and to N A in rabbit femoral arteries (Gisclard et al., 1987). However, it did not alter the contractile responses to PE in rat aortae (Vedernikov et al., 1997), and to N A or PE in rabbit saphenous veins (Gisclard et a l , 1987).  Other estrogens  modulate vascular responses to vasoactive substances as well. Estradiol 17-stearate attenuated the contractile responses to PE in rat female aortae (Paredes-Carbajal et al., 1995), and estradiol  cypionate attenuated contractile responses to U46619 in male and female dog coronary arteries (Karanian & Ramwell, 1996). 17p-Estradiol was also shown to augment contractile responses to vasoactive substances. It augmented contractile responses to N A in rat aortae (Cheng & Gruetter, 1992), rabbit aortae (Miller & Vanhoutte, 1990) and saphenous veins (Rorie & Muldoon, 1979), and increased sensitivity to N A in rat mesenteric arteries (Colucci et al., 1982). It also augmented contractile responses to P E and electrical stimulation in rabbit saphenous veins (Rorie & Muldoon, 1979), and to PGF2« in aortae from female, but not male, rats (Maddox et al., 1987). The 17p-estradiolinduced augmentation of response to N A was not affected by the presence of endothelium in rat aortic rings (Cheng & Gruetter, 1992), but was abolished by removal of the endothelium in rabbit aortic rings (Miller & Vanhoutte, 1990). Opposing results might be due to variations in the species of animal and/or doses of 17p-estradiol. Cheng and Gruetter (1992) implanted rats with s.c. pellets containing 5 mg 17P-estradiol, whereas Miller and Vanhoutte (1990) implanted dogs with pellets containing 100 mg 17P-estradiol.  However, the duration through which  estradiol was released from the pellets was unclear. 17P-Estradiol attenuated (Wellman et al., 1996; Huang et al., 1997a) as well as did not alter (Skarsgard ei. a l , 1997) the pressure-induced myogenic tone in rat vascular preparation ex vivo. Controversial results might be due to differences in the type of blood vessel used and/or protocol of 17p-estradiol administration. Huang and colleagues (1997a) used gracilis muscle arterioles from rats that had received s.c. injections of 17p-estradiol (50 pg/kg) in sesame oil every 48 h for 3 weeks. Skarsgard and colleagues (1997) used second-order middle cerebral arteries from rats that had been treated for 3 weeks with pellets containing 0.5 mg of 17pestradiol, whereas Wellman and colleagues (1996) used coronary arteries from rats that had been treated for 7 weeks with 60 day-release pellets containing 0.25 mg of 17P-estradiol. 19  Biochemical, functional and molecular biological evidence suggest that chronic treatment with 17P-estradiol increases the release of NO and P G I 2 in cardiovascular tissues ex vivo. 17pEstradiol augmented the myogenic responses to L - N A M E in rat middle cerebral arteries (Skarsgard et al., 1997) as well as contractile responses to PE in the presence of L - N A M E (Rahimian et al., 1997) and contractile responses to N -methyl-L-arginine acetate (Bolego et al., 1997) in rat aortae. It also augmented relaxation response to superoxide dismutase in sheep uterine arteries (Veille et al., 1996) and rat aortae (Bolego et al., 1997). These studies indicate that 17p-estradiol augmented the activity of basal N O . Moreover, 17p-estradiol increased the mRNA and activity of NO synthase in hearts and uterine arteries from female guinea pigs after 5 days of treatment. A similar response occurred in hearts from male guinea pig after 10 days of treatment (Weineret al., 1994), It also increased the activity N O synthase in uterine and renal arteries from sheep (Veille et al., 1996), preserved the synthesis of N O in gracilis muscle arterioles from female hypertensive rats (Huang et al., 1997b), and increased the release of P G I 2 in aortae from female rats (Bolego et al., 1997).  1.4.3.3. Human and animal in situ and in vivo studies 1.4.3.3.1. In situ studies Short-term (> 20 min) close arterial infusions of estrogen caused regional vasodilatation. 17P-Estradiol, administered at pharmacological doses, increased coronary arterial flow in dogs, which was attenuated by L - N A M E and iberiotoxin, indicating the involvement of NO and Ca activated K channels, respectively (Node et al., 1997b). 17P-Estradiol also increased the cross+  sectional area and flow of coronary arteries in dogs representing vasodilatation of epicardial and resistance arteries, respectively (Sudhir et al., 1995). balloon-induced  endothelium . denudation,  The vasodilatation was unaffected by  pretreatment  with  L-NAME,  indomethacin,  20  propranolol or ICI 182,780 suggesting no role for endothelium-derived N O , prostanoids, (3adrenoceptors, or traditional intracellular estrogen receptors, respectively. The 17i3-estradiolinduced vasodilatation in epicardial, but not resistance, coronary arteries was attenuated by glibenclamide, which suggests the involvement of ATP-sensitive K channels (Sudhir et al., +  1995). I.v.ihfusioh of estrogen into regional vascular beds for a short period (< 20 min) also modulates vascular responses to vasoactive substances. 17p-Estradiol, infused at physiological doses, converted' the ACh-induced constriction of the  epicardial coronary arteries in  atherosclerotic postmenopausal patients to dilatation (Gilligan et al., 1994b; Collins et al., 1995). The reversal of ACh-induced constriction did not occur in male patients indicating genderdependency of the effects of 17p-estradiol (Collins et al., 1995). 17P-Estradiol also potentiated the vasodilator response to A C h in the coronary arteries of postmenopausal women, which was inhibited by L - N A M E (Guetta et al., 1997). Moreover, it potentiated vasodilator responses to ACh and sodium nitroprusside (SNP) in the brachial (Gilligan et al., 1994a) and forearm (Gilligan et al., 1995) arteries of postmenopausal women with risk factors for atherosclerosis (Gilligan.et al., l-994a, 1995) and women without coronary artery disease (Gilligan et al., 1995).  1.4.3.3.2. Acute studies  .  Acute systemic administration of estrogen was shown to alter systemic cardiovascular variables in humans.  Sublingual administration of 17p-estradiol (1 mg) to postmenopausal  women increased CO, but did not alter M A P or HR at 20 min after administration (Leonardo et al., 1997).  I.v. administration of conjugated equine estrogen (Premarin, 1.25 mg) in  postmenopausal women with normal or atheromatous coronary arteries increased CO, but did not change M A P at 15 min after administration (Sbarouni et al., 1997). It also decreased HR, and  21  did not alter pulmonary artery, pulmonary capillary wedge and right atrial pressures as well as systemic and pulmonary vascular resistances (Sbarouni et al., 1997). Variable effects on HR might be due to differences in the dose and/or preparation of estrogen. Estrogen, administered acutely, was also shown to modulate regional vascular resistance and flow in humans. Sublingual administration of 17(3-estradiol (Volterrani et al., 1995) and i.v. bolus injection of ethinyl estradiol (Reis et al., 1994) caused vasodilatation in the forearm (Volterrani et al., 1995) and coronary (Reis et al., 1994) arteries of postmenopausal women. However, i.v. injection of a high dose (10 mg) of conjugated equine estrogen failed to change forearm vascular resistance (Tagawa et al., 1997). There is a lack of information on the acute effects of estrogen on venous function in humans. The only published study demonstrated that i.v. injection of 17p-estradiol increased venous distensibility in the forearm of healthy premenopausal women (Goodrich & Wood, 1966). Acute systemic administration of estrogen has been shown to modulate systemic cardiovascular variables, ventricular arrhythmias and infarct size in animals. A physiological dose of np-estradiol (1 ug/kg'i.v. bolus) decreased systemic vascular resistance, increased CO and HR, but did not alter M A P (Naden & Rosenfeld, 1985a, 1985b; Magness & Rosenfeld, 1989; Lang et al., 1997) in conscious ovariectomised ewes. However, i.v. injection of a high dose (100 ug/kg) of 17p-estradiol decreased M A P in male rats 4 h later (Shan et al., 1994). 17PEstradiol, given prior to myocardial ischaemia, reduced ventricular arrhythmias in dogs (Node et al., 1997a) and myocardial infarct size in rabbits (Hale et al., 1996) and dogs (Node et al., 1997a).  Administration of conjugated equine estrogen prior to ischaemia also reduced  ventricular arrhythmias during ischaemia and reperfusion in dogs (McHugh et al., 1995). Acute effects of estrogen on body venous tone and cardiac contractility have yet to be studied.  22  Acute systemic administration of estrogen modulated regional vascular resistance in animals. 17P-Estradiol, at a physiological dose (1 pg/kg, i.v.), increased coronary (Lang et al., 1997) and uterine (Killam et al., 1973; Rosenfeld et a l , 1974, 1996; Naden & Rosenfeld, 1985a, 1985b; van Buren et al., 1992) blood flows in conscious ovariectomised (Killam et al., 1973; Naden & Rosenfeld, 1985a, 1985b; van Buren et al., 1992; Rosenfeld et a l , 1996; Lang et al., 1997) and pregnant (Rosenfeld et al.,. 1974, 1996) sheep. The increase of blood flow might be mediated by.endothelium-derived NO, since it was inhibited by L - N A M E in the coronary (Lang et al., 1997) and uterine (van Buren et al., 1992; Rosenfeld et al., 1996) arteries. Acute systemic administration of estrogen to humans or animals also modulated regional responses to vasoconstrictor and vasodilator agents. 17p-Estradiol augmented the vasodilator response to A C h and substance. P in the forearms of postmenopausal women (Tagawa et al., 1997). It also, attenuated the systemic vasoconstrictor responses to angiotensin II (Rosenfeld & Jackson, 1984; Naden & Rosenfeld, 1985a) and N A (Naden & Rosenfeld, 1985a) in conscious ovariectomised ewes. The modulating action of estrogen on vascular responses to vasodilating agents was not limited to 17P-estradiol. Ethinyl estradiol also attenuated the vasoconstrictor response to A C h in the coronary arteries of postmenopausal women (Reis et al., 1994), and converted  ACh-induced  coronary  vasoconstriction  to  vasodilatation  in  anaesthetised  ovariectomised monkeys with atherosclerotic coronary arteries (Williams et al., 1992). Vasodilator response to nitroglycerin was, however, not affected (Williams et al., 1992).  1.4.3.3.3. Chronic studies. Long-term treatment with estrogen has been shown to modulate cardiovascular variables, and systemic as well as regional vascular responses to vasoconstrictor and vasodilator agents in humans and animals.  23  Treatment of postmenopausal women with patches, that released 17|3-estradiol (0.05 mg/day) for 12 weeks, increased CO, decreased M A P , and did not change HR or systemic vascular resistance (Alfie et al., 1997). Treatment with oral 17(3-estradiol (2 mg/day) for a similar period, however, did not alter CO, M A P or HR (Snabe et al., 1997). Supplementation with estradiol valerate in perimenopausal women (2 mg daily) reduced M A P , but did not alter forearm blood flow (Sudhir et al., 1996). Chronic treatment of animals with estrogen also modulates cardiovascular variables, and decreases the incidence of ventricular arrhythmias. 17p-Estradiol (Ueda et al., 1986; Magness et al., 1993), but not.estradiol cypionate (Williams et al., 1994b), decreased M A P , and increased HR. 17p-Estradiol (Magness et al., 1993) as well as estradiol cypionate (Williams et al., 1994b) increased CO, and reduced systemic vascular resistance in ovariectomised sheep (Magness et al., 1993) and monkeys (Williams et al., 1994b). Treatment of ovariectomised rats with s.c. pellets containing 0.5 mg of 17P-estradiol for 3-4 weeks did not alter M A P , HR, CO or TPR (Toba et al., 1991). In contrast, treatment with pellets containing 0.25 mg 17P-estradiol for 4-6 weeks decreased M A P in ovariectomised rats (Wang et al., 1995a). Treatment of ovary-intact guinea pigs with 17p-estradiol increased CO (Hart et al., 1985), MCFP, unstressed blood volume and total vascular capacitance, defined as the volume held in the vasculature at M C F P of 6 mmHg (Davis et al., 1989), but did not alter M A P , H R (Hart et al., 1985; Davis et al., 1989) or total vascular compliance (Davis et al., 1989). In the study of Hart and colleagues (1985), animals were treated with silastic capsules containing 17p-estradiol for 28 days to give a plasma concentrations of 188 ± 73 pg/ml. The study by Davis and colleagues (1989) started with s.c. implantation'of pellets containing 7.5 mg 17p-estradiol, which was later reduced to pellets containing 1 mg 17p-estradiol at an unspecified time during the course of study (60 days). :  24  Similar to acute administrations, long term use of 17|3-estradiol decreased the incidence of ventricular arrhythmias during myocardial ischaemia and reperfusion in dogs (Kim et al., 1996). Long-term treatment with estrogen also increased total blood and total plasma volumes (Hart et al., 1985; Ueda et al., 1986; Davis et al., 1989; Magness et al., 1993). However, it decreased (Magness et al., 1993), or did not alter (Hart et al., 1985; Ueda et al., 1986; Davis et al., 1989; Tpba et al., 1991) haematocrit. It is not clear whether or not the increase in total blood volume was the result of increased total plasma volume alone or total plasma volume along with red cell volume. Estrogen replacement in women has been shown to alter plasma concentrations of vasoactive substances.  Replacement therapy with 17P-estradiol alone (Best et al., 1998) or  concurrent with norethisterone acetate (Rosselli et al., 1995) increased plasma concentrations of N O (measured as nitrite/nitrate). Estrogen supplementation for 8 weeks (estradiol valerate, 2 mg daily) also augmented vasoconstrictor response to N -monomethyl-L-arginine in the forearm of G  perimenopausal women, which may suggest increased basal release of N O (Sudhir et al., 1996). Replacement therapy with 17p-estradiol also decreased the plasma concentrations of endothelin1 (Best e t a l , 1998). Chronic treatment with estrogen may also modulate vascular responses to vasodilator substances.  17p-Estradiol reversed paradoxical constriction to A C h in the atherosclerotic  coronary arteries of cynomolgus monkeys (Williams et al., 1990), and attenuated the systemic vasodilator response to• SNP in premenopausal monkeys (Williams et al., 1994b). However, 17p-estradiol (Gilligan et al., 1995) as well as estradiol valerate (Sudhir et al., 1996) did not change the vasodilator response to A C h or SNP in the forearm artery of postmenopausal and perimenopausal women, respectively.  25  Long-term treatment of animals with estrogen can modulate the development of hypertension as well as systemic vascular responses to vasoconstrictor substances. 17(3-Estradiol attenuated the course of deoxycorticosterone-salt-induced hypertension in intact male and gonadectomised female rats (Crofton & Share, 1997). It also reduced (Toba et al., 1991) or did not change (Conard et al., 1994; Wang et al., 1995a) the systemic pressor response to arginine vasopressin in ovariectomised rats. Moreover, 17P-estradiol attenuated the systemic pressor response to angiotensin II in ovariectomised sheep (Magness et al., 1993), but did not change it in ovariectomised rats (Novak & Kaufman, 1991; Conard et al., 1994). It also did not change the pressor response to N A in ovariectomised rats (Conard et al., 1994), and constrictor response to N A in superficial hand veins of healthy men (Jilma et al., 1994), whereas it enhanced the contractile response to N A in rat mesenteric arteries in vivo (Altura, 1974).  17p-Estradiol  attenuated the vasoconstrictor responses to PE in premenopausal monkeys (Williams et al., 1994b), but enhanced it in rat mesenteric arteries in vivo (Altura, 1974). The existence of estrogen receptors in the cardiovascular system is implicative of direct cardiovascular actions. Classically, estrogen exerts its effects on target tissues by gene induction and stimulation of protein synthesis in a process that requires > 8 h (Clark et al., 1992). However, time courses of acute (< 20 min) effects of estrogens are too short to be attributed to gene induction and de novo protein synthesis.  Moreover, characteristics and magnitude of  estrogen responses may be affected by. gender. The above-mentioned in vivo studies show that estrogen has variable actions on individual cardiovascular variables that may be due to differences in animal models, types and doses of estrogen as well as duration and methods of treatment.  26  1.5. Heart failure Heart failure is a general term used to describe a pathophysiological state in which abnormal cardiac function results in failure of the ventricles to pump blood at a rate commensurate with the metabolic needs of body organs. It is recognised clinically by a diversity of symptoms and signs produced by circulatory and neurohormonal responses to cardiac dysfunction (Poole-Wilson, 1989). The main function of the heart is to transfer blood coming from the low-pressure venous system to the higher-pressure arterial system. Impaired cardiac function results in the failure to empty the venous reservoir leading to reduced delivery of blood into the arterial circulation. Heart failure may be presented as an acute or a chronic state. Generally, the clinical manifestations of acute and chronic heart failure are determined by a difference in the capacity of pulmonary and systemic circulation, and how rapidly the failure develops. Since capacity and pressure of the pulmonary circulation is significantly lower than those of the systemic circulation, a small imbalance in output between the left and right sides of the heart can cause acute pulmonary congestion.  On the other hand, a larger and more  distensible systemic circulation is.able to adjust to a greater imbalance for a more sustained period of time.  Therefore, the clinical picture of chronic heart failure generally reflects  symptoms of systemic congestion, whereas that of acute heart failure reflects symptoms of pulmonary congestion. Experimentation on humans,is limited by ethical considerations.  Therefore, animal  models of human diseases are frequently used to examine the aetiology, mechanism, development, outcome and treatment of human diseases such as heart failure.  Although  experimental models of heart failure can provide answers for questions not easily obtained in humans, no .single model can produce all of the clinical symptoms of heart failure. Several techniques have been used to induce acute or chronic heart failure.  27  1.5.1. Acute heart failure Many techniques have been used to induce acute heart failure in a variety of animals. These include aortic constriction (Larochelle & Ogilvie, 1975a, 1975b), occlusion of the left (Vikhert & Sharov, 1976, Pouleur et al., 1980; Bergmann et al., 1985) or right (Abe et al., 1994) coronary artery, and debilitation of the heart by sodium pentobarbital (Maruyama et al., 1988), propranolol (Maruyama et al., 1988; Wong et al., 1993) overdose, or simultaneous propranolol overdose, volume load and coronary artery occlusion (Kirk et al., 1978). Acute heart failure has also been produced by embolisation of the coronary artery with mercury (Franciosa et al., 1978; Leddy et al., 1983) or plastic. microspheres ( Smiseth & Mjos, 1982; Scholkens et al., 1986; Sweet et al., 1986; Bohn et al., 1995; Wang et al., 1995b) in dogs, embolisation of the coronary artery with plastic microspheres in rats (Gorodetskaya et al., 1990), and combined coronary embolisation with microspheres and i.v. infusion of an ai-adrenoceptor agonist (methoxamine) in dogs (Nakazawa et al.-, 1993).. More recently, acute heart failure was induced by rapid right atrial pacing in pigs (Rutlen et al., 1992), rapid right ventricular pacing in dogs (Walsh et al., 1988; Redfield et al., 1989), and combined rapid right ventricular pacing and volume loading in dogs (Ogilvie & Zborowska-Sluis,. 1992a; Nekooeian et al., 1995). In acute heart failure induced by occlusion of the left coronary artery, CO and ventricular contractility were.lower,, and left ventricular end-diastolic pressure (LVEDP), central venous pressure (CVP) and TPR were higher than pre-occlusion values (Bergmann et al., 1985). Ligation of the right coronary artery, on the other hand, decreased CO, and increased right ventricular end-diastolic pressure, without affecting M A P , pulmonary arterial pressure or L V E D P (Abe et al., 1994). Acute heart failure induced in dogs by a high dose of propranolol alone or simultaneous with a high dose of pentobarbitone, was associated with decreases in left ventricular contractility, M A P , HR.and CO, an increase in L V E D P , and no change in TPR (Maruyama et al., 1988; Wong 28  et al., 1993). Propranolol overdose combined with volume load and occlusion of the left anterior descending coronary artery in dogs increased L V E D P , and decreased M A P and left ventricular contractility (Kirk et al., 1978). Acute heart failure induced by embolisation of the coronary artery with mercury was associated with decreases in M A P , CO and left ventricular contractility, and an increase in L V E D P (Franciosa et al., 1978; Leddy et al., 1983). TPR was, however, increased (Franciosa et al., 1978) or remained unchanged (Leddy et a l , 1983) in this model of acute heart failure. Animals with acute heart failure induced by embolisation of the coronary artery with microspheres had, decreased cardiac contractility, CO and M A P as well as increased L V E D P , pulmonary arterial and right atrial pressures (Smiseth & M J 0 s , 1982; Reikeras et al., 1985, 1986; Scholkens et al., 1986; Sweet et al., 1986; Gorodetskaya et al., 1990; Bohn et al., 1995). Simultaneous embolisation of the coronary artery with microspheres and infusion of an ot]adrenoceptor agonist led to increases in C V P , TPR and L V E D P , a decrease in left ventricular contractility, and no change in M A P (Nakazawa et al., 1993). Animals .with acute, heart failure induced by atrial pacing, ventricular pacing, or combined ventricular pacing and volume loading had reduced M A P and CO, and increased right atrial and pulmonary capillary wedge pressures (Walsh et al., 1988; Redfield et al., 1989; Ogilvie & Zborowska-Sluis, 1992a; Rutlen et al., 1992; Nekooeian et al., 1995). In acute heart failure induced by combined ventricular pacing and volume loading, there were increases in MCFP, venous resistance (Ry) and central blood volume, which comprises volume in the pulmonary circulation and heart (Ogilvie & Zborowska-Sluis, 1992a; Nekooeian et al., 1995).  1.5.2. Chronic heart failure Experimental chronic heart failure has been produced in rat by aortic constriction (Dhalla et al., 1978), aorta-to-vena cava shunt (Flaim et al., 1979), combined arteriovenous shunt and 29  partial occlusion of a renal artery (Noma et al., 1988), coronary artery ligation (Raya et al.,1989; Teerlink et al., 1994a, 1994b; Sanbe et al., 1995), coronary artery embolisation with microspheres (Medvedev et al., 1993), and induction of cardiomyopathy by adriamycin (Mettler et al., 1977). Chronic heart failure has also been produced by embolisation of coronary artery with microspheres (Sabbah et al., 1991) in dogs, and rapid ventricular pacing in sheep (Fitzpatrick et al., 1989), pigs (Chow et al., 1990) and dogs (Armstrong et al., 1986; Ogilvie & Zborowska-Sluis, 19.92b). Rapid right ventricular pacing in dogs and coronary artery ligation in rats are the two most used models of chronic heart failure. Pacing-induced chronic heart failure was shown to develop after ventricular pacing for three to six weeks, two weeks and one week in dogs (Ogilvie & Zborowska-Sluis, 1992b), sheep (Fitzpatrick et al., 1989) and pigs (Chow et al., 1990), respectively:  In rats, chronic heart failure developed three weeks after the ligation of left  coronary artery (Gay et al., 1986). Pacing-induced chronic heart failure was associated  with decreases in cardiac  contractility, M A P and CO as well as increases in pulmonary artery, pulmonary capillary wedge, right atrial and right ventricular end-diastolic pressures, TPR, pulmonary arterial resistance and L V E D P (Armstrong et al., 1986; Fitzpatrick et al., 1989; Chow et al., 1990; Belloni et al., 1992; Ogilvie & Zborowska-Sluis, 1992b). However, in an ovine model of pacing-induced chronic heart failure, the increase of TPR did not reach statistical significance (Fitzpatrick et al., 1989). Pacing-induced chronic heart failure was also associated with increases in MCFP, R and central v  blood volume, and decreases in total vascular compliance, total vascular capacitance and unstressed vascular volume (Ogilvie & Zborowska-Sluis, 1992b). Due to the cost and sophistication involved in experimentation on dogs, coronary artery ligation in rats remains the most widely used model of heart failure. Coronary artery ligation in rats is associated with decreases in M A P , CO, cardiac contractility (Ontkean et al., 1991; Sanbe 30  et al., 1995) as well as total vascular capacitance and compliance (Gay et al., 1986; Raya et al., 1989), and increases in L V E D P (Raya et al., 1989; Ontkean et al., 1991) and M C F P (Gay et al., 1986; Raya et al., 1989). Total blood and total plasma volumes were reported to increase (Raya et al., 1989) or remain unchanged (Gay et al., 1986), but haematocrit was unchanged (Gay et al., 1986; Raya et al., 1989) in this model of chronic heart failure.  1.5.3. Neurohormonal responses in heart failure There have been fewer studies on neurohormonal responses in acute relative to chronic heart failure. Acute, heart failure induced by rapid right ventricular pacing was associated with increases in plasma concentrations of atrial natriuretic peptide and N A (Lee et al., 1989; Redfield et al., 1989; Moe et al., 1991), a decrease in plasma vasopressin (Lee et al., 1989), and no change in plasma renin activity or plasma aldosterone (Lee et al., 1989). Acute heart failure induced by combined rapid right ventricular pacing and volume loading was also associated with increases in plasma concentrations of atrial natriuretic peptide and N A (Nekooeian et al., 1995). Pacing-induced  chronic  heart  failure  was  accompanied  by  increased  plasma  concentrations of N A (Riegger & Liebau, 1982; Riegger et al., 1984; Armstrong et al., 1986; Moe et al., 1991), atrial natriuretic peptide (Riegger et al, 1989; Travill et al., 1992) and vasopressin (Riegger & Liebau, 1982; Travill et al., 1992) as well as increased elevated plasma renin activity (Riegger & Liebau, 1982; Travill et al., 1992).  Plasma renin activity was,  however, suppressed in the early phase of pacing-induced heart failure, a condition that resembles mild heart failure (Riegger, 1991a). The most likely cause of this suppression was the augmented secretion of atrial natriuretic peptide (Riegger, 1991a, 1991b). Plasma concentrations of endothelin is also increased in chronic heart failure induced by pacing (Margulis et al., 1990; Cavero et al., 1990) or constriction of thoracic inferior vena cava (Underwood et al., 1992).  31  Chronic heart failure induced by ligation of the coronary artery in rats was associated with increased plasma renin activity (Schunkert et al., 1993) as well as increased plasma concentrations of endothelin (Teerlink et al., 1994a; Sakai et al., 1996), N A (Hodsman et al., 1988; Deck et al., 1992), angiotensin II (Schunkert et al., 1993), vasopressin (Deck et al., 1992) and atrial natriuretic peptide (Hodsman et al., 1988; Tsunoda et al., 1986). In another study, plasma renin activity and plasrna concentration of vasopressin were, however, unchanged (Hodsman etal., 1988). There is disagreement as to whether or not endothelium-dependent relaxation and basal release of N O is impaired in vessels from rats with chronic heart failure induced by coronary artery ligation. Endothelium-dependent relaxation was not impaired in rings from aortae (Baggia et al., 1997) or small mesenteric arteries (Baggia et al., 1997). However, endothelium-dependent relaxation was impaired in rings from aortae (Ontkean et al., 1991; Teerlink et al., 1993; Nasa et al., 1996;- Toyoshima et al., 1998) and pulmonary arteries (Ontkean et al., 1991; Nasa et al., 1996; Baggia et al., 1997). Basal release of N O decreased in rings from aortae (Nasa et al., 1996) and pulmonary arteries (Ontkean et al., 1991; Baggia et al., 1997), but preserved in rings from aortae (Ontkean et al., 1991; Baggia et al., 1997) and mesenteric arteries (Baggia et al., 1997) . Moreover, basal release of cyclic guanosine monophosphate (cGMP) decreased in aortic rings (Nasa et al., 1996). The endothelium-independent relaxation was not altered in rings from aortae (Ontkean et al., 1991; Baggia et al., 1997), pulmonary arteries (Ontkean et a l , 1991; Baggia et al., 1997) and small mesenteric arteries (Baggia et al., 1997).  1.5.4. Assessment of infarct size , Both in vivo and in vitro methods have been used to quantify the size of myocardial infarct in experimental heart failure. Infarct size has been determined in vivo using computed tomography (Prigent et al., 1991), nuclear magnetic resonance imaging technique (Friedrich et 32  al., 1995) and echocardiography (Burrell et al., 1996). Infarct size has been determined in vitro using masson's trichrome dye (Pfeffer et al., 1979) triphenyltetrazolium chloride dye (Vivaldi et al., 1985; Ytrehus et al., 1994), fluorescent microspheres (Greve & Soetersdal, 1991; Ytrehus et al., 1994) as well as autoradiography with technetium-labeled albumin microspheres (Greve 99m  & Soetersdal, 1991). The use of dyes, fluorescence or radioactivity helps delineate the infarcted and viable tissues. Staining with triphenyltetrazolium chloride is the most used method to determine the size of myocardial infarct. This method involves the interaction of triphenyltetrazolium chloride with nicotinamide adenine dinucleotide and dehydrogenase enzyme, which results in the formation of a stain (triphenylformazan) of red brick colour. Therefore, staining of the infarcted area depends on the presence of the enzyme as well as cofactor (Ytrehus et al., 1994). It was shown that 30 min after occlusion of the coronary artery, the infarcted area of only 50% of the hearts remained unstained with triphenyltetrazolium chloride, while after 3 h of coronary occlusion the infarcted area of all hearts were unstained (Vivaldi et al., 1985). This shows that after 30 min of coronary occlusion, staining with triphenyltetrazolium chloride is not a reliable method for the determination of infarct area.  Greve & Soetersdal (1991) compared the size of myocardial  infarcts determined by triphenyltetrazolium chloride staining, autoradiography and fluorescent microspheres at 5. h after coronary artery occlusion, and concluded that triphenyltetrazolium chloride staining provided the best demarcation line between viable and ischaemic myocardium. However, triphenyltetrazolium chloride staining was criticised for overestimating the infarcted area following 2, 4 and 6 h of ischaemia and 2 h of reperfusion (Barnard et al., 1986). Infarct size has also been determined by a rather simpler method that does not require the use of fluorescence, a dye or radioactivity. This method involves making an incision in the left ventricle so that it can be pressed flat (Chien et a l , 1988; Ontkean et al., 1991; Nishikimi et al., 1992; Baggia et al., 1997). The circumferences of the flat left ventricle and the visualised 33  infarcted area, as judged from both the epicardial and endocardial sides, were outlined on a clear plastic sheet. Then, weights of the areas outlined on the plastic sheet representing infarcted and viable tissues were determined, and the area of infarct was expressed as a percentage of the weight of infarcted area to the weight of left ventricular area on the plastic sheet.  1.5.5. Factors influencing infarct size The area affected by the ligation of the coronary artery is presented into two parts namely, infarct area and area at risk. The infarct area constitutes central part of the affected area, and contains cyanotic and necrotic tissues. The area at risk is the hypoperfused and ischaemic tissue located between the infarct area and viable myocardium (Opie, 1980; Reimer, 1980). The progression from ischaemia to infarction depends on the balance between the oxygen supply and demand in the, area at risk. Factors that can affect such a balance can influence infarct size. The degree of coronary collateral circulation influences the extent and severity infarction (Opie, 1980; Lange & Sobel, 1982), since it acts as a secondary source of oxygen and nutrient for the area at risL  In the presence of an extensive collateral circulation, coronary  occlusion may not produce considerable infarct size. Some animals including dogs and guinea pig have well-developed collateral circulation, while others such as rats are almost completely devoid of it (Maxwell et al., 1987). However, 5 weeks of exercise (one h swimming/day, 5 days/week) reduced infarct size in rats at 48 h after coronary artery ligation; the reduction was attributed to exercise-induced increase in myocardial vascularity (McElory et al., 1978). However, a decrease in arterial oxygen content or coronary perfusion pressure, by virtue of reducing oxygen,supply, would.lead to the extension of infarct size (Gillespie & Sobel, 1977; Reimer, 1980). Haemodynamic parameters such as heart rate, cardiac developed systolic tension, cardiac contractility and systemic vascular resistance also influence infarct size (Gillespie & Sobel, 34  1977; Opie, 1980; Reimer, 1980; Lange & Sobel, 1982). A n increase in any of these factors would increase myocardial oxygen demand and may cause the extension of infarct size. In fact, an increase in HR in coronary ligated dogs elicited by pacing, isoproterenol or atropine was associated with 40%, 72% and 40% increases in infarct size, respectively, as indicated from the increases in serum creatine phosphokinase (Shell & Sobel, 1973). Moreover, an increase in afterload by aortic banding in coronary-ligated rats aggravated infarct size at 7 days after the operation (Nolan et al., .1988). Although to a lesser extent, an increase in preload can also increase infarct size by further dilating a failing myocardium and increasing the myocardial wall tension and oxygen demand (Opie, 1980). However, reduction of afterload below a critical level might increase infarct size by reducing coronary perfusion pressure, which would reduce collateral blood supply to the. area at risk (Reimer, 1980). The effects of drugs on infarct size have yet.to be widely studied in rats with chronic ligation of the coronary artery.. It has been shown that hyaluronidase, cobra venom factor, reserpine and corticosteroids such as hydrocortisone and methylprednisolone decreased infarct size in rats at 21 days after coronary ligation (Maclean et al., 1978). The beneficial effects of hyaluronidase were believed to be due to hydrolysis of interstitial glycoproteins, thereby improving nutrient delivery to the ischaemic myocardium and enhancing the washout of its noxious metabolites. It was suggested that cobra venom factor acted by reducing myocardial injury resulting from the release of lysosomal enzymes by infiltrating polymorphonuclear leukocytes as well as reducing capillary permeability and injury to cell membranes following activation of the complement system.  Reserpine was believed to reduced adrenergically  mediated stimulation of myocardial oxygen consumption through the inhibition of myocardial damage of the release of catecholamines from their stores in cardiac adrenergic nerve endings. The effects of corticosteroids were attributed to the stabilization of cell membrane, prevention or delay of release of lysosomal enzymes (Fox et al., 1976; Spath et a l , 1974), stabilization of 35  phagocytic vacuoles in infiltrating inflammatory cells and thereby reducing heterolytic activity (Libby et al., 1973) or inhibition of the generation of prostaglandins and thromboxanes (Goldstein et al., 1977). More recently, the effects of a number of drugs used for the management of heart failure in humans have been investigated on infarct size in rats with permanent ligation of the coronary artery. Captopril,?a well-known angiotensin converting enzyme inhibitor, was shown not to alter (Pfeffer et al., 1985) or to reduce (van Gilst et al., 1994; Buikema et al., 1997) infarct size at 12 and 8 weeks after coronary ligation, respectively. Neither trandolapril (Yamaguchi et al., 1998), atenolol (Shimada et al., 1995) nor losartan (Sladek et al., 1996) reduced infarct size at 3, 8 or 3 weeks respectively after ligation, whereas ibopamine, a dopamine receptor agonist, reduced infarct size 8 weeks after the operation (van Gilst et al., 1994; Buikema et al., 1997)  1.6. Venous system This thesis will examine the effects of CGS 21680 and 17P-estradiol on cardiovascular variables. The main function of the cardiovascular system is to supply the body with oxygen and nutrients through,maintenance of CO at a rate commensurate with metabolic needs.  CO is  controlled by cardiac parameters namely HR and contractility, arterial parameters including arterial resistance (R ) and arterial compliance, venous parameters such as MCFP, venous A  compliance and Ry, and total blood volume (Greenway, 1982). These variables interact with each other within a closed system, thereby regulating CO at a rate that is proportional to the metabolic need of the body. The venous system is the least understood and least investigated part in the circulation, mainly due to technical difficulties involved with in vivo venous studies. Interest in the venous system has grown during the latter half of this century. Folkow and Mellander (1964) reviewed the functions of veins and concluded that veins were as reactive and regulated as any other 36  circulatory components.  Since then, various techniques have been developed to elucidate the  role of the venous system in cardiovascular homeostasis. The main function of the venous system is the regulation of venous return, which is the rate of blood flow from the periphery back to the heart. Venous return is dependent on the pressure gradient from the peripheral vascular bed to the right atrium. If right atrial pressure increases, then the pressure gradient is reduced, and the flow rate decreases (Rothe, 1986). The venous system contains-75% of the total blood volume (Rothe, 1986), the bulk (75%) of which resides in small veins and venules, and the rest resides in large conduit veins (Milnor, 1990; Rothe, 1983a). Venous system acts as a dynamic reservoir from which blood is pumped by the heart. Alterations in venomotor tone, by passive elastic recoil or active venoconstriction, provide rapid means for the compensatory redistribution of blood volume. Passive redistribution of blood results from changes in arterial pressure, flow or blood volume, whereas active redistribution of blood is due, to changes in the veins' smooth muscle activity, which are controlled by the sympathetic nervous system (Rothe, 1986, 1993a). Relative to arteries, veins have much thinner walls, which contain proportionally less smooth muscle, and offer less resistance, to blood flow (Folkow & Neil, 1971). Moreover, they are less affected by metabolic factors (Lewis & Mellander, 1962; Sharpey-Schafer et al., 1965) than by sympathetic activity (Mellander & Lewis, 1963; Tabrizchi & Pang, 1992).  Drugs  influencing venous tone or reflex control of the venous system have profound effects on venous return, and thereby CO (Pang, 1994).  1.6.1. Venous terminology The venous function in vivo is described by M C F P , venous capacitance, unstressed volume, stressed volume, venous compliance and Ry (Rothe et al., 1983; Pang, 1994).  37  1.6.1.1. Mean circulatory filling pressure MCFP was defined as the pressure that would be measured at all points in the circulation if the heart was stopped suddenly and blood was redistributed instantaneously to an equilibrium level throughout the circulation (Guyton, 1955). It is a measure of fullness of the circulation, and provides the best estimate of the upstream pressure at the level of venules (Rothe, 1993a). It is also a critical component in defining the overall vascular capacitance, and provides a uniquely valuable measure of changes in the overall level of venous tone. M C F P is inversely proportional to overall vascular compliance, but proportional to stressed blood volume (Rothe, 1993a; Pang, 1994). In the absence of a change in blood volume, an increase in M C F P represents an increase in body venous tone (Tabrizchi & Pang, 1992).  1.6.1.2. Venous capacitance Capacitance has been defined for a segment of a vessel as well as whole animals. For a segment of a vessel, it is the relationship between contained volume and the distending pressure (Pang, 1994). In whole animals, venous capacitance is the relationship between M C F P and total blood volume (Rothe, 1993). Total vascular capacitance should be differentiated from the total vascular capacity, which is the amount of blood held in vasculature at a specified MCFP (Shoukas & Sagawa, 1973), and is the sum of the unstressed and stressed blood volumes.  1.6.1.3. Stressed and unstressed blood volumes Stressed blood volume, which constitutes the haemodynamically active portion of total blood volume, is the volume that must be removed from the circulation to decrease the existing value of M C F P to zero. In the absence of a change in total blood volume, a reduction of unstressed volume indicates an increase in stressed volume and vice versa. Unstressed blood volume is the haemodynamically inactive portion of total blood volume that fills the circulation. 38  In other words it is the volume of blood that is held in the system at a M C F P of zero (Pang, 1994).  1.6.1.4. Venous compliance Vascular compliance is a general term that describes the amount of change in volume following a change in stress. In practice, compliance of a segment of a vessel is the slope of plot of contained volume versus distending transmural pressure. The venous compliance in vivo is calculated as the slope of plot of changes in total blood volume versus M C F P (Rothe, 1993 a), and is often normalised to the weight of animals to allow a comparison of values between animals of different weights (Shoukas & Sagawa, 1971).  1.6.1.5. Venous resistance Despite being much smaller than R A , venous resistance is an important factor controlling CO, since the venous bed has a very large cross-sectional area and contains as much as 75% of the total blood volume. For the entire body, it is calculated as the ratio of pressure gradient for venous return, which is M C F P - right atrial pressure, to CO (Rothe, 1993; Pang, 1994). A n increase in Ry not only reduces blood flow to the heart, but also increases upstream distending pressure which leads to the accumulation of blood in small venules (Rothe, 1983a). Accumulation of blood favours capillary filtration and transudation (Rothe, 1983b), which coupled with reduced flow, decreases venous return and CO. The opposite occurs with a reduction in R ... Venous resistance is likely most important in the hepatic venous bed, where a v  pressure gradient of 4-10 mmHg exists between portal venous pressure and C V P (Lautt et al., 1986, 1987).,  39  1.6.2. Methods for studying venous function in vivo Information on the role of the venous system in a whole animal with intact cardiovascular reflexes can not be obtained from in vitro studies that use perfused venous beds or venous preparations, which lack neural and hormonal modulating mechanisms. In addition, large veins are the principal venous vasculature used in vitro, and information obtained may not be representative of those of small veins and venules, which are the primary sites controlling venous capacitance, compliance and resistance (Pang, 1994). Moreover, there is not a prototypical vein to give a response representative of the entire venous system.  Indeed, veins from skin,  splanchnic bed, and skeletal muscle have differential range of control and magnitude of response to reflex sympathetic activity (Rothe, 1983a). There are two general techniques to assess venous function in vivo, namely pump-bypass with reservoir and M C F P techniques. Only the latter will be described.  1.6.2.1. Mean circulatory filling pressure technique MCFP is the equilibrium pressure attained if circulation was stopped suddenly, and blood was made to redistribute instantaneously to a constant pressure.  Circulation is stopped by a  number of methods including arrest of the heart or stopping the venous return. Arrest of the heart is usually achieved by electrical fibrillation (Guyton, 1955) or bolus injection of A C h into the right atrium (Lee et al., 1988; Ogilvie & Zborowska-Sluis, 1992a). Cessation of venous return is achieved by inflating a pneumatic cuff around the pulmonary artery (Samar & Coleman, 1978) or inflating a fluid-filled balloon in the right atrium (Yamamoto et al., 1980; Tabrizchi et al., 1993).  After stopping the circulation, arterial pressure decreases and venous pressure  increases to different plateau values. To equilibrate arterial and venous pressures, blood is rapidly transferred from the arterial to venous side using a pump (Drees & Rothe, 1974). However, MCFP has also been determined mathematically as venous plateau pressure + 40  [(arterial plateau pressure - venous plateau pressure)/ratio of venous to arterial compliance] (Yamamoto et al., 1980; Ogilvie & Zborowska-Sluis, 1992a; Tabrizchi et al., 1993). The values of 30 and 60 have been used as venous to arterial compliance ratio for dogs (Ogilvie & Zborowska-Sluis, 1992a, 1992b) and rats (Yamamoto et al., 1980), respectively.  1.6.3. Measurement of M C F P in rats Yamamoto and colleagues (1980) developed a simple technique for quantifying M C F P in rats. In this method, a water-filled latex balloon-tipped catheter was placed in the right atrium via the right external jugular vein. Circulatory arrest was achieved by inflating the balloon with 0.3 ml of water.  M C F P was measured within 4-5 sec of circulatory arrest before reflex  sympathetic venoconstriction took place. Reproducible M C F P readings could be made as often as 10 times at 10 min intervals. The potential disadvantage of Yamamoto's method is the possible interference of circulation by the implanted atrial balloon, and beating of the heart during circulatory arrest, which displaces, some blood from the pulmonary vascular bed into the systemic circulation (Rothe, 1993). However, Yamamoto and colleagues (1980) reported no significant changes in M A P , CVP, HR or CO compared to the corresponding readings in rats whose M C F P readings were obtained during cardiac arrest. They also reported a lag. of 11 sec between balloon inflation and the onset of venoconstriction in rats. In this thesis the method of Yamamoto et al. (1980) was used for the measurement of MCFP.  1.6.4. Limitations of M C F P technique Although the M C F P technique is perhaps the best method for the assessment of body venous function due to its technical simplicity relative to the pump-bypass reservoir technique (Pang, 1994), it has limitations. By definition, all pressures in the circulation should be equal to 41  M C F P during circulatory arrest; however, there are conditions whereby an equilibrium pressure is not attained.  For example, portal venous pressure was slightly higher than C V P during  circulatory arrest in volume-depleted anaesthetised dogs (Gaddis et al., 1986), in volumedepleted or volume-expanded anaesthetised rats (Cheng & Rankin, 1992), and following i.v. bolus of angiotensin II in anaesthetised rats (Tabrizchi et al., 1993). Moreover, repeated use of A C h to stop the heart in dogs caused pulmonary congestion and atelectasis (Gaddis et al., 1986). M C F P has been widely used to evaluate total body pressure-volume relationship, which requires the measurement of M C F P at different intravascular volumes. However, a change in blood volume alters cardiovascular reflex activity, and causes passive transcapillary fluid shift, which may result in over- or -underestimation of M C F P (Rothe, 1983a; Pang, 1994).  1.7. Objective An important consequence of heart failure is an increase in the venous tone, which is a compensatory mechanism in response to decreased CO. However, the increase in venous tone shifts the circulating blood volume from the peripheral to central circulation, which along with impaired left ventricular systolic function, elevates L V E D P , a measure of preload (Wang et al., 1995b; Francis & Cohn, 1990; Burkoff & Tyberg, 1993). A n increase in L V E D P increases workload of failing heart leading to a further reduction of CO. Vasodilators are widely used.in the management of heart failure (see Patterson & Adams, 1996; Bonarjee & Dickstein, 1996).  They are especially useful in the conditions that heart  failure is associated with increased afterload and/or preload (Haas & Leier, 1994; Braunwald, 1996). Vasodilators directly dilate vascular smooth muscles. The dilatation of arterial resistance vessels reduces R A , thereby diminishes the impedance to cardiac ejection and afterload.  The  decrease of afterload reduces the workload on the failing heart and myocardial oxygen demand as well as improves left ventricular function and CO (Amsterdam et al., 1978). Venodilatation 42  increases venous capacitance thereby shifting blood volume from the central circulation to the peripheral venous system (Packer, 1985; Wang et al., 1995b).  Unloading of the central  circulation reduces ventricular preload, which leads to a decrease in systolic wall tension and oxygen consumption (Sonnenblick & LeJemtal, 1989) as well as an improvement in exercise tolerance (Franciosa et al., 1980). However, they are not useful in isolated diastolic heart failure, and should not be used in heart failure that accompanied with hypotension (Braunwald, 1996).  1.7.1. Effects of CGS 21680 in rats with impaired cardiac function CGS 21680, an adenosine A 2 A receptor agonist, is known to have vasodilator activity in animals (Webb et al., 1991; Tabrizchi, 1997). Due to vasodilating activity, CGS 21680 may play a role in treatment of human heart failure. Prior to investigation of usefulness of CGS 21680 in human heart failure, however, it is necessary to investigate its effects in animal models of human heart failure. Therefore, the objective of first part of this thesis was to investigate the effects of CGS 21680 on haemodynamics.in rat model of heart failure, which is associated with increases in afterload (Ontkean et al., 1991; Sanbe et al., 1995) and preload (Raya et al., 1989; Ontkean et al., 1991). We hypothesized that CGS 21680 would: 1- have arterial as well as venous dilating properties in rats with impaired cardiac function, and 2- increase CO by decreasing R and Ry, A  and increasing HR.  1.7.2. Effects of 17p-estradiol in rats with impaired cardiac function There is a great deal of epidemiological evidence that estrogen replacement therapy in postmenopausal women may have a role in preventing myocardial infarction and cardiac death (Stampfer & Colditz, 1991). These evidence, however, are inconclusive and prone to potential errors, since epidemiological studies can not be controlled so well as the clinical or experimental studies. Therefore, the objective of the second part of this thesis was to investigate the effects of 43  estrogen replacement therapy in rat model of heart failure, where the variables can be better controlled. 17(3-Estradiol, the most abundant naturally-occurring estrogen, has been widely used to investigate the biological effects of estrogens. It was shown to have vasodilator activity in humans (Goodrich & Wood, 1966; Reis et al., 1994; Volterrani et al., 1995; Leonardo et al., 1997) and animals (Rosenfeld et al., 1974, 1996; Naden & Rosenfeld, 1985a, 1985b; Magness & Rosenfeld, 1989; van Buren.et al., 1992; Magness et al., 1993; Williams et al., 1994b). 17(3Estradiol was also shown to increase the release (Weiner et al., 1994; Veille et al., 1996) or activity (Bolego et al., 1997; Rahimian et al., 1997; Skarsgard et al., 1997) of basal N O . Moreover, it was shown to enhance the contraction response to N A , a mixed a-adrenoceptor agonist (Altura, 1974). We hypothesized that 17p-estradiol would; 1- cause arterial as well as venous dilation, 2- increase the'activity of basal NO, and 3- augment the pressor response to N A .  44  2. MATERIALS AND METHODS 2.1. Surgical preparation 2.1.1. Effects of CGS 21680 on haemodynamics in rats with or without impaired cardiac function 2.1.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function Male Sprague-Dawley rats (350-450 g) were anaesthetised with single injections of sodium pentobarbitone (65 mg/kg, i.p.). Polyethylene catheters (PE-50; i.d. 0.58 mm, o.d. 0.965 mm) were inserted into the left and right iliac arteries and veins, right external jugular vein, and left ventricle via the right carotid artery. The venous catheters were used for the administrations of anaesthetic, vehicle and/or drug.  The left and right arterial catheters were used for the  measurement of arterial pressure and withdrawal of a reference blood sample (for the determination of CO), respectively. The left ventricular catheter was used for the injection of radioactively-labelled microspheres for the measurement of CO (See section 2.4). Following the completion of surgery, the rats were stabilised for 20 min before the commencement of experiments:  2.1.1.2. Effects of CGS 21680 on haemodynamics in rats with acute occlusion of the coronary artery  ; : .: Male Sprague-Dawley rats (350-450 g) were anaesthetised with single injections of  thiobutabarbital (Inactin, 100 mg/kg, i.p.). PE-50 catheters were inserted into the left and right iliac arteries and veins, and left ventricle via the right carotid artery. The left venous catheter was advanced into the abdominal inferior vena cava, and used for the measurement of C V P . Moreover, a saline-filled, balloon-tipped catheter was inserted into the right atrium via the right external jugular vein (See section 2.5). The balloon was used to stop the circulation for the 45  measurement of MCFP. A decrease of M A P to 20-25 mmHg within 5-7 sec following inflation of the balloon was taken as an indication of proper placement of the balloon. The rats were then tracheotomised, and ventilated by a small animal ventilator (C.F. Palmer Ltd., U K ) at 60 cycles/min using room air (6-8 ml/cycle). A left thoracotomy was performed at the level of the 4  th  intercostal space.  The heart was exposed and an occluder  (Prolene 6-0 suture) was placed around the left main coronary artery and exteriorised (Johnston et al., 1983). The incision was closed, and the rats were disconnected from the ventilator and allowed to stabilise for 1 h prior to the measurements of cardiovascular variables.  2.1.1.3. Effects of CGS 21680 on haemodynamics in rats with chronic ligation of the coronary artery Male Sprague-Dawley rats (200-235 g) were anaesthetised with halothane (4% in room air for induction and 0.5% for maintenance), and ventilated with a rodent ventilator at 60 cycles/min and 4-6 ml/cycle. A left thoracotomy was performed at the level of the 4 intercostal th  space, and the heart was exposed. The left main coronary artery was ligated at 2-4 mm from its origin using 6-0 Prolene. In the sham-operated rats, the suture was passed around the coronary artery but was not ligated.  Local anaesthetic (2.0 % bupivacaine) and antibiotic powder  (Cicatrin; 250 unit bacitracin and 3300 unit neomycin/g) were applied to the wound, and the chest wall and skin incisions were closed in layers. Afterwards, the rats were recovered from anaesthesia, and housed in single cages under 12 h light/dark cycles with standard rat chow and water ad libitum. Eight weeks later, the rats were anaesthetised with single injections of sodium pentobarbitone (65 mg/kg, i.p.). PE-50 catheters were inserted into the left and right iliac arteries and veins as well as left ventricle via the right carotid artery. A saline-filled balloontipped catheter was also placed in the right atrium via right external jugular vein. The rats were stabilised for 1 h prior to the start of the experiments. 46  2.1.2. Effects of 17(3-estradiol in rats with impaired cardiac function Age-matched (50-60 days) female Sprague-Dawley rats were anaesthetised  with  halothane (4% in room air for induction and 0.5% in room air for maintenance). The rats were implanted s.c. at the back of their neck with 60 days sustained-release pellets containing vehicle or 17p-estradiol (1.5 mg), and ovariectomised through a small midline incision on the skin of the lower back. The skin incision was moved over to the right as well as the left flank areas to allow the resection of both ovaries through small holes. Afterwards, bupivacaine and Cicatrin were applied to the wound, the chest wall and skin incisions were closed in layers, and the rats were recovered from anaesthesia. One additional group of age-matched intact rats was not implanted with pellets, and was used as a control for the measurement of serum concentration of 17(3estradiol. A l l the rats were individually housed in single cages under 12 h light/dark cycles with standard rat chow and water ad libitum. One week later, under halothane anaesthesia (4% in room air for induction and 0.5% in room air for maintenance), the vehicle-treated rats were given either sham-operation (V-S) or ligation of the left main coronary artery (V-CL). The 17p-estradiol-treated rats were subjected to left main coronary artery ligation (E-CL), whereas the intact rats were not given sham-operation or coronary artery ligation. A l l the rats were individually housed as previously described. Seven weeks later,, the. rats were anaesthetised with single injections of sodium pentobarbitone (65 mg/kg, i.p.). Catheters were inserted into the left and right iliac arteries, left and right iliac veins and left ventricle. In some rats, a saline-filled balloon-tipped catheter was also placed in the right atrium for determination of M C F P and Ry. Some rats were also used ex vivo studies whereby, the thoracic aortae, pulmonary arteries and portal veins from V-S, V - C L and E - C L rats were isolated, and dissected free of adhering fat and connective tissues.  A  pulmonary artery ring, a thoracic aortic ring (0.5 cm long) and a longitudinal portal vein strip  47  from each rat were suspended between two stainless steel hooks in separate organ baths for the measurements of isometric tension.  2.2. Instrumentation A l l catheters were filled with heparinised (25 IU/ml) normal saline (0.9% NaCl). The body temperature was maintained at 37°C via a heating pad connected to a thermistemp temperature controller (Model 71, Yellow Spring Instrument Co. Inc., O H , USA).  Arterial  pressure, L V E D P and C V P were recorded with a pressure transducer (Model PD23B, Gould Statham, C A , USA). The rate of rise of left ventricular pressure (+dP/dt) was quantified using an electronic differentiator (Model 7P20C, Grass Instruments Co., M A , USA). HR was counted from the arterial pressure upstroke, or derived from the upstroke of arterial pulse pressure via a tachograph (Model 7P4G, Grass Instruments Co., M A , USA) connected to a polygraph (Model 79D, Grass Instruments Co., M A , USA). CO was measured through injections of radioactivelylabelled microspheres (See section 2.4).  M C F P was measured at 5-7 sec after transiently  stopping the circulation through inflation of the balloon placed in the right atrium. Isometric tension was measured in the aortic rings, pulmonary artery rings and portal vein strips at a resting tension of 1.0, 0.5 and 0.5 g, respectively, using force-displacement transducers (Model FT-03-C, Grass Instrument Company, Quincy, M A , USA) connected to a polygraph (Model 79D, Grass Instruments Co., M A , USA).  The experiments were performed in organ  baths (20 ml) containing, Krebs Henseleit solution (pH 7.4, 37°C, bubbled with 95% 0 and 5% 2  C 0 ) with the following composition (mM): NaCl 118, glucose 11, KC1 4.7, C a C l 2.5, N a H C 0 2  2  25, K H 2 P O 4 , 1.2, M g C l 2 6 H 0 1.2. 2  3  Since the portal vein displayed spontaneous contractile  activity, force signals were integrated electronically (model B7P10, Grass Instruments Co., M A , USA) over 2 min intervals on a separate channel.  48  2.3. Experimental designs and protocols 2.3.1. Effects of CGS 21680 on haemodynamics in rats with or without impaired cardiac function 2.3.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function The selectivity of CGS 21680 was examined in two groups of rats (n = 5 each). The rats were infused for30 min with;l,3-dipropy-8-cyclopentylxanthine (DPCPX, a selective adenosine A i receptor antagonist, 1 ug/kg.min) (Bruns et al., 1987), or dimethyl sulfoxide (DMSO, vehicle, 9.0 ul/kg.min). At 15 min after the start of infusion with D P C P X or DMSO, rats were infused with CGS 21680 (1.0 [ig/kg.min) for 15 min. For the remainder of the study, rats were divided into eight groups (n = 6 each). A timecontrol group received a continuous infusion of normal saline (0.0193 ml/min) for 25 min as well as a second infusion of normal saline (0.037 ml/kg.min) for the last 15 min of the experiment. Three groups received a continuous infusion of normal saline for 25 min and an infusion of a single dose of CGS 21680 (0.1, 0.3 or 1.0 ug/kg.min) for the last 15 min. Two additional groups received continuous infusion of hexamethonium (200 ug/kg.min) for 25 min and further infusions of either normal saline (0.037 ml/kg.min) or CGS 21680 (0.3 u.g/kg.min) for the last 15 min. The last two groups received a continuous infusion of PE (7 p-g/kg.min) for 25 min and further infusions of either normal saline (0.037 ml/min) or CGS 21680 (0.3 lag/kg.min) for the last 15 min. Measurements of haemodynamics were performed twice, once when M A P was stabilised after surgery and once 25 min later, when animals had received infusions of normal saline and/or a drug. ; Afterwards, the rats were sacrificed by a bolus injection of KC1, and hearts were excised for the counting of radioactivity.  49  2.3.1.2. Effects of CGS 21680 on haemodynamics in rats with acute occlusion of the coronary artery Rats were assigned into 5 groups (n = 6 each). Two groups were either sham-operated or coronary artery occluded, and.received an infusion of normal saline (0.018 ml/min). Three additional groups were coronary artery-occluded and given an infusion of a single dose of CGS 21680 (0.1, 0.3 or 1.0 ug/kg.min). Following stabilisation, cardiovascular variables (CO, M A P , R , L V E D P , left ventricular A  +dP/dt, MCFP and Rv) were measured.  The occluder around the coronary artery was then  tightened in four,groups to, occlude the coronary artery (Johnston et al., 1983).  This was  followed by a second measurement of cardiovascular parameters at 90 min after the coronary artery occlusion, after which animals were infused with vehicle or CGS 21680. At 14-15 min into the infusion of normal saline or CGS 21680, a third measurement of cardiovascular variables was performed.  At the end of experiments, rats were sacrificed by sodium  pentobarbitone overdose, the hearts were excised and the occluded zones were quantified (See section 2.6).  2.3.1.3. Effects of CGS 21680 on haemodynamics in rats with chronic ligation of the coronary artery Rats.were assigned.to 6 groups (n = 6 each). Two groups were either sham-operated or coronary artery-ligated, and given normal saline (0.037 ml/kg.min). Three groups were coronary artery-ligated, and received an infusion of CGS 21680 (0.1, 0.3 or 1.0 ug/kg.min). Another group was coronary artery-ligated and given SNP (4 ug/kg.min). Following stabilisation, cardiovascular variables (CO, M A P , R , L V E D P , left ventricular A  +dP/dt, MCFP and Ry) were measured. Afterwards, the rats were infused with vehicle, CGS 21680 or SNP. At 14-15 min into the infusion of vehicle, CGS 21680 or SNP, a second 50  measurement of cardiovascular variables was performed. At the end of experiments, the rats were killed, the hearts and lungs were removed and weighed, and surface areas of myocardial infarcts were determined (See section 2.7).  2.3.2. Effects of 17(3-estradiol in rats with impaired cardiac function 2.3.2.1. Effects of 17p-estradiol on the activity of basal N O in rats with chronic ligation of the coronary artery Three groups of ovariectomised rats (V-S, V - C L and E-CL) and one group of intact rats were used (n = 6 each). A blood sample (0.6 ml) was taken from each rat for the measurement of serum 17p-estradiol.  Afterwards, M A P , CO, HR, R  A  and L V E D P were measured in the  ovariectomised rats. This was followed by the construction of dose-MAP response curves for N A , ACh, SNP and L - N A M E . N A (0.1, 0.3, 0.9 and 1.8 ug/kg), ACh (0.8, 2.8 and 7.2 ug/kg) or SNP (1, 3 and 9 pg/kg) were injected as i.v. boluses at intervals of 1-5 min to allow complete recovery of M A P to the pre-injection levels. L - N A M E (2, 4 and 8 mg/kg) was injected as i.v. boluses at intervals of 10 min with no recovery of M A P . At the end of experiments, rats were sacrificed, and lungs were cleaned of surrounding tissues and weighed. The hearts were excised, ventricles were weighed, and surface areas of myocardial infarcts were quantified.  2.3.2.2. Effects of 17p-estradiol on the activity of basal N O in blood vessels from rats with chronic ligation of the coronary artery Three groups of ovariectomised rats (V-S, V - C L and E-CL) and one group of intact rats were used (n = 6 each). A blood sample (0.6 ml) was taken from each rat for the measurement of serum 17p-estradiol. Afterwards, cardiovascular variables (MAP, CO, HR, L V E D P , R and left A  ventricular +dP/dt) were measured in the ovariectomised rats.  A pulmonary artery ring, a  51  thoracic aortic ring and a longitudinal portal vein strip from each rat were used for the measurement of isometric tension ex vivo. A l l tissues were equilibrated for 1 h with 3 washouts at 20 min intervals., PE (10" M) was added to all the baths. At the plateau phase of constriction to PE, a 5  cumulative concentration-response curve to A C h (10" to 10 M ) was constructed. Afterwards, 9  -4  the tissues were washed and re-equilibrated for 1 h. The vessels were again constricted with PE followed by relaxation with SNP (10~ to 10 9  -4  M). The tissues were again washed, and re-  equijibratedfor 1 h. Finally they were constricted with PE (10" M) and then exposed to L 6  NAME(10" M). 4  2.3.2.3. Effects of 17P-estradiol on M C F P and Ry in rats with chronic ligation of the coronary artery Eight groups of rats (n = 6 each) consisting of two groups of intact rats, and two groups each of the ovariectomised V-S, V - C L and E-CL rats were used. After stabilisation, a blood sample (0.6 ml) was taken from each rat for the measurement of serum 17|3-estradiol. Cardiovascular parameters (MAP, CO, HR, R , L V E D P , MCFP, R and left ventricular +dP/dt) A  v  were then measured in the ovariectomised rats. Afterwards, one group of each of the V-S, V - C L and E-CL was given normal saline (0.018 ml/min), and the second group of these rats were given N A (0.5 u.g/kg.min). At 14-15 min into the infusion of saline or N A , cardiovascular variables were again measured. At the end of the experiments, rats were sacrificed, lungs and ventricles were weighed, and the myocardial infarcts were quantified (See section 2.7).  2.4. Measurement of CO CO was measured using radioactively-labelled microspheres (Pang, 1983). A suspension (25000-30000 in 150 ul) of radioactively-labelled microspheres (15 u M diameter, New England 52  Nuclear) was suspended in ficoll (200 pi), vortexed, and injected into the left ventricle over a period of 10 sec. This was followed by a flush (200 pi) of normal saline over 10 sec. Blood was withdrawn from the right femoral artery for one min (0.35 ml/min) starting at 15 sec before the injection of microspheres using an infusion/withdrawal pump (Harvard pump Model 940, M A , USA). In the study of the effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function (Section 2.3.1.1), C o as well as " S n 57  3  labeled microspheres were used for the measurement of CO. The order of microspheres injections was reversed so that in half of the experiments Co-labeled microspheres and in the 57  113  other half 57  113  Sn-labeled microspheres were used first. In the remaining studies, either  Sn or  Co-labelled microspheres were used. The withdrawn blood, syringes used for injections of  microspheres arid collections of blood as well as test tubes used for holding the microsphere samples were counted for radioactivity at 160 kiloelectronvolt (Kev) with a Searle 1185 gamma counter (Nuclear-Chicago, IL, USA). In the study of the effects of CGS 21680 on coronary arterial conductance (Section 2.3.1.1), the C o counts in the hearts were corrected for spillover 57  from  113  Sn counts.  2.5. Measurement of MCFP MCFP was measured by stopping the circulation through injection of saline into the balloon placed in the right atrium. As circulation stopped, M A P decreased and CVP increased. Since circulatory pressure was not equilibrated through the transfer of blood from the arterial to venous side, MCFP at 5-7 sec after circulatory arrest was mathematically corrected, by taking into account the differential arterial and venous pressures, and the ratio of venous to arterial compliance (See section 2.10).  53  2.6. Measurement of total blood volume Total blood volume was measured using the radioactively-labelled red blood cell technique (Filep, 1997). Briefly, C r (Amersham)-labelled red blood cells (0.3 ml) was injected 5l  as i.v. bolus. Five min later, blood (0.3 ml) was withdrawn from a femoral arterial catheter and counted for radioactivity using a Searle 1185 dual channel automatic gamma counter. Red cell volume (RCV), total blood volume (TBV) and total plasma volume (TPV) were calculated from the following formulae: .  R C V (ml) -  T B V (ml) =  injected radioactivity (cpm) x Hct cpm/ml of withdrawn blood sample x 100  R C V (ml) x 100 Hct  TPV (ml) = T B V (ml) - R C V (ml)  2.7. Assessment of occluded zone The occluded zone was quantified using the method described by Johnston and colleagues (1983). Hearts were perfused via the aortae with normal saline to remove blood in the coronary vessels. This was followed by perfusion with cardiogreen dye (10 mg/ml). The non-perfused region remained pale red, while the perfused tissue appeared green. The weights of the occluded zone and the ventricles were recorded. The occluded zone was calculated as a percentage of non-perfused weight to total ventricular weight.  2.8. Assessment of surface area of infarct A modification of the method of Chien and colleagues (1988) was used to quantify the area of infarct. Briefly, after cutting the atria away, the ventricles were cleaned of blood, and a  54  saline-filled balloon was placed in the left ventricle. The balloon was then inflated and sealed, and the ventricles were placed in 100% formalin. Fixation in formalin helps to preserve size of the heart, and reduces over- or underestimation of the infarct area. After 24 h, in a blind design, the right ventricle was trimmed away, and an incision was made in the left ventricle so that it could be flattened and traced. The circumferences of the left ventricle and area of infarct were outlined on a plastic sheet from both endocardial and epicardial surfaces over a source of light, which sharpens the demarcation of the areas with or without infarct. The area of infarct was calculated as a percentage of left ventricular surface area, estimated by the proportional weights of the areas marked on the plastic sheet. The areas of infarct in the endocardial and epicardial surfaces were averaged.  2.9. Measurement of serum 17p-estradioI Blood samples were allowed to clot for at least 30 min, followed by centrifugation at 10,000 rpm for 10 min to separate the serum, which was stored at -20°C. Serum concentrations of 17p-estradiol were measured using a I-labelled radioimmunoassay kit (ICN Biomedicals, l25  Inc., Costa, Mesa, C A , USA).  The intra-assay and inter-assay coefficients of variation were  4.7% and 9.1%, respectively, and the limit of detection was 10 pg/ml. A l l samples, including the standard curve, were run in duplicate and the average was reported.  2.10. Chemicals CGS 21680 and D P C P X were purchased from Research Biochemicals International (Natick, M A , USA). N A , ACh, L - N A M E , PE and hexamethonium were obtained from Sigma Chemicals Co. (St. Louis, M O , USA). SNP was obtained from Fisher Scientific Co. (NJ, USA). Pellets containing 17P-estradiol or vehicle were obtained from Innovative Research of America  55  (Sarasota, FL, USA). PE, hexamethonium, CGS 21680, N A , A C h and L - N A M E were dissolved in normal saline, and D P C P X was dissolved in DMSO.  2.11. Calculations and statistical analysis M A P was calculated as diastolic arterial pressure plus one third of the arterial pulse pressure. CO, coronary arterial flow, coronary arterial conductance, R A , M C F P and Ry were calculated as follows:  „ ^ , ,, . rate of withdrawal of blood (ml/min) x total injected radioactivity (cpm) CO (ml/min) = cpm in the withdrawn blood Coronary arterial flow (ml/min) =  rate of withdrawal of blood (ml/min) x cpm in the heart cpm in the withdrawn blood  Coronary arterial conductance (ml/mmHg.min)  coronary arterial flow (ml/min) M A P (mmHg)  n , • , ,x M A P (mmHg) R A (mmHg.min/ml) = — CO (ml/min) TT  MCFP  ( m m  H g ) = VPP +  FAPC-m)  -VPPQnmHg) 60  Where F A P (final arterial pressure) and VPP (venous plateau pressure) are taken at 5-7 sec after the circulatory stop, and 60 is the ratio of venous to arterial compliance.  , „ . • MCFP (mmHg)-CVP (mmHg) Rv (mmHg.min/ml) = — — CO (ml/min) w  56  Due to placement of the balloon in the right atrium, it was not possible to measure right atrial pressure. Therefore, CVP, the pressure measured in the abdominal inferior vena cava, was used to calculate Ry. This is legitimate, since mean CVP is nearly identical to mean right atrial pressure (for review see Rothe, 1993). In ex vivo study on blood vessels, relaxation responses to A C h and SNP, and contraction to L - N A M E were calculated as % contractile response to PE. Maximal relaxation response ( E  max  ) and EC50 to A C h and SNP were computed from individual  dose-response curves using GraphPad Prism statistical software (GraphPad Software, version 2.0, San Diego, C A , USA). Mortality, reported as the percentage of total number of animals used in each group, was analysed by a non-parametric test using a fourfold contingency table. The remaining data were reported as mean ± S E M , and analysed by one way analysis of variance (ANOVA).  Where  significant differences were obtained with A N O V A , the source of the difference was located by Duncan's multiple range or Newman-Keuls tests using P < 0.05 as the criterion for statistical significance.  57  3. RESULTS 3.1. Effects of CGS 21680 on haemodynamics in rats with or without impaired cardiac function 3.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function 3.1.1.1. Selectivity of CGS 21680 Baseline M A P and HR of groups receiving vehicle (DMSO) or adenosine A i receptor antagonist (DPCPX) were 111 ± 3 and 110 + 6 mmHg, and 390 ± 15 and 376 ± 8 beats/min, respectively. DMSO or DPCPX did not change M A P or HR significantly. CGS 21680 (1.0 ug/kg.min) reduced M A P in DPCPX and DMSO-treated rats to 65 + 5 and 70 ± 8 mmHg, and increased HR to 435 ± 17 beats/min and 420 ± 16 beats/min, respectively.  3.1.1.2. Effects of CGS 21680 on haemodynamics Baseline values of M A P , HR, R , CO, coronary arterial flow or coronary arterial A  conductance of all groups.of rats are shown in Table 1. These values were not significantly different among rats treated normal saline or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min. They were also not significantly different among rats treated with normal saline, CGS 21680 at 0.3 ug/kg.min, hexamethonium (200 ug/kg.min) or CGS 21680 (0.3 ug/kg.min) in the presence of hexamethonium. Furthermore, these values were not significantly different among rats treated with.normal saline,. CGS. 21680 at 0.3 ug/kg.min, PE (7 ug/kg.min) or CGS 21680 (0.3 ug/kg.min) in the presence of PE. CGS 21680 (0.1, 0.3 and 1.0 ug/kg.min), in a dose-dependent manner, decreased M A P and R , and increased CO, HR as well as coronary arterial flow and A  conductance (Figs. 1-3).  58  Hexamethonium (200 pg/kg.min) reduced M A P , HR and CO, but did not change R A , and coronary arterial flow or conductance (Figs. 4-6). In the presence of hexamethonium, CGS 21680 (0.3 pg/kg.min) reduced M A P and R , increased coronary arterial flow and conductance, A  but did not alter CO or HR (Figs. 4-6). PE (7 pg/kg.min) increased M A P and R , decreased CO and HR, but did not alter A  coronary arterial-flow or conductance (Figs. 7-9).  In the presence of PE, CGS 21680 (0.3  pg/kg.min) reduced M A P and R A , and increased CO, HR as well as coronary arterial flow and conductance (Figs. 7-9).  59  NO  CO <u o CU CU CaU  o  u  cn 'cn l-i _  C  •g  8  £  -  -S  00  ON  o CU  NO  ;g  CN  g  .  g  * -tJ  O n ^ a a  &, C3 3 O  CU C3  _  +1  +1  +1  +1  +1  O  O  CN  o CN  NO ^H  oo  CO  o oo  g  PH  •  a u  g s 'g ob  O  £ O  o T3  — '3 < a a  S 'S3 S -£ 3  CU  O JH  -3 c3 CU  °  C3  CU  o  cd *  OH ^  c3  3  cu  cn  +1  ON  00 ON  CO  CN  o o  +1  +1  +1  o  CN  1—1  CO  CO ©  T—H  CO © ©  ON  +1  © +1  O  NO  r—(  CO  rt  II  «  CO  -a  ro CO  CO  a u  OH  au -a a a  CN  CN  O  in © ©  +1  +1  +1  co  ON ON  o CN  NO ON  CO  CN  o o  *—i  CN ©  +1  +1  +1  +l  +1  00  CN CO  ON 00  CN CO  ^_  in  CN  CO  in p o  >—i  +1  +1  +1  +1  © +1  © © © +1  T—<  CO  o  CO  ON  T—1  ,—1  a u  ON  ^s  ON  CO  CO  , — i  NO  g x  CO  00  u  o  CU  C4_  8 w  © +1 © iri  ©  +1 CO ©  rj  © +1 ON CN ©  CO o  m © ©  CO  CN  "* '—1  NO ©  +1  +1  +1  +1  +1  +1  00  NO 00  m CN  00  CO  co CO ©  CO  in p o  00  +1  +1  +1  +1  ON 00  CN CN  CN  in  < tf  tf  o  o  ©  cn CN  U J! ^  ^ - N  CN © ©  o o  g ° °r CU  •a oo  ©  Tj"  CO  g O £  00 +H  g  +1 CO  +1  00  CO  NO  ©  r-~ o o  OH  CU CU  « s  m|  ©  +1 ©  o CN  o  CU cu  .a cs  "3 s < cn J—i  ©  +1  > 5  ^ u g J3 a ' •§ -2 o  CU ~  ON  +1  CU ' £  \E3  a  S 8  m © ©  +1  ^H  CN  cn  NO  CO  i:> a  ctf  O  ©  ,  o o  ^  -a- g^,  C/3  —  C a •B oo g-B  CU  w  ON  £  U  g  g g  ••  +1  X> O  +1  IT)  o  u T3S «Ue M  CN  <=> co'  ^  5 3  o  CO  =$_  "H" co g B  „ CU c3 M  ^H  © © ©  CN  u  co ob O  oh  •a g  ^  o  o o  -  w  . g CL,  •a o u "S •a a cu  o  a "5  CO  +1 00 .•  CO  © ©  00  ©  +1 ©  CO  o oo  _^ g ^es aa ~c — 3NO cn H g g CN i o-  <  o O  X  tin  o  o u  ©  NO  A 0  00  X  -20  -40  -60  0.2  B  -,  0.0  -0.2  -0.4  -0,6  -0.8  GO  r/3  o  o.  m GO  Fig. 1. Changes (mean ± S E M , n = 6) from the baseline A) mean arterial pressure (MAP) and B) arterial resistance (R ) in rats without impaired cardiac function treated with saline (0.9% NaCl) or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the saline-treated group. A  61  40  A  * 20  O  u  0  on  to •O  •U  CN  oo  O  u  m oo  O U  Fig. 2. Changes (mean ± S E M , n = 6) from the baseline A) cardiac output (CO) and B) heart rate (HR) in rats without impaired cardiac function treated with -saline (0.9% NaCl) or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the saline-treated group.  62  <U  S  13 GO  <-H GO  O  CN  .' . cn  GO  ' G O  O  U  U  :C O  Fig. 3. Changes (mean ± S E M , n = 6) from the baseline A) coronary arterial flow (CF) and B) coronary arterial conductance (CC) in rats without impaired cardiac function treated with saline (0.9% NaCl) or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the saline-treated group. ;  63  A o  -20  -40  -60  0.2  B  -0.8  CN GO  Id </5  O  u  Figure 4. Changes (mean ± S E M , n = 6) from the baseline A) mean arterial pressure (MAP) and B ) arterial resistance (RA) in rats without impaired cardiac function treated with saline (0.9% NaCl), CGS 21680 (CGS2, 0.3 ug/kg.min), hexamethonium (Hxm, 200 ug/kg.min) and CGS2 in the presence of Hxm (CGS2-Hxm; shown as changes from the baseline prior to infusion of Hxm). * Significantly (P < 0.05) different from the saline-treated group. Significantly different from the Hxm-treated group. A  64  25  A  Fig. 5. Changes (mean ± S E M , n = 6) from the baseline A) cardiac output (CO) and B) heart rate (HR) in rats without impaired cardiac function treated with saline (0.9% NaCl), CGS 21680 (CGS2, 0.3 ug/kg.min), hexamethonium (Hxm, 200 ug/kg.min).and CGS2 in the presence of Hxm (CGS2-Hxm; shown as changes from the baseline prior to infusion of Hxm). * Significantly (P < 0.05) different from the saline-treated group.  65  5.0  2.5  u <  0.0  -2.5 1  0.15  -1  0.10  -  B  0.05  u U  0.00  -0.05  Fig. 6. Changes (mean ± S E M , n = 6) from the baseline A) coronary arterial flow (CF) and B) coronary arterial conductance (CC) in rats without impaired cardiac function treated with saline (0.9% NaCl), CGS 21680 (CGS2, 0.3 ug/kg.min), hexamethonium (Hxm, 200 ug/kg.min) and CGS2 in the presence of Hxm (CGS2-Hxm; shown as changes from the baseline prior to infusion of Hxm). * Significantly (P < 0.05) different from the saline-treated group. Significantly different from the Hxm-treated group. A  66  m a.  S O  •  CN GO  ^  - S  GO  ;•  U  CN GO  w  R  PH  U  Fig. 7. Changes (mean ± S E M , n = 6) from the baseline A) mean arterial pressure (MAP) and B) arterial resistance (RA) in rats without impaired cardiac function treated with saline (0.9% NaCl), CGS 21680 (CGS2, 0.3 ug/kg.min), phenylephrine (PE, 7 ug/kg.min) and CGS 21680 in the presence of PE (CGS2-PE; shown as changes from the baseline prior to infusion of PE). * Significantly (P < 0.05) different from the saline-treated group. Significantly different from the PE-treated group. A  67  25 n  o u  A  0  < -25  J  Fig. 8. Changes (mean ± S E M , n = 6) from the baseline A) cardiac output (CO) and B) heart rate (HR) in rats without impaired cardiac function treated with saline (0.9% NaCl), CGS 21680 (CGS2, 0.3 ug/kg.min), phenylephrine (PE, 7 ug/kg.min) and CGS 21680 in the presence of PE (CGS2-PE; shown as changes from the baseline prior to infusion of PE). * Significantly (P < 0.05) different from the saline-treated group. Significantly different from the PE-treated group. A  68  Fig. 9. Changes (mean ± S E M , n = 6) from the baseline A) coronary arterial flow (CF) and B) coronary arterial conductance (CC) in rats without impaired cardiac function treated with saline (0.9% NaCl), CGS 21680 (CGS2, 0.3 ug/kg.min), phenylephrine (PE, 7 ug/kg.min) and CGS 21680 in the presence of PE (CGS2-PE; shown as changes from the baseline prior to infusion of PE). * Significantly (P < 0.05) different from the saline-treated group. Significantly different from the PE-treated group. A  69  3.1.2. Effects of CGS 21680 in rats with acute occlusion of the coronary artery Baseline values of M A P , CO, R , HR, L V E D P , MCFP or R were similar among the A  v  five groups of rats (Table 2). At 90 min after the occlusion of left main coronary artery, the coronary artery-occluded rats had higher L V E D P , MCFP and R , lower M A P , CO and left v  ventricular +dP/dt, but similar R or HR relative to the corresponding values of sham-operated A  rats (Table 3).  There was no mortality in the sham-operated rats, but the coronary artery-  occluded rats had a 50% mortality. There were no occluded zones in the sham-operated rats. The occluded zones were similar among the various groups of coronary artery-occluded groups treated with the vehicle or CGS 21680 at 0.1, 0.3 or 1.0 pg/kg.min (35 ± 2, 36 ± 2, 36 ± 3 and 37 ± 3 %, respectively).  :  Compared, to the coronary artery-occluded rats treated with vehicle, the low (0.1 pg/kg.min) and medium (0.3 pg/kg.min) doses of CGS 21680 did not alter any cardiovascular variables except for R , which was lowered by the medium dose.  The highest dose (1.0  A  pg/kg.min) of CGS 21680 reduced M A P , R , L V E D P , MCFP and R , increased CO and HR, A  v  but did not alter left ventricular +dP/dt (Figs. 10-13).  70  CD  I" <  <  + P-  n  -d  |  CD  r <  >  o o  a  to CT EQ . 9 3 H ON < OO o CD P 3 P O 3 §• '  >  -a  e 3 a  CT  p  CD er  LO O cr  P  i—.  00  i-l  O 4^ O  o o o  Hp d  H-  o  NO LO  4^  NO H© Lo  -3.1  CD 3' CD 3 o  cyo  H© bo  LO 00 L/l  1.17  00  H-  H-  NO  o  L/l H4^  d  OO ON  cc  H-  LO  xj  4^  P 3o O !-! °> CTQ t3 C CD Hi-iD CTQ 53 O 3 5' cr ^ 3 c7 ^5 CD h+5 C D i O o | 11  O  oo  C  O 4^ LO Hp d  o  LO -J  —1  L/l  4^  NO Hp  to xj  i xj Hp bo  LO L/l Hi — '  I—*  1H - 1  o d  xj 4^ H-  i—.  oo  LO HLO  3  O <  4^  LO  L/l K-  -J -J  o d o  o o  o LO ON  o o o  LO  H-  4^  NO H-  O  to 14© bo  LO ON O H4^  H-  o d o  H-  LO i — '  ON  M  n  o  (50  H-  14-  tO  LO L/l  LO  OO O N HLO  ©  o  4^ bo H-  O io  to LO  LO  ON  H-  O  4^ bo H-  O  to  i—>  H-  o  4^  LH-O o o  NO  i to  _ tO  LO L/l o H-  d  3  n  C  O CTQ <*> <' LO ^ 3 5' g |-l C D hH O C^ C/J T3 C D O CD r > - ^ g oo >o 3 <' CD i=l CD oo g i-l 5 ^ O O- CD o 3 I—I i O 3^ CD s? a O CD " • &. o l-l- S2. 3 f j o 13  -o  O N H-  NO LO  LO  L/l  L/l H-  N O HLO  H-  00 to H-  o  w  n o t/5 i  to  O 1  o o  S3  o d o  ON  0.05  LO  ON O  o  -o  ON  to  LO  Hd  to  4^  1.15  O  cT  O ^ 5 3.3 GO g ^ 3 6 — CD P CD £ Q O o- -1 3> 3 ii CD CD 3 to » 3 « 3 2. p o 3 ° ^~ C ^ i-l r* i» o^ p. C -D t« ^ O  xj  cr CD  P-  d  CD o° i-lD C oo I-I C D  p  CD / i r v—; CD  3 P  3 s <» CD C D- p i-l '< 3 3 3 2. X o <D ffq 3 C cr o' 3 | cT 3 ° O ^ w 3i o i-t CD -  I  n o cr  r~4~  CO  00  O 3' 3 5' ^ P  S 2.  B. CD oo  CD  3. ? (JOG.  * Oq'  <  3  + ft ft  o  X  r  >  O O  >  o  O P 3  3  Ho © o 4^  NO o o Hto  to  4^  NO H© to  NO  •1.19  C D <-t C D  o 4^  -2.9  A p o  PR  Hp  H^o  Ho ©  oo H-  O N H-  C/5  4^ 35  p  ||  3  I'  2  O 3"  CD CO  <  OQ  i-i O 3  *T3  o  -J  to 00  —  1  oo H-  H-  H-  © ©  00  o IO  o  *  ?0  1  H-  — i> ©  U) H4^  *  * —» to 4i> ff ©  I—i o  H4i. #  O N 4^ H-  to  o I  *  CD  < CD P  3' CD 3 o  o © o H-  o © o  lO © H-  Hp  to  — 1 H-  bo  *  c/o  4^ © Hp  NO  *  ^3 3^  4^ O Ho ©  to H-  *  —J o H-  to  *  o o o 1  i—'  CD CD » -i CD  o  ON  to  NO  bo  o  (O 00 4^ O H-  © ©  tO  *  o  ON H-  *  *  ^ 1 to  H-  o to *  H-  Hp  ND  -J to  HtO  H*  C/5 R £ < — ^ H O Q "Q CD Hw  o t/3  o  *  o> H-  H-  H-  4^  4^ H#  ON ON H-  *  o n 1  S3  o to *  bo H-  0.09  tO to IO  H-  1.24  o © o  H-  -J  0.8*  o o  bS  H-  1>J  1.32  3  o  3"  OX)  <  0  -10 A  -15 0.2  riHg.m  IT  J  -I  B  0.1 0.0 -o.i -  <  <  -0.2 -0.3 GO  >i GO  > I  o  a  u  CN GO  a  ui o  CO GO  a  u  Fig. 10. Changes (mean ± S E M , n = 6) from the pre-treatment values of A) mean arterial pressure (MAP) and B ) arterial resistance (RA) in rats with acute sham operation treated with vehicle (S-V), and rats with acute occlusion of the coronary artery treated with vehicle (O-V) or CGS 21680 at 0.1, 0.3 or 1.0 pg/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the O-V group.  73  20  10  o u  <  0  -10  40  20 03  0  -20 GO  > i GO  >  6  O U  CN GO  O  u  GO  o u  Fig. 11. Changes (mean ± S E M , n = 6) from the pre-treatment values of A) cardiac output (CO) and B) heart rate (HR) in rats with acute sham operation treated with vehicle (S-V), and rats with acute occlusion of the coronary artery treated with vehicle (O-V) or CGS 21680 at 0.1, 0.3 or 1.0 pg/kg.min (CGS1, CGS2 or CGS3, respectively).* Significantly (P < 0.05) different from the OV group.  74  '1.0  J2P  0.0  PH Q  -1.0  <  -2.0  I ~5b  A  250.0  5  + <  -250.0  -500.0  >• 00  > 6  oo O u  6  CN  m  O U  O U  00  6  00  6  Fig. 12. Changes (mean ± S E M , n = 6) from the pre-treatment values of A) left ventricular enddiastolic pressure (LVEDP) and B) rate of rise of left ventricular pressure (+dP/dt) in rats with acute sham operation treated with vehicle (S-V), and rats with acute occlusion of the coronary artery treated with vehicle (O-V) or CGS 21680 at 0.1, 0.3 or 1.0 pg/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the O-V group.  75  PH  U  -0.5  < -0.04  > oo  >  oo O O  CN  OO  O  cn oo  O  I  O Fig. 13. Changes (mean ± S E M , n = 6) from the pre-treatment values of A) mean circulatory filling pressure (MCFP) and B) venous resistance (Ry) in rats with acute sham operation treated with vehicle (S-V), and rats with acute occlusion of the coronary artery treated with vehicle (OV) or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the O-V group.  76  3.1.3. Effects of CGS 21680 in rats with chronic ligation of the coronary artery There was no mortality or myocardial infarct in the sham-operated group. Ligation of the left main coronary artery caused 50% mortality within 8 weeks after the ligation. A l l six groups of the coronary artery-ligated rats had similar surface areas of myocardial infarct. Body weight or ventricular weight (normalised to body weight) were similar among the sham-operated and coronary artery-ligated groups.  Lung weight (normalised to body weight) in each of the  coronary artery-ligated.groups was, however, higher than that in the sham group (Table 4). Compared to-the sham-operated rats, all six groups of coronary artery ligated-rats had lower baseline M A P , CO and left ventricular +dP/dt, higher L V E D P , M C F P and R , but similar v  R A or HR at 8 weeks following ligation (Table 5). Relative to vehicle treatment, acute treatment of coronary artery-ligated rats with CGS 21680 (0.1 pg/kg.min) did not alter any of the cardiovascular variables (Figs. 14-17). CGS 21680 (0.3 and 1.0 pg/kg.min) reduced M A P , R , L V E D P and R , increased CO and HR, but A  v  did not alter left ventricular +dP/dt (Figs. 14-17).  M C F P was reduced by the high (1.0  pg/kg.min),- but not the medium (0.3 pg/kg.min), dose of CGS 21680 (Fig. 17A). SNP (4.0 pg/kg.min), which caused a similar depressor response to CGS 21680 (0.3 pg/kg.min), decreased R , L V E D P , MCFP and R , increased CO, but did not alter +dP/dt and A  v  HR (Figs. 18-21).' The effects of SNP were similar to those of CGS 21680 except for HR, which was increased by CGS 21680 but not SNP, and MCFP, which was reduced by SNP but not CGS 21680.  77  Table 4. Values (means ± SEM, n = 6) of body weight (BW; g), ventricular weight (VW; g/kg body weight), lung weight (LW; g/kg body weight), and surface area of infarct (IA; % of left ventricular surface area) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V), CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively) or sodium nitroprusside (CL-SNP, 4.0 ug/kg.min).  S-V  CL-V  BW  482 ± 2  473 ± 9  VW  2.8 + 0.1  LW  3.4 ± 0 . 1  IA  —  CL-CGS1  CL-CGS2  CL-CGS3  CL-SNP  501 ± 2  493 + 9  486 + 7  480 + 20  3.1+0.1  3.0 ± 0 . 1  3:3 ± 0 . 2  3.0 ±0.1  3.0 ±0.1  4.6 ±0.2*  4.5 ± 0.2*  5.1 ±0.2*  . 4.5 ± 0 . 1 *  5.0 ± 0 . 3 *  31.7 ± 2 . 0  33.6 ± 2 . 2  34.4 ± 1.8  34.1 ± 1.5  33.2 ± 1.4  * Significantly (P < 0.05) different from the S-V group.  78  <  00  Si  ON  3  1  Xgl  a u  -  13 _>  u  o  JZ  s-  CO  C/5  to u  -  a y  cn  o3 c «  1 ? 1•n. 2  .a §1 ^3  O  CU w tf cn co +1 • r cu Si «-H <U w  o  3  O  cu  cu  Si  /  u  -H NO  0.2*  0.03  0.004 0.064  00  -w  -H  +1  -H  00  NO  CO  NO CO  NO  N O,  *N O  *C O  -H  -H  CO  NO NO  00  O  d  -H  CN  -H O CO  * d  -H  NO  *  d  -H  co  *  *  -H'  -H 00  -H  -H  -H  -H  00  CN  CN  NO  CN OO  NO  CO  00  41  NO CN  -H in ON CO CO  o o d o  * >n CN  in  o o d  -H  -H  o  in  o  0  o  \C  u  ,  0  IT)  CN  -H O  -H  CN  ON  00  d IT) CN  1—i  -H  d  in  -H in  CO  CO  00  d  -H  CO  in  NO  in -H in 00  3 3 3  O s-, 00 CU -3 NO <E cn ^ c0 CN  cn O  a to to cu >  "cu  tf  O  Si  00  > I  CO  a o  3.  o d  ft  4^  in  o  «  Si CU  X!  in  CO  ~5b  % a5 to £ •s a aj s o& § U cu «as SI  H  00  CO  00  Si  cu  3  O U  co  cn cn  ft  O' cO Si  ^ CU  00 K3  CN  # CO  O U  § to ^ .2 a* 3 to cu  in -H  CU  CO  CU •~ 3  CU 3  ro  >  Sf.  *  cn  ft  CO  CO  ft  sS <^u  * <u  -H  0.003*  CN  1  CN CO  3  -H  ft  o  3  f  -H CO  3  3  3 cu SJ >  m  Si  tf £ O  "S?  NO  -H 00 in  *  a  cu t-  I 3  o  101*  u  3  S^ i  — •°  NO  3562  -J  *  0.6*  4-T  - r t is 3 5  CU  1  O  si "~ Oi  C  -H  in  CU  II  03 ^  NO"  a u  > o o  cu  CU "~  CO  o d  0.055  *->  cn '—  £ j3  -H  00  o  201*  3  a a ft "ob.2  cn CU cn .  -H  NO OO  NO  4?  00  3166  O  3§  3  -H  -ft CN  0.4*  cn  CO • / - ^ CO  .2  CO  in  -H  0.5*  co W ^ >  co  CU cu 'cn CO >> cn S-i Si CO ft  s o >- o  §a  *-i  -H CN IO CO  O  0.4*  tf ^  -4—•  S  -H  NO CO  CN  CO  ab x *a aa U  p  -H NO NO  00  0.9*  CU  -H ON  d  *  a a *a  cO ;3 „ o  73  u  *  1.40  k H  I  o  CN  *  0.05  /  -  00 3.  *  0.06  <u  S i C+H Si O  .2  z  Si  M cn -3 <u r3 3  •C  *  rh  1.21  B  to B a .a 3 ob  0.05  CO  cu <u <-> 3 ci,  1.26  rrS cn cn  1.2.1  tf  M  <  Q O  u  < tf  tf  >  P-H  5  U  T3  tf  +  o o d 4)  o CO o d  -*-> 3 cu Si  ct! in  o d V 3 CO o  '3 > tf  op  'co  *  A  bO  a  0.0  X  bb  a.  -0.5  i  •1.0  > GO  Fig. 14. Changes (mean ± S E M , n = 6) from the baseline A) mean arterial pressure (MAP) and B ) arterial resistance (RA) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V), or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the C L - V group.  80  35  25  '5  s  £  15  o u  43  cn  CO  CN CO  CO  u  U  u  o U  o U  au  > GO  Fig. 15. Changes (mean ± S E M , n = 6) from the baseline A) cardiac output (CO) and B) heart rate (HR) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V) or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the C L - V group.  81  1.0  -I  A  0.0 -  1  -1.0 -  PH  -2.0 -  Q  -3.0 -  <  -4.0 -5.0 300.0  o "5a  B  200.0 100.0 0.0  a; + <  -100.0 -200.0 CN CO  >• CO  >  o U  u.  co  a  •t-Y  u  CO CO  a u  Fig. 16. Changes (mean ± S E M , n = 6) from the baseline A) left ventricular end-diastolic pressure (LVEDP) and B) rate of rise of left ventricular pressure (+dP/dt) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V), or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the C L - V group.  82  B  a •  >  0.00  -0.02 J  -0.04  J  oo  >  >i oo  O U  I  U  u  CN  CO  OO  oo  o U  oU  •  i  Fig. 17. Changes (mean ± S E M , n - 6) from the baseline A) mean circulatory filling pressure (MCFP) and B) venous resistance (R ) in rats with chronic sham operation treated with the vehicle (S-V), and rats with chronic ligation of the coronary artery treated with the vehicle (CLV), or CGS 21680 at 0.1, 0.3 or 1.0 ug/kg.min (CGS1, CGS2 or CGS3, respectively). * Significantly (P < 0.05) different from the C L - V group. v  83  A 10  bfl  0  -10  -20  B  0.50  |  0.25  0.00  -0.25  -0.50  J  >•  00  Fig. 18. Changes (mean ± S E M , n = 6) from the baseline A) mean arterial pressure (MAP) and B ) arterial resistance (RA) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V), CGS 21680 (CLCGS2, 0.3 ug/kg.min) or sodium nitroprusside (CL-SNP, 4.0 ug/kg.min). * Significantly (P < 0.05) different from the C L - V group.  84  Fig. 19. Changes (mean ± S E M , n = 6) from the baseline A) cardiac output (CO) and B) heart rate (HR) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V), CGS 21680 (CL-CGS2, 0.3 ug/kg.min) or sodium nitroprusside (CL-SNP, 4.0 ug/kg.min). * Significantly (P < 0.05) different from the C L - V group. Significantly different from the CL-CGS2 group. A  85  A  300.0 -,  ^  200.0  100.0  0.0 CN t/5  >i  > I  CJ  O  CJ I  i—1  u  CO I  U  Fig. 20. Changes (mean ± S E M , n = 6) from the baseline A ) left ventricular end-diastolic pressure (LVEDP) and B) rate of rise of left ventricular pressure (+dP/dt) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V), CGS 21680 (CL-CGS2, 0.3 ug/kg.min) or sodium nitroprusside (CL-SNP, 4.0 ug/kg.min). * Significantly (P < 0.05) different from the C L - V group.  86  I  0.005  0.000  >  Pi <  -0.005 -0.010  J  CN GO  > I  GO  >i u  PH  o  CO  U U  Fig. 21. Changes (mean ± S E M , n = 6) from baseline A) mean circulatory filling pressure (MCFP) and B) venous resistance (Ry) in rats with chronic sham operation treated with vehicle (S-V), and rats with chronic ligation of the coronary artery treated with vehicle (CL-V), CGS 21680 (CL-CGS2, 0.3 ug/kg.min) or sodium nitroprusside (CL-SNP, 4.0 (.ig/kg.min). * Significantly (P < 0.05) different from the C L - V group. Significantly different from the C L CGS2 group. A  87  3.2. Effects of 17p-estradiol in rats with impaired cardiac function 3.2.1. Effects of 17p-estradiol on the activity of basal N O in rats with chronic ligation of the coronary artery There were no significant differences in the surface area of myocardial infarct, mortality, ventricular, lung and body weights, serum concentrations of 17p-estradiol or baseline values of M A P , CO, R A , HR, left ventricular +dP/dt and L V E D P between the two groups of shamoperated rats treated- with vehicle, coronary artery-ligated rats treated with vehicle or coronary artery-ligated rats treated with 17p-estradiol in the present study and the next one (Section 3.2.2). Therefore, these values for groups with the same treatments in the two studies were pooled Serum concentration of 17P-estradiol in intact rats (n = 12) was 208 ± 40 pg/ml. It was reduced by ovariectomy to 109 ± 10 and 100 ± 8 pg/ml, respectively, in the sham-operated rats (n = 12) arid coronary artery-ligated rats treated with vehicle (n = 12). Treatment of coronary artery-ligated rats (n = 12) with 17p-estradiol restored serum 17p-estradiol to 223 ± 1 0 pg/ml at 7 weeks after ligation. There was no myocardial infarct or mortality in the sham-operated group treated with vehicle. The coronary-ligated groups treated with vehicle or 17P-estradiol had similar surface areas of infarct (Table 6) or mortality: 10 out of 22 and 6 out of 18 rats died in the coronary artery-ligated rats treated with vehicle or 17p-estradiol, respectively. Ligation of the coronary artery increased the wet lung weight, but did not alter the ventricular weight in the vehicletreated group.  Treatment with 17P-estradiol abolished the coronary artery ligation-induced  increase in wet lung weight, but did not alter the ventricular weight. Body weight gain was not affected by coronary artery ligation, but was prevented by 17P-estradiol treatment (Table 6). Relative to sham-operated rats treated with vehicle, coronary-ligated rats treated with vehicle had lower M A P , CO and left ventricular +dP/dt, higher L V E D P and R , and similar HR A  88  (Table 7). 17(3-Estradiol reduced L V E D P and R , increased CO, but did not alter the other A  cardiovascular variables in rats with coronary ligation (Table 7). Curve analysis shows that coronary artery ligated rats (n = 6) had attenuated pressor responses to L - N A M E and depressor responses to A C h and SNP, but similar pressor responses to N A relative to those of sham-operated rats (n = 6) (Figs. 22 & 23). Treatment of coronaryligated rats with 17(3-estradiol (n = 6) increased the pressor responses to L - N A M E , further reduced depressor responses to ACh, but did not alter pressor responses to N A or depressor responses to SNP.  89  Table 6. Values (means ± SEM, n = 12) of surface areas of myocardial infarct, and ventricular, wet lung and body weights in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17p-estradiol (ECL).  Infarct area (%)  V-S  V-CL  —  32.8 ± 1.1  E-CL 31.5 ± 0.6  Ventricular weight (g)  0.91+0.02  1.06 ±0.04  0.98 ± 0.02  Wet lung weight (g)  1.33 + 0.04  2.1 ±0.20*  1.38±0.04  Body weight (g), ,.  337 ± 1 3  329 ± 12  246 ± 5*  * Significantly (P < 0.05) different from the V-S groups. group.  A  A  Significantly different from the V - C L  Table 7. Baseline" values'(mean ± S E M , n = 12) of mean arterial pressure (MAP), cardiac output (CO), arterial resistance (RA), heart rate ( H R ) , left ventricular end-diastolic pressure (LVEDP) and rate of rise of left ventricular pressure (+dP/dt) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (VCL) or 17p-estradiol (E-CL).  M A P (mmHg) CO (ml/min) R A (mmHg.miri/rhl) HR (beats/min) ' L V E D P (mmHg) +dP/dt (mmHg/sec)  V-S  V-CL  E-CL  106 ± 1  81 ± 3 *  76 ± 2*  83 ± 2  56 ± 1*  62±2*  1.28 ± 0.03 • 354 ± 9  1.44 ±0.04* 340 ± 7  A  1.23 ± 0 . 0 4 323 ± 10  -1.0 ± 0 . 4  8.9 ± 0 . 5 *  5.0±0.7*  4695 ± 8 4  3192 ±106*  3137 ± 1 1 6 *  * Significantly (P < 0.05) different from the V-S group. group. Relative to baseline atmospheric pressure.  A  A  Significantly different from the V - C L  1  90  o  J  — - ,  2  •  ,  4  8  L - N A M E (mg/kg)  Fig. 22. Effects (n = 6) of i.v. bolus injections of A) noradrenaline (mean + SEM) and B) N nitro-L-arginine methyl ester (L-NAME) (mean ± confidence limit) on mean arterial pressure (MAP; mmHg) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17p-estradiol (E-CL). The L - N A M E data were log-transformed to render normality of distribution. * Curve significantly (P < 0.05) different from the V-S group. Curve significantly different from the V - C L group. A  91  A  -20 -, 00  <  -40 H  -60 0.8  2.4 Acetylcholine (ixg/kg)  7.2  B -14 M  -28  ^  -42  <  -56  •  V-S  •  V-CL  A  E-CL  -70 1 3 ". Sodium nitroprusside (|ig/kg)  9  Fig. 23. Effects (n = 6) of i.v. bolus injections of A) acetylcholine (mean ± confidence limit) and B) sodium nitroprusside (mean ± SEM) on mean arterial pressure (MAP, mmHg) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17(3-estradiol (E-CL). The M A P responses to acetylcholine have been log-transformed to render normality of distribution. * Curve significantly (P < 0.05) different from the V-S group. Curve significantly different from the V - C L group. A  92  3.2.2. Effects of 17(3-estradiol on the activity of basal N O in blood vessels from rats with chronic ligation of the coronary artery Baseline values of haemodynamics as well as surface area of myocardial infarct, mortality, ventricular, body and lung weights from each of the two groups of sham-operated rats, coronary artery ligated rats treated with vehicle or coronary artery ligated rats treated with 17(3estradiol were pooled with the previous study, and presented in Section 3.2.1. Relative to the responses from sham-operated rats treated with vehicle, ligation of the coronary artery in rats treated.with vehicle did not alter the constriction to PE prior to exposure to ACh, SNP or L - N A M E in aortae, pulmonary arteries or portal veins (Table 8). Relative to vehicle treatment in coronary artery-ligated rats, 17p-estradiol significantly increased the constrictor responses to PE in the aortae prior to exposure to A C h or SNP, but not L - N A M E . Relative to vehicle treatment in coronary artery-ligated rats, 17p-estradiol significantly reduced the constrictor responses to PE •in. the portal veins prior to exposure to ACh or SNP, but not L N A M E . However, treatment of coronary artery-ligated rats with 17p-estradiol did not affect the constriction to PE in the pulmonary arteries (Table 8). ACh caused similar concentration-dependent relaxations (similar E  m a x  and EC50) in the  aortae, pulmonary arteries or portal veins from the sham-operated and coronary artery-ligated rats treated with vehicle (table 9)." Relative to vehicle treatment in coronary-ligated rats, 17Pestradiol increased E  to A C h in the portal veins, reduced the response to A C h in the aortae,  m a x  but did not alter it in the pulmonary arteries (Table 8). The aortae, pulmonary arteries or portal veins from the three groups of rats had similar EC50 values to ACh.  Neither coronary artery  ligation nor treatment with 17p-estradiol altered the relaxation responses to SNP in any of the vessels (Table 8). " s  '  93  Relative to responses from sham-operated rats treated with vehicle, ligation of the coronary artery in rats treated with vehicle reduced constriction to L - N A M E in the aortae, pulmonary arteries and portal veins, which reached statistical significance in the aortae and portal veins (Fig. 24). 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UJ  co i  >  U  >  i  CJi  tLl  Fig. 24. Contractile responses (mean ± S E M , n = 6) to N -nitro-L-arginine methyl ester (10" M) in phenylephrine-preconstricted aortae, pulmonary arteries and portal veins from rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17p-estradiol (E-CL). * Significantly (P < 0.05) different from the V-S group. Significantly different from the V - C L group. A  97  3.2.3. Effects of 17P-estradiol on MCFP and Rv in rats with chronic ligation of the coronary artery There were no significant differences in the surface area of myocardial infarct, ventricular, wet lung and body weights, haematocrit, red cell volume, total plasma volume, total blood volume or baseline haemodynamics from the two groups of sham-operated rats treated with vehicle, coronary artery-ligated rats treated with vehicle or coronary artery-ligated rats treated with 17P-estradiol. Therefore, these values for groups with the same treatments were pooled (Table 10 & 1-1). Serum concentration of 17p-estradiol in intact rats was 213 ± 24 pg/ml. Ovariectomy reduced it to 100 + 7 and 99 ± 5 pg/ml, respectively, in the sham-operated rats (n = 12) and coronary artery-ligated rats treated with vehicle (n = 12). Treatment of coronary arteryligated rats (n = 12) with 17p-estradiol restored serum 17p-estradiol to 223 ± 9 pg/ml. There was ho myocardial infarct in sham-operated rats treated with vehicle. Ligation of the coronary artery produced similar surface areas of myocardial infarct in coronary arteryligated rats treated with vehicle or 17P-estradiol. Relative to sham-operation, coronary artery ligation in" vehicle-treated rats increased the wet lung weight, and did not alter the body or ventricular weight. However, relative to vehicle treatment in coronary artery-ligated rats, 17(3estradiol prevented the increase in wet lung weight, did not affect the ventricular weight, and prevented the body weight gain. Relative to sham-operation, ligation of the coronary artery increased red cell volume, total plasma volume and total blood volume, but did not alter haematocrit.  Relative to treatment with vehicle, treatment of coronary artery-ligated rats with  17p-estradiol decreased haematocrit and red cell volume, increased total plasma volume, but did not alter total blood volume (Table 10). Compared to sham-operated rats treated with vehicle, coronary artery-ligated rats treated with vehicle had lower M A P , CO and left ventricular +dP/dt, higher R , L V E D P , M C F P and R , A  v  98  and similar H R (Table 11). Relative to treatment with vehicle, treatment of coronary arteryligated rats with 17|3-estradiol reduced R A , L V E D P , M C F P and Ry, increased CO, but did not alter M A P , left ventricular +dP/dt or H R (Table 11). Infusion of N A into sham-operated rats treated with the vehicle increased M A P , CO, HR, left ventricular +dP/dt, M C F P and R , but did not alter R or L V E D P (Figs. 25-28). N A caused v  A  similar cardiovascular responses in sham-operated rats and coronary artery-ligated rats treated with vehicle (Figs.. 25-28).  Treatment of coronary artery-ligated rats with 17p-estradiol  augmented the effects of N A on M A P , R , M C F P and R , but did not alter the N A effects on the A  other parameters (Figs. 25-28).  v  ,  99  Table 10. Values (mean ± S E M , n = 12) of surface areas of myocardial infarct, ventricular, wet lung and body weights, haematocrit, and red cell, total plasma and total blood volumes in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17(3-estradiol (E-CL). V-CL  E-CL  33.1 ± 0 . 8  32.0 ± 0 . 9  0.95 ± 0.02  0.99 ± 0.02  0.96 ± 0.02  1.35 ±0.02  1.88 ±0.10*  1.35 ± 0 . 0 2  V-S Infarct area (%)  —  Ventricular weight (g) Wet lung weight (g)  ~  Body weight (g)  322 ± 8  318 ± 1 0  269±10  Haematocrit (%)  47 ± 1  49 ± 2  41± 1  Red cell volume (ml)  7.8 ± 0 . 2  9.8 ± 0.4*  8.1 ± 0 . 5  A  Total plasma volume (ml)  8.9 ± 0 . 3  10.2 ±0.4*  11.5 ± 0.4  A  16.7 ± 0 . 5  20.0 ±0.6*  19.6 ± 0 . 8  Total blood volume (ml)  Significantly (P < 0.05) different from the V-S group. group.  A  A  A  A  Significantly different from the V - C L  Table 11. Baseline (mean ± S E M , n = 12) mean arterial pressure (MAP), cardiac output (CO), arterial resistance (RA), heart rate (HR), left ventricular end-diastolic pressure (LVEDP), rate of rise of left ventricular pressure (+dP/dt), mean circulatory filling pressure (MCFP) and venous resistance, (Ry) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17|3-estradiol (E-CL).  M A P (mmHg) CO (ml/min)  ' V-S  V-CL  E-CL  112 ±-2  78 ± 4 *  75 ± 3 *  89 ± 2  55 + 2*  63 ± 3 *  A  R A (mmHg.min/ml)  1.28 ±0.04  1.43 ±0.03*  1.20±0.06  HR (beats/min)  338 ± 7  352 £6  336 ± 6  A  L V E D P (mmHg)  -3.3-0.6  8.0 ± 1.0*  5.0±0.5*  +dP/dt (mmHg/sec)  4542 ± 74  3146 ±129*  3250 ± 1 1 5 *  5.2 ±0.1  7.3 ±0.2*  5.6 ± 0.2  0.037 ± 0.002  0.080 ±0.003*  0.052 ± 0.004  1  M C F P (mmHg) R (mmHg.min/ml) v  * Significantly (P < 0.05) different from the V-S group. group. Relative to baseline atmospheric pressure.  A  A  A  A  Significantly different from the V - C L  1  100  A  *A  Fig. 25. Effects (mean ± S E M , n = 6) of normal saline (NS; 0.018 ml/min) and noradrenaline (NA; 0.5 |ag/kg.min) on A) mean arterial pressure (MAP; mmHg) and B) arterial resistance (R ; mmHg min/ml) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17(3-estradiol (E-CL). V S-NS and V-S-NA are vehicle-treated rats with chronic sham operation receiving NS and N A , respectively. V-CL-NS and V - C L - N A are vehicle-treated rats with chronic ligation of the coronary artery receiving NS and N A , respectively. E-CL-NS and E - C L - N A are 17P-estradioltreated rats with chronic ligation of the coronary artery receiving NS and N A , respectively. * Significantly (P < 0.05) different from the corresponding NS-treated groups. Significantly different from the V - C L - N A group. A  A  101  20  A  ULii JiI  -i  15 -  1  10 -  O  5 -  o  <  0 -5 60 45  Ira  3 0  CU  B  1  15 -\  0  •15  CO  on CO  I  CO I  >  >  U  CO  I  U I  >  >  I  U I  u I  Fig. 26. Effects (mean ± S E M , n = 6) of normal saline (NS; 0.018 ml/min) and noradrenaline (NA; 0.5 ug/kg.min) on A) cardiac output (CO; ml/min) and B) heart rate (HR; beats/min) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17p-estradiol (E-CL). V-S-NS and V - S - N A are vehicle-treated rats with chronic sham operation receiving NS and N A , respectively. V - C L - N S and V - C L - N A are vehicle-treated rats with chronic ligation of the coronary artery receiving NS and N A , respectively. E-CL-NS and E - C L - N A are 17P-estradiol-treated rats with chronic ligation of the coronary artery receiving NS or N A , respectively. * Significantly (P < 0.05) different from the corresponding NS-treated groups.  102  l n  A  Fig. 27. Effects (mean ± S E M , n = 6) of normal saline (NS; 0.018 ml/min) and noradrenaline (NA; 0.5 ug/kg.min) on A) left ventricular end-diastolic pressure (LVEDP; mmHg) and B) rate of rise of left ventricular pressure (+dP/dt; mmHg/sec) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17p-estradiol (E-CL). V-S-NS and V-S-NA are vehicle-treated rats with chronic sham operation receiving NS or N A respectively. V-CL-NS and V - C L - N A are vehicletreated rats with chronic ligation of the coronary artery receiving NS or N A respectively. E - C L NS and E - C L - N A are 17P-estradiol-treated rat with chronic ligation of the coronary artery receiving NS or N A respectively. * Significantly (P < 0.05) different from respective NS-treated group.  103  0.00 00 I  < I  CO  00  >  >  I  I  oo  oo  <  O  O  u  1  >  >  W •  Fig. 28. Effects (mean ± S E M , n = 6) of normal saline (NS; 0.018 ml/min) and noradrenaline (NA; 0.5 ia.g/kg.min) on A) mean circulatory filling pressure (MCFP; mmHg) and B) venous resistance (R ; mmHg.min/ml) in rats with chronic sham operation treated with vehicle (V-S), and rats with chronic ligation of the coronary artery treated with vehicle (V-CL) or 17p-estradiol (E-CL). V-S-NS'and V - S - N A are vehicle-treated rats with chronic sham operation receiving NS and N A , respectively. V-CL-NS-and V - C L - N A are vehicle-treated rats with chronic ligation of the coronary artery receiving NS and N A , respectively. E-CL-NS and E - C L - N A are 17(3estradiol-treated rats with chronic ligation of the coronary artery receiving NS or N A , respectively. * Significantly (P < 0.05) different from the corresponding NS-treated groups. Significantly different from the V - C L - N A group. v  A  104  4. DISCUSSION 4.1. Effects of CGS 21680 on haemodynamics in rats with or without impaired cardiac function 4.1.1. Effects of CGS 21680 on haemodynamics and coronary arterial conductance in rats without impaired cardiac function In vivo and in vitro studies have shown that adenosine is a dilator of arterial as well as venous vessels. Adenosine relaxed precontracted dog saphenous (Verhaeghe, 1977), rat portal (Sjoberg & Wahlstrom, 1975) and human dorsal hand (Ford et al., 1992) veins as well as dog hind limb venous bed (Cotterrell & Karim, 1982). Moreover, it increased the volume of adipose tissue in dogs suggesting the dilatation of capacitance vessels (Sollevi & Fredholm, 1981). Furthermore, adenosine reduced M C F P in rats (Glick et al., 1992; Tabrizchi, 1997).  The  vasodilator effect of adenosine was partly mediated via the activation of adenosine A 2 receptors (Tabrizchi, 1997).  .  CGS 21680 was reported to be a selective A 2 adenosine receptor agonist. In binding studies it was shown to be 170-fold more selective for A 2 than A i receptors (Jarvis et al., 1989). In the present study, selectivity of CGS 21680 for adenosine A 2 receptors was tested by observing its effects on HR in rats with or without pretreatment with DPCPX, a selective adenosine A i receptor antagonist.  In binding studies DPCPX was shown to have 740 folds  selectivity, for A i over A 2 receptors (Bruns et al., 1987). It was assumed i f CGS 21680 loses selectivity at the highest dose (1 pg/kg.min) used in the present study, then its effect on HR should be different in the presence of DPCPX. (response to question 5 of ext. examiner). D P C P X (1 pg/kg:min) was shown to block the bradycardic effects of N -cyclopentyladenosine, a 6  selective adenosine. A | receptor agonist (Kuan et al., 1992). In the present study, D P C P X (1 pg/kg.min) did not affect the. hypotensive or tachycardic responses to CGS 21680 (1 pg/kg.min).  105  These results indicate that the cardiovascular effects of CGS 21680 in the present study were not due to the activation of adenosine A i receptors. CGS 21680 (0.1, 0.3 and 1,0 ug/kg.min) dose-dependently decreased M A P via the reduction of R . Our observations are in agreement with reports which showed that CGS 21680 A  reduced M A P and R (Hutchison et al., 1989; Fozard & Carruthers, 1993b). The depressor and A  arterial dilator, effects  of, CGS 21680 prevailed following  ganglionic blockade with  hexamethonium, or elevation of M A P with the infusion of PE. CGS 21680 also increased HR and CO in a dose-dependent manner. CO is determined by a number of variables that include HR, cardiac contractility, resistances, compliances and blood volume. The increase in CO elicited by CGS 21680 was likely in part due to the reduction in R and increase in HR. The CGS 21680-induced increases in H R and CO persisted during the A  infusion of PE, but were abolished following ganglionic blockade with hexamethonium. The absence of HR response to CGS 21680 in the presence of hexamethonium indicates that tachycardic responses to CGS 21680 in intact rats as well as the PE-treated rats were due to hypotension-induced,reflex sympathetic activation. Others have also shown that the tachycardic effect of CGS 21680 is reflex-mediated. For example, CGS 21680 did not cause tachycardia in pithed rats (Fozard & Carruthers, 1993b) and isolated rat hearts (Hutchison et al., 1989; Conti et al., 1993). In addition, tachycardic responses to CGS 21680 in intact rats were inhibited by pretreatment with metoprolol ((3-adrenoceptor antagonist) (Webb et al., 1991). Moreover, CGS 21680 did not affect automaticity in isolated rat right ventricles (Hernandez et al., 1994). CGS 21680 dose-dependently increased coronary arterial flow and conductance. CGS 21680 was reported to increase coronary arterial flow in rats (Hutchison et al., 1989; Lewis & Hourani, 1997), and caused coronary vasodilatation in isolated perfused guinea pigs hearts (Vials & Burnstock, 1993; Fel.schiet al., 1994).  The coronary vasodilator effects of CGS 21680  persisted following treatment with hexamethonium or PE. 106  4.1.2. Effects of CGS 21680 on haemodynamics in rats with impaired cardiac function 4.1.2.1. Effects of acute occlusion of the coronary artery on haemodynamics Relative to sham-operation, acute occlusion of the coronary artery reduced M A P , CO, cardiac contractility, and increased L V E D P , MCFP and Ry suggesting the impairment of cardiac function.  Reductions in M A P , CO and cardiac contractility, and increase in L V E D P are the  general changes associated with acute heart failure elicited by various procedures such as occlusion of the left coronary artery (Bergmann et al., 1985), cardiac depression following propranolol or pentobarbitone overdose (Maruyama et al., 1988; Wong et a l , 1993), and coronary embolisation (Franciosa et al., 1978; Smiseth & Mjos, 1982; Leddy et al., 1983; Reikeras et al., 1985, 1986; Scholkens et al., 1986; Sweet et al., 1986; Gorodetskaya et al., 1990; Bohn et al., .1995) (See section 1.5.1). Moreover, increments in MCFP and Ry have been reported in dogs with acute heart failure induced by combined ventricular pacing and volume loading (Ogilvie & Zborowska-Sluis, 1992a; Nekooeian et al., 1995).  4.1.2.2. Effects of chronic ligation of the coronary artery on haemodynamics The effects of CGS 216.80 on haemodynamics were also investigated in a rat model of impaired cardiac function induced by chronic ligation of the coronary artery. Chronic coronary artery ligation in rats has been reported to cause chronic heart failure (Gay et al., 1986; Drexler et a l , 1987, 1992a; Raya et al., 1989; Ontkean et al., 1991; Drexler & Lu, 1992; Sanbe et al., 1995). The haemodynamic profile on this model includes reduced M A P (Ontkean et a l , 1991; Sanbe et al., 1995), CO (Chien et al., 1988; Sanbe et al., 1995) and left ventricular +dP/dt (Ontkean et al., 1991; Sanbe.et al., 1995) as well as increased L V E D P (Raya et al., 1989; Ontkean et al., 1991) and MCFP (Gay et a l , 1986; Raya et al., 1989). In the present study, rats subjected to chronic ligation of the left main coronary artery had lower M A P , CO and left ventricular +dP/dt as. well as higher MCFP, L V E D P and wet lung weights at 8 weeks after 107  ligation of the coronary artery indicating chronic heart failure. They also had a higher R , which v  has never been reported.  4.1.2.3. Effects of CGS 21680 on haemodynamics In the present models of acute and chronic heart failure, reductions in CO are accompanied by increases in M C F P and R . The increase in M C F P may be due to an increase in v  blood volume or venous tone. The elevation in Ry indicates increased venous tone, which is likely a result of cardiovascular adjustment in response to reduced CO and M A P . Increased Ry not only reduces venous return, but also leads to an increase in upstream distending pressure and accumulation of,blood in the peripheral venous bed, with the degree of distension depending on the upstream vascular compliance.  Increased Ry also favours capillary filtration and fluid  transudation, and subsequent peripheral edema (Rothe, 1983a). The use of a yenodilatpr drug in chronic heart failure would be expected to shift the circulating blood volume from the central to peripheral circulation, thereby reducing right or left ventricular, preload (ehd-diastolic volume). A decrease in left ventricular preload, as indicated from the reduced L V E D P , would reduce developed systolic wall tension, thereby decreasing myocardial oxygen consumption (Sonnenblick & LeJemtel, 1989). A reduction in Ry would also facilitate the flow of venous return, which if coupled with a reduction of impedance to left ventricular ejection and afterload (RA), would increase CO. In the present study, CGS 21680 decreased M A P , R , L V E D P , M C F P and R A  with either acute or chronic heart failure. Reductions of R , M C F P and R A  v  v  in rats  show that CGS  21680 is an arterial as well as venous dilator. Reductions of M A P and R A by CGS 21680 have been reported (Hutchison et al., 1989; Fozard & Carruthers, 1993b). A reduction in R reduces A  the impedance to left ventricular ejection (afterload).  Such an effect would augment left  ventricular function and thus CO (Amsterdam et al., 1978). There are few studies on the effects 108  of CGS 21680 on venous function.  CGS 21680 has been shown to relax precontracted rat  femoral veins in vitro (Abiru et al., 1995). It was also shown to increase M C F P , but not to affect Rv in rats without heart failure (Tabrizchi, 1997). The increase in M C F P by CGS 21680 was attributed to hypotension-induced reflex sympathetic activation, since CGS 21680 reduced M C F P in the presence of ganglion blockade (Tabrizchi, 1997). It seems that CGS 21680 is capable of reducing M C F P ,and Ry in acute or chronic heart failure, where venous tone is elevated. .  ,  The venodilator property of CGS 21680 is most likely responsible for the decrease in LVEDP.  In support of such a view, Wang and colleagues (1995b) demonstrated that  nitroglycerin and enalapril decreased L V E D P by venodilatation in acute heart failure.  In  contrast, hydralazine, which lacks venodilator activity, did not affect the L V E D P at a dose that caused a similar reduction in M A P as nitroglycerin and enalapril (Wang et al., 1995b). Moreover, Raya and colleagues (1989) demonstrated that captopril decreased M C F P and L V E D P in rats with chronic heart failure, but hydralazine failed to do so (Raya et al., 1989). CGS 21680 also increased HR and CO in animals with acute heart failure or chronic heart failure. The increase, in H R was likely the result of reflex sympathetic activation. Such an increase in HR is not favoured in rats with acute or chronic heart failure, since increase of HR by ventricular pacing, isoproterenol or atropin was associated with expansion of infarct size at 24 h after ligation of coronary artery in dogs (Shell & Sobel, 1973). The increase in CO by CGS 21680 was likely due to an increase in HR and decreases in R A and Ry. The haemodynamic effects of CGS 21680 (0.3 pg/kg.min) were compared with those of SNP (4 pg/kg.min), which caused, a similar decrease in M A P . Relative to CGS 21680, SNP elicited similar decreases in R , Ry and L V E D P , a similar increase in CO, and a similar lack of A  effect on left ventricular contractility. SNP, however, reduced MCFP, and did not significantly increase HR, whereas CGS 21680 (0.3 pg/kg.min) did not alter MCFP, but increased HR. . ...  109  The effects of SNP have been investigated on the arterial, but not venous, circulation of animals with either acute or chronic heart failure.  SNP reduced M A P in canine models of  chronic heart failure induced by embolisation of coronary artery with plastic microspheres (Kono et al., 1994) or rapid right ventricular pacing (Himura et al., 1994). It also reduced M A P in canine models of acute heart failure induced by embolisation of coronary artery by mercury (Franciosa et al., 1978), or by coronary artery occlusion (Pouleur, et al., 1980).  SNP also  reduced L V E D P in these models of acute or chronic heart failure (Kono et al., 1994; Himura et al., 1994; Pouleur, et.al., 1980; Franciosa et al.,. 1978). It also reduced L V E D P in a dogs with acute heart failure induced by embolisation of coronary artery with plastic microspheres (Yamamoto et a l , 1995; Nagano et al., 1997). However, SNP failed to change left ventricular contractility in chronic (Himura et al., 1994) or acute (Pouleur, et al., 1980; Yamamoto et al., 1995; Nagano et al, 1997) heart failure. SNP has been shown to. cause variable effects on HR and CO in animals with heart failure. SNP reduced;(Himura .et.al,, 1994) or did not change (Kono et al., 1994) HR in chronic heart failure. It increased (Franciosa et al., 1978), or did not change (Pouleur et al., 1980; Yamamoto et al., 1995; Nagano et al., 1997) H R in acute heart failure. SNP was also shown to have no influence on CO in chronic heart failure (Himura et al., 1994; Kono et al., 1994), and increased (Pouleur et al., 1980; Franciosa et al., 1978) or decreased (Yamamoto et al., 1995; Nagano et al., 1997). it in acute,heart failure. The differential effects of SNP on H R or CO may have been due to differences in the preparations of heart failure or doses of SNP. In rats with acute heart failure, the high (1.0 ug/kg.min) but not the medium (0.3 ug/kg.min) dose of CGS 21680 lowered M A P , L V E D P , R and MCFP, and increased CO and v  HR. In rats with chronic heart failure, the medium dose (0.3 ug/kg.min) of CGS 21680 reduced M A P , L V E D P and Ry,-and increased CO and HR as well. However, M C F P was reduced only by the high dose (1.0 ug/kg.min) in chrOnic heart failure. These results show that a relatively 110  higher dose of CGS 21680 was required to elicit a response in rats with acute heart failure than in rats with chronic heart failure. The reason for this difference is unclear. The two models of heart failure are different in many aspects. In contrast to rats with chronic heart failure, rats with acute failure were given acute thoracotomy in addition to ligation of the left coronary artery. Major surgery was shown to be associated with increased plasma concentrations of N A (Butler et al., 1977), epinephrine (Butler et al., 1977) plasma renin activity (Yun et al., 1978), and vasopressin (Moran & Zimmerman 1967; Ukai et al., 1968).  Therefore, one would expect  greater release of neurohormonal agents following acute than chronic heart failure. Moreover, chronic heart failure is associated with structural changes in the cardiovascular system (Thuillez et al., 1995; Ganguly et al., 1997.; Mulder et al., 1997), which are unlikely to occur in acute heart failure. Furthermore, experiments on rats with acute or chronic heart failure were performed under two different barbiturate anaesthetics. Inactin has a longer duration of action than that of sodium pentobarbitone (Wayforth, 1980), and was used to minimise the effects of changes in levels of anaesthesia on cardiovascular parameters in acute heart failure experiments, which took a longer time. Whether or not these anaesthetics were involved in the differential response to CGS 21680 in acute and chronic heart failure is not clear. The comparative effects of these anaesthetics on cardiovascular parameters have not been documented.  However, at doses  comparable to those used in the present study, Inactin and sodium pentobarbitone caused similar reductions in M A P (Cupples et al., 1981) or renal flow (Koeppen et a l , 1979) in rat. To summarise, these results show that acute occlusion and chronic ligation of the left main coronary artery in rats resulted in acute heart failure and chronic heart failure, respectively. Both models were associated with decreased M A P , CO and cardiac contractility as well as increased MCFP, R and L V E D P . Acute administration of CGS 21680, a selective adenosine v  A 2 A receptor agonist, increased CO. via reductions in R , Rv and an increase in HR. A  4.2. Effects of 17(3-estradiol in rats with impaired cardiac function 4.2.1. Effects of 17p-estradiol on the activity of basal N O in rats with chronic ligation of the coronary artery Ligation of the left main coronary artery in ovariectomised rats resulted in chronic heart failure characterised by decreased M A P , CO and left ventricular +dP/dt as well as increased R A , L V E D P and wet lung weight at seven weeks after ligation. These cardiovascular changes are consistent with those reported in.rats .with chronic heart failure elicited by ligation of the coronary artery (Sanbe et al., 1995).  :  Ovariectomy reduced serum concentrations of 17p-estradiol to approximately half of those in age-matched intact rats. The sustained-release pellets restored serum 17P-estradiol to concentrations comparable to those of intact rats. The first estrus cycle in rats occurs at the age of 37 days (Baker, 1979). In the present study, rats were ovariectomised and implanted with estrogen  pellets at :13-23 days post-estrus, therefore,  estrogen  treatment represented a  replacement therapy. Treatment with 17p-estradiol decreased the body and wet lung weights. A reduction in body weight in response to 17p-estradiol has been. reported (Davis et al., 1989; Meyer et al., 1997). Reduction.in the lung weight.could be due to lower body weight and/or lower pulmonary congestion as indicated by reduced L V E D P . 17p-Estradiol did not significantly alter the mortality, surface area of myocardial infarct or ventricular weight. However, in myocardial ischaemia and reperfusion studies in dog (Node et al., 1997a) and rabbit, (Hale et al., 1996), 17p-estradiol reduced the size of myocardial infarct. Condition of the present study, using rats with permanent ligation of the coronary artery was, however, different from those of brief myocardial ischaemia followed by reperfusion, whereby 17p-estradiol could gain access to ischaemic tissue during the reperfusion phase.  112  17p-Estradiol increased CO in the present study. 17p-Estradiol was shown to increase CO in animals that did not have heart failure (Magness et al., 1993, Williams et al., 1994b) as well as male transsexuals (Slater et al., 1986). 17p-Estradiol also reduced R A as well as L V E D P indicating reductions in afterload and preload, respectively. The former was due to the dilatation of arterial resistance vessels and the latter was likely due to the dilatation of capacitance vessels, since myocardial contractility was unaltered.  It is of interest that captopril (angiotensin  converting enzyme inhibitor) was shown to cause dilatation of capacitance vessels, which reduced left ventricular preload, and dilatation of resistance vessels, which reduced afterload (Raya et al., 1989), Captopril,is also known to improve left ventricular function, and increase survival in heart failure.  . . . . .  Depressor responses to A C h and SNP were attenuated in rats with chronic heart failure. Vasodilator response to A C h was decreased in epicardial coronary arteries from dogs with chronic heart failure induced by rapid ventricular pacing (Wang et al., 1994), and perfused hindquarters of .rats with chronic, heart failure induced by ligation of the coronary artery (Drexler & Lu, 1992). Vasodilatation to A C h was also decreased in the forearms of patients with heart failure (Kubo et al., 1991). Decreased depressor response to A C h is not necessarily indicative of decreased release of NO, since A C h has been shown to dilate resistance arteries primarily via the release of an endothelium-dependent hyperpolarising factor (EDHF), which is distinct from N O or prostanoids (for .review see Garland et al., 1995).  It has been suggested that impaired  vasodilator response to A C h could be due to abnormal production of cyclooxygenase-dependent vasoconstricting factor, impaired endothelial release of NO, and/or decreased vascular smooth muscle response to cGMP (Katz et al., 1993). Reduced response to cGMP likely contributed to the decreased depressor response to A C h in this study, since depressor responses to SNP, a nitrovasodilatpr, ,were .also, reduced in rats with chronic heart failure. Similar to our findings, epicardial coronary flow responses to nitroglycerin were attenuated in dogs with chronic heart 113  failure induced by rapid ventricular pacing (Wang et al., 1994) or coronary embolisation (Knecht et al., 1997). Chronic heart failure did not alter the pressor responses to N A , but reduced that to L NAME.  Attenuated contractile responses to PE have been reported in isolated perfused  mesenteric arteries from rats with chronic heart failure (Stassen et al., 1997).  Attenuated  response to L-NAME.is.suggestive of reduced involvement of NO in the regulation of vascular tone. Reduced pressor response to L - N A M E has also been reported in canine (Eisner et al., 1991) and ovine (Rademaker et al., 1996) chronic heart failure induced by rapid right ventricular pacing.  Furthermore, the release of nitrite was decreased in isolated coronary arteries and  microvessels from dogs with chronic heart failure induced by rapid ventricular pacing (Wang et al., 1994).. Basal release of NO, was, however, preserved in perfused hindquarters of rats with chronic heart failure (Drexler & Lu, 1992) and radial arteries of patients with congestive heart failure (Drexler etal., 1992b). Chronic administration of,17P-estradiol to rats with chronic heart failure did not alter the pressor response to N A - i n the present, study. There are no published studies on the chronic effects of estrogens on pressor.responses to N A in animals with chronic heart failure. There is, ;  however, no consensus on the effects of estrogen on the pressor responses to N A or other aadrenoceptor. agonists in animals without heart failure. Chronic treatment with 17p-estradiol or mestranol did not alter the pressor responses to N A in conscious, ovariectomised rats (Shiverick et al., 1983; Conard et al., 1994). In contrast, chronic treatment of monkeys with 17p-estradiol attenuated pressor responses to PE (Williams et al., 1994b). Chronic treatment with 17P-estradiol further reduced depressor responses to ACh. It is unclear whether Or not the attenuation was due to the reduction of ACh-stimulated release of NO or the elusive EDHF. There are conflicting reports on the chronic effects of 17p-estradiol on the  114  vasodilator response to ACh. Chronic 17(3-estradiol treatment restored ACh-induced dilatation in atherosclerotic coronary arteries in monkeys (Williams et al., 1990, 1994a), but did not change it in the human forearm (Sudhir et al., 1996). 17(3-Estradiol did not alter the depressor responses to SNP. There are also conflicting reports  regarding the  chronic effects  of 17(3-estradiol on vasodilatation response  to  nitrovasodilators. 17(3-Estradiol potentiated the SNP-induced vasodilatation in the forearm of women with risk factors for atherosclerosis and impaired vascular function (Gilligan et al., 1994a). It also potentiated the nitrbglycerin-induced vasodilatation in the brachial artery of male to female.transsexuals (McCrohon et al., 1997). However, 17(3-estradiol attenuated the SNPinduced vasodilator response in the coronary artery of cynomolgus monkeys (Williams et al., 1992), and did not affect it in the coronary artery of atherosclerotic women (Gilligan et al., 1994b). Chronic i7(3-estradidl treatment increased the pressor responses to L - N A M E in rats with chronic heart failure. This may indicate that 17p-estradiol increased the release and/or reduced the breakdown of NO.  In fact, estrogen was shown to increase the expression of NO synthase  (Weiner et al., 1994) as well as inhibit the production of superoxide (Arnal et al., 1996), which inactivates NO. This study, however, can not confirm or reject either of these possibilities. The finding is also i n agreement with reports that 17p-estradiol increased the plasma concentration of NO metabolites in dogs (Kim et al., 1996) and women (Rosselli et al., 1995). The results of this study show that restoration of serum concentrations of 17p-estradiol has favourable cardiovascular actions through reductions, in. cardiac preload (LVEDP) and afterload (RA). Furthermore, estradiol increased the pressor response to L - N A M E , which implies increased vasodilator role of NO. • The estradiol-induced decreases in preload and afterload might therefore be due to restoration of the. vasodilating role of NO.  Reductions of preload and 115  afterload might lead to a decreased myocardial work and increased myocardial efficiency, which are of vital importance in heart failure. To summarise, the present study indicates that ligation of the left main coronary artery caused chronic heart failure characterised by decreased M A P , CO and left ventricular +dP/dt, and increased L V E D P and R A . This model of chronic heart failure was also associated with attenuated depressor responses.to A C h and SNP, attenuated pressor responses to L - N A M E , and no change in pressor responses to N A . Chronic treatment with 17p-estradiol reduced L V E D P and R , restored pressor responses to L - N A M E , and further reduced depressor responses to ACh A  in rats with chronic heart failure. 17P-Estradiol also increased CO, but did not alter M A P , left ventricular +dP/dt o r H R . . The.results show that estrogen reduces preload as well as afterload, and restores the vasodilator role of basal N O in ovariectomised rats with chronic heart failure.  4.2.2. Effects of 17p-estradiol on the activity of basal NO in blood vessels from rats with chronic ligation of the coronary artery  .  • . .Contractile responses; to PE in the aorta, pulmonary artery or portal vein were not affected by ligation of the coronary,artery. However, treatment of coronary artery-ligated rats with 17p-estradiol enhanced PE-induced contraction in the aorta, did not alter the response in the pulmonary artery and attenuated it in the portal vein. Chronic treatment with 17p-estradiol may differentially affect the contractile response of various blood vessels to PE. 17p-Estradiol did not alter ex vivo contractile responses to PE in rat aortic rings (Vedernikov et al., 1997). On the other hand, it augmented (Rorie & Muldoon, 1979), or did not affect (Gisclard et al., 1987) the contractile response to PE in rabbit saphenous vein. It is not clear why 17P-estradiol inhibited contraction to PE in the portal vein, but not aorta or pulmonary artery in the present study. The portal vein, is, however, functionally distinct from the aorta or pulmonary artery. In contrast to  116  the aorta or pulmonary artery, the portal vein has spontaneous action potential and spontaneous contractile activity. .Furthermore, relative to the aorta, the portal vein is more dependent on external calcium for spontaneous as well as sustained contractions (Sutter, 1976, 1990). Since 17p-estradiol was shown to have calcium antagonistic action in vascular smooth muscles (Jiang et al., 1991), it is tempting to speculate that reduced constriction to PE in the portal vein from 17p-estradiol-treated rats in the present study is related to the portal vein's susceptibility to calcium antagonism.  .  .  :  Coronary artery ligation did not affect the endothelium-dependent relaxation to A C h in the aorta, pulmonary artery or portal vein. The extent of impairment of endothelium-dependent relaxation in rat model of chronic heart failure depends on the particular vasculature and the time of assessment after coronary artery ligation (Teerlink et al., 1993; Vanhoutte, 1996; Baggia et al., 1997) . ACh-induced relaxation was not impaired in the aorta at weeks 1 (Teerlink et al., 1994b) and 10 (Baggia et al., 1.997).after coronary artery ligation. It was also not impaired in small mesenteric artery at week.10 after ligation (Baggia et al., 1997). However, chronic heart failure impaired ACh-induced relaxation in aorta at weeks 6 (Teerlink et al., 1993), 8 (Toyoshima et al., 1998) , 10 (Ontkean et al., 1991) and 12 (Nasa et al., 1996) following coronary artery ligation. The ACh-induced relaxation was also impaired in pulmonary artery at weeks 10 and 12 after coronary artery ligation (Ontkean et al., 1991; Nasa et a l , 1996; Baggia et al., 1997). In contrast to our use of the main pulmonary artery, Ontkean et al. (1991) and Baggia et al. (1997) used hilar pulmonary arteries, which are smaller and embedded in the lung parenchyma. Chronic treatment of coronary  artery-ligated  rats with  17p-estradiol  enhanced  endothelium-dependent relaxation to A C h in the portal vein, but not in the aorta or pulmonary artery. These results are in accordance with those obtained from vascular tissues of healthy animals, which showed that 17p-estradiol did not affect (Vedernikov et al., 1997; Bolego et al.,  117  1997) or enhanced (Gisclard et al., 1988; Cheng et al., 1994) endothelium-dependent relaxation ex vivo. Neither ligation of the coronary artery nor treatment of coronary artery-ligated rats with 17p-estradiol altered the relaxation to SNP in the aorta, pulmonary artery or portal vein. Lack of effect of coronary artery ligation on endothelium-independent relaxation has been reported (Ontkean et al., 1991; Teerlink et al., 1994b; Nasa et al., 1996; Toyoshima et al., 1998). There are no published reports on the effect of chronic estrogen treatment on endothelium-independent relaxation in isolated blood vessels from animals with chronic heart failure. Chronic treatment with  17P-estradiol, however, had no effect  on endothelium-independent  relaxation in  vasculatures from healthy animals (Vedernikov et al., 1997; Bolego et al., 1997). Ligation of the coronary artery reduced the contractile responses to L - N A M E in the aorta, portal vein and pulmonary artery, which reached statistical significance in the aorta and portal vein. These findings suggest that the activity of basal N O was impaired in the aorta and portal vein. Both.reduced (Ontkean et al., 1991; Teerlink et al., 1994b; Nasa et al., 1996; Toyoshima et al., 1998) and preserved (Ontkean et al., 1991) activity of basal cGMP or NO have been reported in vessels from rats with chronic heart failure. It has been speculated that decreased basal release of NO in chronic heart failure might be related to a reduction in CO, which reduces shear stress on vascular endothelial cells (Vanhoutte, 1996). Treatment with 17f3-estradiol restored contractile responses to L - N A M E in the aorta, pulmonary artery and portal vein from rats with chronic heart failure suggesting the augmentation of the activity of basal N O in these vessels. Increased activity of basal N O might be due to an increase in the release and/or a decrease in the breakdown of the compound. 17(3Estradiol was reported to increase the expression of constitutive (Weiner et al., 1994; Kleinert et al., 1998) and inducible (Binko & Majewski, 1998) isoforms of NO synthase, and to inhibit the production of superoxide (Amal.et al., 1996), which degrades NO. 118  In summary, the present study indicates that aorta, pulmonary artery'and portal vein from rats with chronic heart failure had relaxation responses to A C h or SNP similar to those in the corresponding vessels from sham-operated rats. Relative to those from sham-operated rats, aorta and portal vein from rats with chronic heart failure had reduced responses to L - N A M E suggesting the impaired activity of basal endothelium-derived N O . Chronic administration of 17(3-estradiol to. rats with chronic heart failure did not.affect the relaxation to SNP in any vessel, increased relaxation to A C h in-the portal vein, attenuated relaxation to A C h in the aorta, and augmented contractile responses to L - N A M E in all three types of vessels. These results indicate that 17(3-estradiol enhances the activity of basal N O in the aorta, pulmonary artery and portal vein from rats with chronic heart failure.  4.2.3. Effects of 17p-estradiol on M C F P and Rv in rats with chronic ligation of the coronary artery  v.-  ., •  The results of the present study show that ligation of the coronary artery in rats resulted in chronic heart failure characterised by reduced M A P , CO and left ventricular +dP/dt, and increased R , L V E D P , MCFP, R and wet lung weight at 7 weeks after coronary ligation. These A  v  changes are consistent with those previously reported in the same model of chronic heart failure (Gay et al., 1986; Sanbe et.al., 1995). Relative to the values in sham-operated rats, total blood volume, total plasma volume and red cell volume increased, and haematocrit remained unchanged at 7 weeks following ligation. Total blood and total plasma volumes were reported to increase (Raya et al., 1989), but haematocrit was shown to remain unchanged (Gay et al., 1986; Raya et al., 1989) in this.model,of chronic heart failure The effects of estradiol on M A P , CO, R , L V E D P and lung weight were discussed earlier A  (see section 4.2.1.). Similar to results of the previous two studies, 17p-estradiol also did not  119  affect the surface area of infarct. Treatment with 17[3-estradiol reduced red cell volume and haematocrit, further increased total plasma volume, but did not change total blood volume. Chronic treatment with 17 (3-estradiol was also shown to increase total plasma volume in sheep (Ueda et al., 1986; Magness et al., 1993) and guinea pig (Hart et al., 1985; Davis et al., 1989) that did not have heart failure. Reduced red cell volume by chronic estrogen treatment was reported in male transsexuals (Slater et al., 1986) and Japanese quail (Gibbins & Robinson, 1982).  Chronic treatment with 17P-estradiol was reported to decrease haematocrit in sheep  (Ueda et al., 1986; Davis et al., 1989; Magness et al., 1993), or not to affect it in guinea pigs (Hart et al., 1985) or rats (Toba et al., 1991). The lack of a change in total blood volume by 17pestradiol in the present study is the resultant of a decrease in red cell volume and an increase in total plasma volume. The mechanisms by which 17p-estradiol increased total plasma volume, and decreased red cell volume are not clear. The increase in total plasma volume might be due to an increase in ;  the plasma concentrations of arginine vasopressin (antidiuretic hormone).  Seven days of  treatment of rats-wifh silicone elastomer implants containing 17p-estradiol (1 mg) was associated with an increase in the plasma concentrations of arginine vasopressin (Peysner & Forsling, 1989).  As well, treatment of postmenopausal women with 17p-estradiol for 14 days (0.1  mg/day) increased plasma concentrations of arginine vasopressin (Stachenfeld et al., 1998). On the other hand, treatment of rats with pellets containing 0.5 or 5.0 mg 17p-estradiol did not change the plasma concentration of arginine vasopressin (Barron et al., 1986). The increase in total plasma volume might not be due to salt retention, since 17P-estradiol did not alter (Peysner & Forsling, 1989; Stachenfeld et al., 1998), or even reduced (Barron et al., 1986) the plasma concentration of-Na . +  The decrease in red cell volume might be due to a decrease in  erythropoietin production, since low doses of estradiol benzoate (2.5-10.0 ug/day for 5 days)  120  inhibited the production of erythropoietin in female rats exposed to various intensities of hypoxia (Peschle et al., 1973). There are no published reports on the effect of estrogens on the venous system in animals with chronic heart failure. Treatment of guinea pigs without heart failure with 17P-estradiol (125 )j,g per day), for. 55 days resulted in an increase in MCFP, which was attributed to an increase in total blood'volume (Davis et al., 1989). The present study shows that chronic 17pestradiol treatment reduced M C F P in rats with heart failure. In the absence of a change in total blood volume, a reduction in M C F P reflects a decrease in venous compliance (Grodins, 1959). Chronic 17p-estradiol treatment also reduced Rv in rats with heart failure indicating the dilatation of venous resistance vessels. Venodilatation would be expected to shift the circulating blood volume from the central to peripheral circulation, which reduces pulmonary congestion and edema as well.as left ventricular preload, characterised by L V E D P .  It is of interest that  L V E D P as well as wet lung weight were reduced by 17P-estradiol. Reduced preload would reduce developed systolic wall tension as well as myocardial oxygen consumption (Sonnenblick & LeJemtel,-. 198.9),.thereby improving cardiac function.  Reduced Rv would also facilitate  venous return to the heart.,: The increase in CO by 17p-estradiol could therefore be due to reductions in Rv-ahd afterload^ which reduces the impedance to left ventricular ejection. There are no published studies on the mechanisms by which 17P-estradiol causes venodilatation.  Pretreatment  with glibenclamide attenuated the  17p-estradiol-induced  vasodilatation of-the canine epicardial coronary artery in vivo (Sudhir et al., 1995) suggesting that the arterial vasodilator activity of 17p-estradiol may be partly mediated by the opening of ATP-sensitive K channels. '17p-Estradiol also relaxed precontracted arterial rings in vitro (Bell +  et al., 1995; Jiang et al., 1991; Belfort et al., 1996) via blockade of C a  2+  channels (Jiang et a l ,  1991). Moreover, 17p-estradiol was shown to increase the release of endothelium-derived N O 121  (McNeill et al., 1996) and formation of c A M P as well as cGMP (Miigge et al., 1993). It is not known i f similar mechanisms underlie the dilator effect of 17(3-estradiol on the venous system. Ligation of the coronary artery did not alter the responses of M A P , RA, Rv, MCFP, CO, HR, L V E D P or left ventricular +dP/dt to N A . However, treatment with 17(3-estradiol enhanced M A P , R , R V and MCFP, but not CO, HR, L V E D P or left ventricular +dP/dt, responses to N A . A  17p-Estradiol was shown to enhance the pressor response to N A in rat mesenteric arteries in vivo (Altura, 1975). It also enhanced ex vivo contractile responses to N A in rat aorta (Cheng & Gruetter, 1992) as well as. rabbit uterine artery (Graham & Sani, 1971), saphenous vein (Rorie & Muldoon, 1979) and aorta (Miller & Vanhoutte, 1990). The mechanisms by which 17p-estradiol increased responsiveness to N A are unclear. 17p-Estradiol was reported to decrease the release of N A (Blum et al., 1996) as well as increase the affinity (Colucci et al., 1982) and density (Roberts et-al.,:.1977; Colucci etal., 1982) of a i-adrenoceptors.  Therefore, it is tempting to  speculate that 17p-estradiol might have enhanced responses to N A in the arterial and venous circulation by decreasing the release of N A leading to the up-regulation of ai-adrenoceptors. Contrary to the present findings, treatment with 17P-estradiol pellets (2.5 pg/day) for 21 days (Conard et al., 1994) or mestranol (15 pg i.p.) every two weeks (Shiverick et al., 1983) did not alter the pressor responses to i.v. bolus injections of N A in conscious, ovariectomised rats. Moreover, acute intramuscular injection of 17P-estradiol (10 mg) in healthy men did not affect the pressor responses to i.v. bolus injections of N A in superficial hand veins (Jilma et al., 1994). Discrepancies in responses to N A among various studies might be due to differences in dose and/or duration of estrogen therapy, methods of N A administration, and pathophysiological state of animals (healthy versus chronic heart failure)... To summarise, these results demonstrate that ligation of the coronary artery in rats resulted in chronic heart failure characterised by lower M A P , CO and left ventricular 122  contractility as we'll as higher R A , L V E D P , MCFP and Ry relative to the corresponding readings in sham-operated rats. Coronary artery ligation did not alter the cardiovascular responses to N A . Chronic 17(3-estradiol treatment of rats with heart failure reduced R A as well as L V E D P , MCFP and R reflecting the dilatation of resistance as well as capacitance vessels. Furthermore, 17(3v  estradiol enhanced the constrictor responses to N A in the arterial as well as venous circulation.  123  5. SUMMARY AND CONCLUSION 5.1. Effects of CGS 21680 on haemodynamics in rats with or without impaired cardiac function CGS 21680, an A2A receptor agonist with 170-fold selectivity for A2 relative to A) receptors, dose-dependently reduced M A P and R A , and increased CO, H R as well as coronary arterial flow and conductance in pentobarbitone-anaesthetised rats without heart failure. The (  effects of CGS 21680 on CO and HR, but not the other variables, were attenuated by ganglionic blockade, which indicate that these effects were mediated by the activation of autonomic reflexes. The haemodynamic effects of CGS 21680 were also examined in rats with either acute heart failure induced by acute occlusion of the left main coronary artery, or chronic heart failure induced by chronic ligation of the artery. Rats with acute heart failure had decreased M A P , CO and left ventricular +dP/dt, and increased L V E D P , M C F P and R at 90 min after coronary artery v  occlusion. Rats with chronic heart failure had decreased M A P , CO and left ventricular +dP/dt, and increased L V E D P , M C F P and Ry at week 8 after coronary artery ligation. CGS 21680 at 1.0 |Lig/kg.min in acute heart failure and at 0.3 and 1.0 ug/kg.min in' chronic heart failure increased CO and HR, and decreased M A P , R A , L V E D P and Ry. CGS 21680 (1.0 ug/kg.min)'also reduced MCFP in both acute and chronic heart failure. Thus, a lower dose of CGS 21680 was generally required to elicit cardiovascular responses in chronic heart failure. The CGS 21680-induced decrease in M A P was due to a reduction in R A . The increase in CO might have been due to decreases in R A (afterload) and Ry as well as an increase in HR. The haemodynamic effects of CGS 21680 (0.3 ug/kg.min) were also compared with those of a similar depressor dose of SNP (4.0 ug/kg.min) in chronic heart failure. SNP increased CO, and decreased M A P , R , L V E D P , MCFP as well as Ry. CGS 21680 and SNP had a very similar A  haemodynamic profile, however, SNP caused less tachycardia than CGS 21680.  124  5.2. Effects of 17P-estradiol in rats with impaired cardiac function Ligation of the left main coronary artery in ovariectomised rats resulted in chronic heart failure at 7 weeks after the ligation, which was characterised by decreased M A P , CO, left ventricular +dP/dt, and increased RA, L V E D P , MCFP and Ry. Chronic heart failure also reduced pressor responses to L - N A M E , which suggests reduced involvement of NO in the regulation of vascular tone.  The attenuated pressor response to L - N A M E might be a consequence of  decreased CO, and subsequent reduced shear stress on the vascular endothelium. Chronic heart failure also attenuated depressor response to ACh, which might be due to reduced responsiveness to cGMP, since depressor response to SNP was also reduced in rats with chronic heart failure. The aorta, pulmonary artery and portal vein from rats with chronic heart failure had attenuated ex vivo contractile response to L - N A M E , which reached statistical significance in the aorta and portal vein. Reduced contraction to L - N A M E implicates reduced activity of basal N O in these vessels. Chronic heart failure did not alter the ex vivo relaxation to A C h or SNP, or contraction to PE, in the.aorta, pulmonary artery or portal vein. The first estrus cycle in rats occurs at the age of 37 days. At the age of 50-60 days, rats were ovariectomised and implanted with pellets containing 17p-estradiol (1.5 mg), which restored serum 17p-estradiol to concentrations comparable to those of intact rats. Therefore, 17P-estradiol treatment represents estrogen replacement therapy.  17P-Estradiol reduced R A ,  MCFP, R , L V E D P and .wet lung weight, and increased CO. In the absence of a change in total v  blood volume, the reduction in MCFP denotes a decrease in venous compliance. The decreases in R and L V E D P reflect reductions of afterload and preload, respectively. The reductions in R A  A  as well as Rv would be expected to facilitate venous return and CO. Lower lung weight might be due to lower body weight and/or lower pulmonary congestion resulting from venodilatation.  125  17(3-Estradiol did not affect left ventricular contractility, mortality or surface area of myocardial infarct. Chronic treatment with 17p-estradiol reduced the depressor response to A C h , and increased pressor response to L - N A M E as well as M A P , R , M C F P and Ry responses to i.v. A  infusion of N A . 17p-Estradiol was reported to decrease the release of N A as well as increase the affinity and density of ai-adrenoceptors. Therefore, 17P-estradiol-induced enhancement of the in vivo arterial and venous constrictor response to N A might be a result of the up-regulation of a,| -adrenoceptors. 17p-Estradiol attenuated ex vivo contraction to PE in the portal vein, augmented it in the aorta, but did not affect it in the pulmonary artery. 17p-Estradiol enhanced ex vivo relaxation response to ACh' in' the portal vein, attenuated it in the aorta, but did not affect it in the pulmonary artery. The reason for differential ex vivo responses to PE and ACh in these vessels is not clear. 17P-Estradiol did not affect ex vivo relaxation to SNP in the aorta, pulmonary artery or portal vein, but increased contractile responses to L - N A M E in all three types of vessels. Increased contraction to L - N A M E in vivo as well as ex vivo might be due to an increase in the release of NO and/or inhibition of its breakdown.  126  6. R E F E R E N C E S  Abe, Y., K . Kotoh, P. H . Deleuze, M ; Miyama, G. J. Cooper, D. Y . 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