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Vascular B-adrenoceptors and pressor response to B-adrenoceptor antagonists Abdelrahman, Aly Mohamed Omar 1991

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VASCULAR B-ADRENOCEPTORS AND PRESSOR RESPONSE TO 6-ADRENOCEPTOR ANTAGONISTS By ALY MOHAMED OMAR ABDELRAHMAN MB.BCH., Cairo University, 1981 M.Sc, Cairo University, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics) We accept this thesis as confirming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1991 © A l y Abdelrahman, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference arid study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P ^QLV IA^CL CjrG)Oy^ The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract 6-adrenoceptors have been classifi e d into two subtypes, a 6^-subtype found in the heart and a 62-subtype found in the vasculature. However, there i s evidence that B^-adrenoceptors may also be present in the vasculature. We have examined the role of B-adrenoceptors in the vas-culature, f i r s t in the resistance blood vessels and second in the venous system. Fi r s t l y , we studied the effects of isoprenaline (mixed 6), isoprenaline plus ICI 118,551 (B^) isoprenaline plus atenolol (B2) and isoprenaline plus both ICI 118,551 and atenolol on haemodynamics in pentobarbital-anaesthetized rats using the radioactive microsphere tech-nique. This study showed that B 2- but not B^-adrenoceptor stimulation reduced total peripheral resistance (TPR) and mean art e r i a l pressure (MAP). Both B^- and B2-adrenoceptor stimulation increased coronary and skeletal muscle vascular conductances. Secondly, we examined the effect of iso-prenaline on MAP, heart rate (HR) and mean circulatory f i l l i n g pressure (MCFP) in conscious rats. Isoprenaline was infused into intact, hexamethonium-pretreated or nor-adrenaline-pretreated rats. Our results show that under normal conditions, isoprenaline decreased MAP and increased HR and MCFP. Hexamethonium pretreatment did not affect the tachycardic and hypotensive effects of isoprenaline but i t abolished the increase in MCFP indicating that this increase was due to reflex venoconstriction. Under conditions of high venous tone, isoprenaline decreased MAP and MCFP and i i i increased HR. Therefore, our results show that B-adreno-ceptor stimulation mediates direct venodilatation in the presence of a high venous tone and reflex-mediated venocon-str i c t i o n under normal conditions. Paradoxical pressor responses to S-adrenoceptor ant-agonists have been reported in some c l i n i c a l and experimental conditions. The mechanisms underlying this phenomenon are not known. We examined the conditions under which ^-adrenoceptor antagonists produced a pressor response. F i r s t l y , we examined the haemodynamic changes which occur during a-adrenoceptor blockade by phentolamine, and after the development of the pressor response to 6-adrenoceptor antagonists in urethane-anaesthetized rats and in conscious rats. In urethane-anaesthetized rats, propranolol reversed the increase in conductance induced by phentolamine in skeletal muscle and skin and i t also decreased renal vascular conductance. In conscious rats, propranolol or atenolol reversed the increase in conductance induced by phentolamine in skeletal muscle and in addition, i t decreased conductance in the intestinal, renal and cutaneous vasculature. The inhibition of angiotensin converting enzyme by captopril attenuated the ^-adrenoceptor antagonist-induced pressor response demonstrating the importance of the renin-angiotensin system in the production of this response. Secondly, experiments were done to investigate the effects of anaesthetic agents on pressor response to 6-adrenoceptor antagonists. Our results show iv that anaesthetic agents have variable effects on 6-adrenoceptor antagonist-induced pressor responses. Urethane did not alter the pressor response to 6-adrenoceptor antagonists. Halothane and ketamine, on the other hand, attenuated the pressor response while 6-adrenoceptor antagonists did not produce a pressor response with pentobarbital, amobarbital or chloralose. It was further shown in pentobarbital-anaesthetized rats that phentolamine increased arteriovenous conductance and reduced MAP with no effects on other vascular beds and propranolol then did not have any effects on vascular conductance, TPR or MAP. An infusion of adrenaline partially restored the pressor response to both propranolol and atenolol showing the importance of adrenaline in the production of a pressor response. Thirdly. dose-response curves for propranolol, atenolol or ICI 118,551 in the presence of both noradrenaline and phentolamine were constructed in the isolated rat pulmonary artery. A l l three 6-adrenoceptor antagonists completely restored the phentolamine-induced relaxation response. Therefore, our in vitro and in vivo results are in accordance with a possible interaction of a-and 6-adrenoceptor antagonists which leads to subsequent stimulation of the a-adrenoceptors in the presence of adrenaline. The mechanism of this interaction is not clear. The results of in vivo studies show that additional factors such as the renin-angiotensin system may also be involved in this pressor response. V TABLE OF CONTENTS CHAPTER Page ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES X LIST OF ABREVIATIONS xiv ACKNOWLEDGEMENTS XV GENERAL OVERVIEW 1 1 INTRODUCTION 2 1.1 The sympathetic nervous system 2 1.2 Classification of adrenoceptors 4 1.3 6-adrenoceptors 5 1.3.1 Classification 5 1.3.2 Identification and characterization 6 1.3.2.1 Binding studies 6 1.3.2.2 Purification and isolation 7 1.3.2.3 Cloning and sequencing 9 1.3.3 Cellular signalling mechanisms 11 1.3.4 Localization of B-adrenoceptors in the heart 13 1.3.5 6-adrenoceptor agonists 16 1.3.6 6-adrenoceptor antagonists 18 1.3.6.1 Introduction 18 1.3.6.2 Classifications 19 1.3.6.3 Chemical structure 21 1.3.6.4 Selective 6-adrenoceptor antagonists 22 1.3.6.4.1 62-adrenoceptor antagonists 22 1.3.6.4.2 6i~adrenoceptor antagonists 23 1.4 a-adrenoceptors 25 1.4.1 Classification 25 1.4.1.1 a^-adrenoceptors 26 1.4.1.2 a 2-adrenoceptors 27 1.4.2 Identification and characterization 28 1.4.2.1 Binding studies 28 1.4.2.2 Purification and isolation 29 1.4.2.3 Cloning and sequencing 3 0 1.4.2.3.1. a^-adrenoceptors 30 1.4.2.3.2. 0:2-adrenoceptors 3 1 1.4.3 Molecular mechanisms 32 1.4.3.1 a^-adrenoceptors 32 1.4.3.2 a 2-adrenoceptors 34 1.4.4 a-adrenoceptor antagonists 35 v i CHAPTER Page 1.4.5 Vascular a-adrenoceptors 37 1.5 Aim of the thesis 38 1.5.1 Role of 6-adrenoceptors in the vasculature 38 1.5.2 Pressor response to 6-adrenoceptor antagonists 42 2 MATERIALS AND METHODS 46 2.1 Preparation of the rats 46 2.1.1 In vivo experiments 46 2.1.1.1 Measurements of MAP and HR 46 2.1.1.2 Measurements of the MCFP 46 2.1.1.3 Measurements of CO and BF 47 2.1.2 In vitro experiments 47 2.2 Experimental protocol 48 2.2.1 Selectivity of atenolol and ICI 118,551 48 2.2.2 6^ - and 62-adrenoceptor stimulation on haemo-dynamics in pentobarbital-anaesthetized rats 49 2.2.3 Effect of isoprenaline on MCFP 49 2.2.4 Pressor response to 6-adrenoceptor antagonists 50 2.2.4.1 Haemodynamic changes in urethane-anaesthetized rats 51 2.2.4.2 Haemodynamic changes in conscious rats 51 2.2.4.3 Effects of anaesthetic agents 52 2.2.4.3.1 Effects of urethane, pentobarbital and halothane on dose response curves to propranolol, atenolol and ICI 118,551 52 2.2.4.3.2 Effects of pentobarbital, amobarbital, ketamine and chloralose on i.v. bolus of propranolol 53 2.2.4.3.3 Effects of adrenaline on i.v. propranolol and atenolol in pentobarbital-anaesthetized rats 53 2.2.4.3.4 Haemodynamic changes in pentobarbital-anaes-thetized rats 54 2.2.4.4 Effects of captopril 54 2.2.5 In vitro cumulative dose-response curves for 6-adrenoceptor antagonists 55 2.3 The microsphere technique 55 2.3.1 Method 55 2.3.2 Calculations 57 2.4 Measurements of MCFP 58 2.5 St a t i s t i c a l analysis 58 v i i CHAPTER Page 2.6 Drugs 59 3 RESULTS 61 3.1 Selectivity of atenolol and ICI 118,551 61 3.2 Effects of 6]_- and 63-adrenoceptor stimulation 63 3.2.1 Effects on MAP, TPR, CO and HR 63 3.2.2 Effects on blood flow and vascular conductances 66 3.3 Effects of isoprenaline on MCFP 77 3.1 Selectivity of atenolol and ICI 118,551 61 3.4 Pressor response to 6-adrenoceptor antagonists 81 3.4.1 Haemodynamic changes in urethane-anaesthetized rats 86 3.4.1.1 Effects on MAP, TPR, CO and HR 86 3.4.1.2 Effects on blood flow and vascular conductance 89 3.4.2 Haemodynamic changes in conscious rats 98 3.4.2.1 Effects on MAP, TPR, CO and HR 98 3.4.2.2 Effects on blood flow and vascular conductance 98 3.4.3 Effects of anaesthetic agents 103 3.4.3.1 Effects of urethane, pentobarbital and halothane on dose response curves to 6-blockers 103 3.4.3.2 Effects of pentobarbital, amobarbital, ketamine and chloralose on i.v. bolus of propranolol 111 3.4.3.3 Effect of adrenaline on i.v. bolus of pro-pranolol and atenolol in pentobarbital anaes-thetized rats 111 3.4.3.4 Haemodynamic changes in pentobarbital anaes-rats 117 3.4.4 Effects of captopril 117 3.5 In vitro dose response curves of 6-blockers 128 4 DISCUSSION 132 4.1 Selectivity of atenolol and ICI 118,551 132 4.2 Role of 6]_- and 62-adrenoceptors i - n t n e vasculature 132 4.3 Role of 6-adrenoceptors in the venous system 138 4.4 Pressor response to 6-adrenoceptor antagonists in phentolamine-treated rats 145 4.4.1 Haemodynamic changes 145 4.4.2 Effects of anaesthetic agents 152 v i i i Chapter Page 4.5 Reversal of a-adrenoceptor blockade by 6-adrenoceptor antagonists in the isolated rat pulmonary artery 157 4.6 Interaction between a-and 6-adrenoceptor antagonists 159 4.7 Conclusions 160 References 162 ix LIST OF TABLES Table Page 1 Effect of atenolol and ICI 118,551 on the ED 5 0 62 values for the chronotropic effect of dobutamine and the vasodepressor effect of salbutamol in pento-barbital-anaesthetized rats. 2 Control values of MAP, HR and MCFP in conscious rats. 78 3 MAP prior to and after the infusion of phentolamine 110 in rats anaesthetized with urethane, pentobarbital or halothane. X LIST OF FIGURES Figure Page 1 Effects of normal saline, isoprenaline, ICI 118,551 64 with isoprenaline, atenolol with isoprenaline and both ICI 118,551 and atenolol with isoprenaline on TPR and MAP in pentobarbital anaesthetized rats. 2 Effects of normal saline, isoprenaline, ICI 118,551 65 with isoprenaline, atenolol with isoprenaline and both ICI 118,551 and atenolol with isoprenaline on HR and CO in pentobarbital anaesthetized rats. 3 Effects of normal saline on distribution of blood 68 flow and vascular conductance in pentobarbital-anaesthetized rats. 4 Effects of isoprenaline on distribution of blood 70 flow and vascular conductance in pentobarbital-anaesthetized rats. 5 Effects of isoprenaline on distribution of blood 72 flow and vascular conductance in pentobarbital-anaesthetized rats pretreated with ICI 118,551. 6 Effects of isoprenaline on distribution of blood 74 flow and vascular conductance in pentobarbital-anaesthetized rats pretreated with atenolol. 7 Effects of isoprenaline on distribution of blood 76 flow and vascular conductance in pentobarbital-anaesthetized rats pretreated with ICI 118,551 and atenolol. 8 Dose-response curves for the effects of isoprenaline 80 or saline on MAP, HR and MCFP in conscious rats. 9 Dose-response curves for the effects of isoprenaline 83 or saline on MAP, HR and MCFP in conscious, hexa-methonium treated rats. 10 Dose-response curves for the effects of isoprenaline 85 or saline on MAP, HR and MCFP in conscious, noradren-aline-treated rats. 11 Effects of normal saline, phentolamine, propranolol 87 in the presence of phentolamine and saline in the presence of phentolamine on TPR and MAP in urethane-anaesthetized rats. xi Figure Page 12 Effects of normal saline, phentolamine, propranolol 88 in the presence of phentolamine and saline in the presence of phentolamine on CO and HR in urethane-anaesthetized rats. 13 Effects of normal saline on distribution of blood 91 flow and vascular conductance in urethane-anaes-thetized rats. 14 Effects of phentolamine on distribution of blood 93 flow and vascular conductance in urethane-anaes-thetized rats. 15 Effects of propranolol on distribution of blood 95 flow and vascular conductance in phentolamine-treated urethane-anaesthetized rats. 16 Effects of saline on distribution of blood flow 97 and vascular conductance in phentolamine-treated urethane-anaesthetized rats. 17 Effects of normal saline, phentolamine, propranolol 99 in the presence of phentolamine and atenolol in the presence of phentolamine on TPR and MAP in conscious rats. 18 Effects of normal saline, phentolamine, propanolol 100 in the presence of phentolamine and atenolol in the presence of phentolamine on CO and HR in conscious rats. 19 Effects of normal saline on distribution of blood 102 flow and vascular conductance in conscious rats. 20 Effects of phentolamine on distribution of blood 105 flow and vascular conductance in conscious rats. 21 Effects of propranolol on distribution of blood 107 flow and vascular conductance in phentolamine-treated conscious rats. 22 Effects of atenolol on distribution of blood flow 109 and vascular conductance in phentolamine-treated conscious rats. 23 Dose-response curves for propranolol on MAP in 112 groups of urethane, pentobarbital and halothane anaesthetized rats pretreated with phentolamine. x i i Figure Page 24 Dose-response curves for ICI 118,551 on MAP in 113 groups of urethane, pentobarbital and halothane anaesthetized rats pretreated with phentolamine. 25 Dose-response curves for atenolol on MAP in 114 groups of urethane, pentobarbital and halothane anaesthetized rats pretreated with phentolamine. 26 MAP in groups of rats anaesthetized with pento- 115 barbital, amobarbital, ketamine and chloralose during control conditions, phentolamine infusion and propranolol in the presence of phentolamine. 27 MAP in pentobarbital anaesthetized rats during 116 control conditions, adrenaline infusion, phento-lamine and adrenaline infusions and propranolol or atenolol injections in the presence of phentolamine and adrenaline. 28 TPR and MAP during control conditions, the infusion 118 of phentolamine and the injection of propranolol in the presence of phentolamine in pentobarbital-anaes-thetized rats. 29 CO and TPR during control conditions, the infusion 119 of phentolamine and the injection of propranolol in the presence of phentolamine in pentobarbital-anaesthetized rats. 30 Distribution of blood flow in pentobarbital-anaes- 121 thetized rats during control conditions, the i n -fusion of saline and the injection of saline in the presence of saline infusion. 31 Vascular conductance in pentobarbital-anaesthetized 123 rats during control conditions, the infusion of saline and the injection of saline in the presence of saline infusion. 3 2 Distribution of blood flow in pentobarbital-anaes- 12 5 thetized rats during control conditions, the i n -fusion of phentolamine and the injection of pro-pranolol in the presence of phentolamine infusion. 33 Vascular conductance in pentobarbital-anaesthetized 127 rats during control conditions, the infusion of phentolamine and the injection of propranolol in the presence of phentolamine infusion. Figure Page 34 MAP during control conditions, the injection of 129 captopril, the infusion of phentolamine and the injection of 6-adrenoceptor antagonists in the presence of phentolamine in conscious rats. 35 Maximum developed force in isolated rat pulmonary 130 artery stimulated by noradrenaline, in the presence of phento-lamine and both phentolamine and 6-adrenoceptor antagonists. 3 6 Dose-response curves for 6-adrenoceptor antagonists 131 on the isolated rat pulmonary artery in the presence of noradrenaline and phentolamine. xiv LIST OF ABBREVIATIONS Adrenaline A Cardiac output CO Count per minute cpm Heart rate HR Hour(s) h International Unit I.U. Intravenous I.V. Mean art e r i a l pressure MAP Mean circulatory f i l l i n g pressure MCFP Molecular weight Mr Minute min Noradrenaline NA Seconds s Standard error SE Subcutaneous sc Total peripheral resistance TPR Venous plateau pressure VPP XV ACKNOWLE DGEMENTS I would like to express my appreciation to a l l the members of the department of Pharmacology and Therapeutics. I am especially grateful for Dr. M.C. Sutter, Dr. M.J.A. Walker, Dr. R. Tabrizchi, Su l i n Lim and Caroline Bruce for their support and advice. I would like to thank Dr. Y-X Wang and H. Nguyen for their assistance in some of these studies. I am grateful to the B.C. Heart Foundation and the University Of British Columbia for their financial support. I am most grateful to my supervisor, Dr. C.C.Y. Pang whose constant encouragement and advice as well as her careful guidance have been invaluble. 1 GENERAL OVERVIEW The cardiovascular system plays a special role in li v i n g animals and in man. It i s the transport system for the delivery of oxygen and removal of carbon dioxide. It delivers nutrients from the gastrointestinal tract to a l l the body parts, carries waste products of cellular metabolism to the kidney and other excretory organs, transports electrolytes and hormones, maintains body temperature and transports ce l l s and immune substances. The cardiovascular system i s mainly under the control of the sympathetic nervous system, the renin angiotensin system and the vasopressin system. Stimulation of the sympathetic nervous system involves the release of catecholamines from the nerve terminals and from the adrenal medulla. Catecholamines stimulate either a- or 6-adrenoceptors in blood vessels causing either contraction or relaxation, respectively. The aim of this study i s to define the role of 6-adrenoceptors in the different ar t e r i a l beds in the rat. In addition, we studied the role of 6-adrenoceptors in the venous system. The use of 6-blockers in the presence of phentolamine was associated with a pressor response in animals and in humans. We also examined the conditions under which this pressor response was produced. 2 1. INTRODUCTION 1.1. The sympathetic nervous system The general anatomical features of the autonomic chain have been known since the end of the seventeenth century. The ganglia, communicating filaments with the spinal cord, and i t s branches to the visceral organs were described by Wil l i s (1644). However, at that time, the ganglionated chain was thought to arise from the cranial nerves. In the eighteenth century, the sympathetic ganglia was considered to arise from the spinal cord and this peripheral system was thought to be the site of involuntary action and associated with anabolic and metabolic aspects of digestion and circulation. In the nineteenth century, Gaskell (1886) outlined the anatomy of the peripheral autonomic nervous system and was able to construct the concept of bulbar, thoracolumbar and sacral outflows of involuntary nerves to the ganglia. The concept of a chemical substance being released from the nerve endings was f i r s t proposed by Dubois-Reymond (1860). In 1895, Oliver and Schafer showed that extracts from the suprarenal glands produced striking physiological effects upon the heart and arteries. This was followed by studies which showed the similarities in responses between the injections of suprarenal extracts and stimulation of the sympathetic nervous system (Lewandowsky, 1898; Langley, 1901). The active substance of the suprarenal gland was f i r s t . isolated by Abel and Crawford (1897) and was termed "epinephrin". Later, Takamine (1902) isolated this substance in a more purified form and he named i t "adrenalin". In 1905, E l l i o t proposed that stimulation of sympathetic nerves resulted in the release of minute amounts of an adrenaline-like substance. The concept of a receptive substance was introduced in 1905 by Langley who proposed that effector c e l l s have excitatory and inhibitory receptive substances and that the response to nerve stimulation depended on the type of substance present. In 1921, Loewi demonstrated that nerve endings, when stimulated, release a chemical substance which acts on the target tissue to produce a response. In the same year, Cannon and U r i d i l (1921)- showed that stimulation of the sympathetic hepatic nerves released a substance which was adrenaline-like and they called i t "sympathin". Later in 1933, Cannon and Rosenblueth proposed that "sympathin" i s present in two forms, an excitatory form (sympathin E) and an inhibitory form (sympathin I). Extracts from the spleen and heart were found to contain a substance which resembled noradrenaline (Euler, 1946 a, b) . In 1949, Peart showed that noradrenaline was the substance released by sympathetic nerve stimulation. Preganglionic fibres of the sympathetic nervous system arise from the intermediolateral columns of the spinal cord of a l l the thoracic and the upper three lumbar segments. These fibres synapse with the postganglionic fibres in the ganglia which are found at three locations; paravertebral, prevertebral and terminal. The postganglionic fibres innervate the various tissues and organs (Lefkowitz et a l . , 1990). Blaschko (1939) proposed the synthetic steps of nor-adrenaline and adrenaline from tyrosine which involve the hydroxylation of tyrosine to DOPA by the enzyme tyrosine hydroxylase, followed by decarboxylation of DOPA to dopamine by the decarboxylase enzyme. These two steps occur in the cytoplasm and are followed by the active transport of dopamine into the adrenergic vesicles and i t s conversion to noradrenaline by the enzyme dopamine 6-hydroxylase. In the adrenal medulla, noradrenaline i s methylated to adrenaline by the enzyme phenylethanolamine-N-methyl transferase. Catecholamines are stored at the terminals of the adrenergic nerve endings in vesicles together with ATP in the ratio of 4:1 as well as the chromogranins. The actions of cate-cholamines are terminated by either uptake or metabolic transformation. Uptake is divided into neuronal uptake or uptake-1 and extraneuronal uptake or uptake-2. Metabolic transformations of catecholamines involve two enzymes, monoamine oxidase (MAO) and catechol-O-methyltransferase (Lefkowitz et a l . , 1990). 1.2. Classification of adrenoceptors Dale (1906) introduced the use of the receptor concept in connection with the sympathetic nervous system when he studied the sympatholytic action of ergot alkaloids. Adrenotropic receptors were considered to be of two classes, those which predominantly mediate excitatory actions and others which predominantly mediate inhibitory actions. He showed that many of the excitatory actions of adrenaline, but not the cardioaccelerator effects were blocked by ergot alkaloids while the inhibitory effects were not. In 1948, Ahlquist showed that adrenoceptors cannot be simply c l a s s i -fied as excitatory and inhibitory. He cl a s s i f i e d adreno-ceptors by ranking the order of potency of six sympa-thomimetic agonists on different functions. He found that there were only two ranks of potencies for these sympatho-mimetic amines and concluded that there were two subtypes of adrenoceptors. The one which i s predominantly excitatory except in the intestine, was named alpha and the other which is predominantly inhibitory, except in the heart, was named beta (Ahlquist, 1948). 1.3. B-adrenoceptors 1.3.1. Classification Lands et a l . (1967) classified B-adrenoceptors into two subtypes, a B^-subtype found in the heart and adipose tissue and a B2-subtype found in the vasculature, bronchial and other smooth muscles. This classification was based on the relative potency of a series of sympathomimetic amines. The B^-adrenoceptor subtype has the following relative sensitivity of, ISO > A = NA while the B2-adrenoceptor sub-type has that of ISO > A » NA. Pharmacological studies using selective antagonists for both subtypes and ligand binding studies have confirmed this classification. A third subtype, B3, was cloned and i s postulated to be the receptor which mediates catecholamine actions on metabolic rate (Emorine et a l . , 1989). 1.3.2. Identification and characterization of B-adreno- ceptors 1.3.2.1. Binding studies The f i r s t successful direct radioligand experiments for B-adrenoceptors date back to 1974 with the ligands (-) [3H] dihydroalprenolol (DHA) (Lefkowitz et a l . , 1974), (±)[ 1 2 5I] hydroxybenzylpindolol (IHYP) (Aurbach et a l . , 1974) and (±)[3H] propranolol (Levitzki et a l . , 1974). Tritiated compounds have the disadvantage of having low specific radioactivity and therefore requiring high amounts of proteins for binding (Engel et a l . , 1981). Sporn and Molinoff (1976) reported that IHYP has highly nonspecific binding properties and binds to both B- and a-adrenoceptors in the rat cortical membrane. IHYP was also shown to bind to serotonin binding sites in rat cortex (Dickinson et a l . , 1981) . This shows that new compounds which are more selective and with higher specific radioactivity were needed. Engel et a l . (1981) introduced (±)[ 1 2 5Iodo] cyanopindolol which had no a f f i n i t y for a-adrenoceptors nor serotonin receptors. In addition to the radioligand 6-adrenoceptor antagonists, the agonists (±)[3H] hydroxybenzylisoprenaline (Lefkowitz and Williams, 1977, Williams and Lefkowitz, 1977) and (±)[3H] isoprenaline (Malchoff and Marinetti, 1976) were used. Recently, more selective radioligand binding antagonists [3H]-ICI 118, 551 (Lemoine et a l . , 1985) and (-) [ H] bisprolol (Kaumann and Lemoine, 1985) , were used for the study of 6 2 - and B^-adrenoceptors, respectively. Binding studies were used to examine B^adrenoceptor subtypes in different tissues via the determination of the relative potencies of the agonists isoprenaline, adrenaline and noradrenaline in competing with nonselective radioligand antagonists such as [3H] DHA and [ 1 2 5 I ] HYP. Binding studies are also useful in estimating the relative distribution of B^- and 62-adrenoceptors in different tissues. Tissues used for binding studies included intact chicken erythrocytes and erythrocyte ghosts (Malchoff and Marinetti, 1975), frog erythrocytes (Mukherjee et a l . , 1975), rat lung membranes (Barnett et a l . , 1978), rabbit lung membranes (Rugg et a l . , 1978; Brodde, 1986), kitten heart (Kaumann and Lemoine, 1985), guinea pig lungs, l e f t ventricular and, rat cortical membranes (Engel et a l . , 1981), rat live r , cat soleus muscle and l e f t ventricle (Minneman et a l . , 1979a), human heart membranes (Waelbroeck et a l . , 1983; Heitz et a l . , 1983), guinea pig trachea, dog heart and lung membranes (Manalan et a l . , 1981) and rat brain regions which included cortex, caudate, cerebellum, hippocampus and diencephalon (Minneman et a l . , 1979b). 1.3.2.2. Purification and isolation Two techniques were used to try to purify and characterize adrenoceptors: a f f i n i t y or photoaffinity labeling and a f f i n i t y chromatography. It i s to be noted 8 that these techniques purify only the 6-adrenergic binding site which i s not necessarily identical with the receptor. Various values for the molecular masses (Mr) were shown to exist in different species. Purification of the frog erythrocyte 6-adrenergic binding sites was performed in 1981 They were shown to be composed of a polypeptide of Mr = 58,000d (Shorr et a l . , 1981). The turkey erythrocyte 6-adrenergic binding site was shown by a f f i n i t y chromatography to have two peptides with Mr of 40,000 and 45,000 in the ratio of 4:1 (Shorr et a l . , 1982). Af f i n i t y labeling showed that in rat reticulocytes and in frog and turkey erythrocytes, predominant peptides with Mr = 65,000 + 53,000, 58,000 and 45,000 + 39,000, were present, respectively (Lavin et a l . , 1982). In duck erythyrocytes two polypeptides with Mr = 45,000 + 48,500 were photolabeled in a ratio of 4 : 1 (Rashidbaigi and Ruoho, 1981) while in pigeon erythrocytes there were three photolabeled binding units of Mr = 53,000 + 46,000 + 45,000, in the ratio of 5 (53,000): 2(46,000 + 45,000) (Rashidbaigi and Ruoho, 1982). 62-adrenergic binding sites purified from lung membranes of hamster, guinea pig and rat contain a peptide of Mr = 64,000 (Benovic et a l . , 1984). 62-adrenergic binding sites of guinea pig lung have a photolabeled peptide of Mr = 67,000 (Burgermeister et a l . , 1983), while those of canine lung membranes have a peptide of Mr = 52,000-53,000 (Homey et a l . , 1983). Rat hepatic B2-adrenergic binding site have a peptide of Mr = 67,000 (Graziano et a l . , 1985). &i~ adrenoceptors from human, canine, porcine and rat l e f t ventricle has a binding subunit with Mr = 62,000 (Stiles et a l . , 1983). Rat fat ce l l s considered to contain 6^-adrenoceptor binding subunits have a peptide of Mr = 67,000 (Cubero and Malbon, 1984). Venter (1987) concluded that both &i~ and 62-adrenoceptors share many structural features in common including a molecular mass of 68,000 and an isoelectric point of 5. The results of target size analysis show that &y- and 62-adrenoceptors have a molecular mass of 140,000 and 120,000, respectively. 1.3.2.3. Cloning and sequencing 6-adrenoceptors belong to the family of guanine nu-cleotide binding proteins [(G)-linked receptors]. The genes encoding human (Frielle et a l . , 1987) and turkey (Yarden et a l . , 1986) 6i-adrenoceptors and human (Kobilka et a l . , 1987a) and hamster (Dixon et a l . , 1986) 62-adrenoceptors have been cloned and sequenced. In addition, the gene for human 63 adrenoceptor has been recently cloned and sequenced (Emorine et a l . , 1989). A l l 6-adrenoceptor subtypes have seven hydrophobic a-helical membrane spanning domains of 20-28 amino acid residues. These are connected to an amino terminal which i s located extracellularly and to an intracellular carboxyl end. Three hydrophilic extracellular and three intracellular loops connect these a-helices. The 6-adrenoceptor also has two sites of N-linked glycosylation near the amino terminus. The human 6^, 6 2 and 63 adrenoceptors consist of 477, 413 (Frielle et a l . , 1989) and 402 (Emorine et a l . , 1989) amino acids, respectively. The percentage of sequence homology between 6 ^ - and 6 2 -adrenoceptors i s 54% (Frielle et a l . , 1989), while sequence homology between 6 3 - and 6 ^ - or 62-adrenoceptors ^ s 50.7% and 45.5%, respectively (Emorine et a l . , 1989). Similarities between the different 6-adrenoceptor subtypes are highest within the transmembrane domains. In comparing 6 ^ - and 62-adrenoceptors, the amino and carboxy termini of the receptors are less conserved. The f i r s t two cytoplasmic loops are conserved while the extracellular loops are not. The third cytoplasmic loop of 61-adrenoceptors i s 27 amino acid residues longer and contains 16 additional proline residues (Frielle et a l . , 1989). Chimeric receptor construction suggests that the amino acid residues within the a-helix IV-VII especially helix IV determine 6 -adrenoceptor agonist subtype specificity while amino acid residues in other helices are less important. Amino acid residues within helices II-VII may be involved in determining 6 ^ - vs 62-adrenoceptor antagonist specificity (Frielle et a l . , 1989). Strader et a l . , (1989) presented a model for the ligand binding domain of the receptor: (i) the ligand binding pocket l i e s within the transmembrane domain, (ii) the ligand is attached to the receptor by an ionic interaction between the carboxylate side chain of Asp 113 on helix III and the amino group of the ligand, a hydrogen bond between helix V serine 204 and 207 residues and the catechol hydroxyl groups and hydrophobic interactions between the helix VI Phe 290 and the aromatic portion of the ligand. It has also been suggested that cysteine residues within the extracellular loops may stabilize the ligand binding pocket via disulfide linkages (Raymond et a l . , 1990). The amine and carboxyl ends of the third intracellular loop, especially the amino residues (222-229) and (258-270), were demonstrated to be important in the coupling of 6-adrenoceptor to G protein (Strader et a l . , 1989). The potential sites of phosphory-lation by cAMP dependent kinase have been suggested to be present in the third intracellular loop and in the carboxyl terminus while the potential site of phosphorylation by 6-adrenoceptor kinase i s a serine and threonine-rich region near the carboxyl terminus (Deblasi, 1989). 1.3.3. Cellular signalling mechanisms The role of cAMP in the mediation of the effects of adrenaline was f i r s t suggested by Rail and Sutherland (1958) who showed that adrenaline increased accumulation of cAMP in fractions of liver homogenates and in particular prepa-rations from heart and skeletal muscle. This was followed by studying different tissues where accumulation of cAMP was increased by adrenaline, including the brain, especially the cerebellum, the lungs, spleen, epididymal fat and erythro-cytes (Klainer et a l . , 1962). It i s now generally agreed that the secondary messenger of 6-adrenoceptors in a l l tissues i s cAMP. The 6-adrenoceptor i s coupled to a guanine nucleotide binding protein (Gs) which activates adenyl cyclase (Stiles, 1989) . The G s protein i s a heterotrimer formed of a GTP-binding and hydrolysing unit which i s the a-subunit, plus a 6 and a i subunit. The G protein i s present in three con-formational states. The f i r s t is the G protein bound to GDP (inactive form) . This i s followed by the release of GDP converting the inactive form into a quite transient (empty) state. GTP then enters this empty state and an active form of the G protein i s produced which returns back to the i n -active state when GTP is hydrolysed (Bourne et a l . , 1991). G protein i s normally present tightly bound to GDP. 6 -adrenoceptor stimulation speeds the G activation cycle and GDP i s replaced with GTP. GTP causes dissociation of the G protein to an a-subunit and to the 6 f subunits. The former Will activate adenyl cyclase which in turn increases the level of cAMP (Gilman, 1990). In addition, the a-subunit has a GTPase activity that permits slow hydrolysis of bound GTP to GDP (Weiss et a l . , 1988). Although i t appears that 67 subunits do not activate adenyl cyclase, they are pro-posed to be important for several reasons. First, they can stabilize the alpha GDP form, thereby allowing GDP to dissociate slowly at a rate of 100 fold less than in the presence of a-subunit alone. This means that the 67 subunits dampen signal transmission in the resting state and act as "noise" suppressors (Bourne et a l . , 1991). Second, their dissociation allows the receptor to act as a catalyst (Birnbaumer, 1990). Third, their presence allows activation of G proteins, since receptors do not recognize the GDP-a complex alone (Birnbaumer, 1990). Another view i s that a l -though association of E>i subunits with a-subunits i s impor-tant for G protein activation (i.e catalysis of GTP binding), a-subunits alone can also interact with the receptor but G protein activation then i s inefficient (Weiss et a l . , 1988). G protein acts as an amplifier of the ligand effect since a few receptors are capable of activating many G protein molecules. G proteins also allow reversal of the action of the ligand as they have an internal turnoff mechanism whereby the a-subunit hydrolyzes GTP to GDP (Birnbaumer, 1990). The cAMP protein kinase consists of two different types of subunits, a regulatory (R) subunit which i s the binding site for cAMP and a catalytic (C) subunit. The enzyme usually exists as an inactive tetramer, R2C2- cAMP binds with high a f f i n i t y to the R subunit which decreases the af f i n i t y of the R subunit for the C subunit and leads to the dissociation of an R2-(cAMP)4 dimer and two free C subunits that are catalytically active (Taylor et a l . , 1988). 1.3.4. Localization of B-adrenoceptors in the Heart. Lands et a l . (1967) originally c l a s s i f i e d the 6-adrenoceptors in the heart as B^-adrenoceptors. The 6^ -adrenoceptors were proposed to mediate increases in heart rate, contractility and conduction velocity in the atrioventricular node, His Purkinje system and the ventricles (Lefkowitz et a l . , 1990). However, results from pharmacological and binding studies suggest that 6 -adrenoceptors in the heart are not homogeneous in nature. Pharmacological studies using cat hearts in vivo and in vitro showed that i t was possible that both 6 ^ - and 6 2 -adrenoceptors are both present and mediate chronotropic responses (Carlsson et a l . , 1972). 6-adrenoceptor subtypes were proposed to mediate different degrees of chronotropic and inotropic responses. In anaesthetized cats, the 6 ^ -adrenoceptor agonist H 80/62 produced equal inotropic and chronotropic effects at a given dose while the 62-adreno-ceptor agonist, terbutaline, produced significantly greater chronotropic than inotropic response. This was interpreted to indicate that both 6 ^ - and 62-adrenoceptors are present in the sinoatrial node, and in the myocardium of the ventricles, but with different relative distributions. The 61-adrenoceptors are the predominant type in both regions with 6 1 : 6 2 concentration ratio higher in ventricle than in sinoatrial node (Carlsson et a l . , 1977). In the isolated rat atria, salbutamol showed a relatively greater effect on rate than on contractile force (Farmer et a l . , 1970). Soterenol, another 62-adrenoceptor selective agonist was shown to have similar effects,, as salbutamol, on rate and contractile force of guinea pig a t r i a l strips (Brittain et a l . , 1970). Using p A 2 values for the selective 6 ^ -adrenoceptor antagonist atenolol and the selective 6 2 -adrenoceptor antagonist a-methylpropranolol, i t was shown that B2-adrenoceptors are present in addition to B^-adrenoceptors in cat but not guinea pig atrium (O'Donnell and Wanstall, 1979a). However, employing the B 2-selective agonist procaterol, a minor population of B2-adrenoceptors was revealed in the guinea pig (Johansson and Persson, 1983; O'Donnell and Wanstall, 1985) but not rabbit atrium (Costin et a l i , 1983). Bryan et a l . (1981) showed that only B--adrenoceptors are involved in mediation of chronotropic effects in the rat atrium. However O'Donnell and Wanstall (1985) showed variable results within the same species in which some rat atria contain a small population of 6 2-adrenoceptors while others did not. They concluded that the comparative importance of B2-adrenoceptors in chronotropic responses in atria are cat > guinea pig > rat > rabbit. Binding studies showed that the right atria of cat and guinea pig hearts contained both Bi~ and B 2-adrenergic binding sites in the ratio of 75 : 25 while the ventricles contained only B^-type (Hedberg et a l . , 1980; Engel et a l . , 1981). In the rabbit, the ratio of B^ to B 2 for the right and l e f t atrium is 72 : 28 and 82 : 18, respectively, while the ventricles contain mainly B^-adrenoceptor subtype (Brodde et a l . , 1982). In humans, there i s a mixture of both Bf- and 6 2-adrenergic binding sites in both the atrium and ventricle in the ratio of 65 : 35 (Heitz et a l . , 1983), while in the rat the ratio of e>-± to fl2 i s 83 : 17 (Minneman et a l . , 1979b). In mammals, the role of B^-adrenoceptors i s the same for different regions in the heart while that for 62-adrenoceptors i s sinoatrial node > atrium > ventricle (Kaumann et a l . , 1989). The role of 62-adrenoceptors in mediating increased a t r i a l force i s enhanced in patients chronically treated with selective 6i~adrenoceptor ant-agonists (Hall et a l . , 1988). Recently, 63-adrenoceptors have been suggested to be present in the heart; their role is also more important in the sinoatrial node, less in the atrium, and even less in the ventricle. The proposal for the existence of 63-adrenoceptors in the heart i s based on the results of studies using partial agonists such as (-) pindolol and CGP-12177 (Kaumann et a l . , 1989). (-)Pindolol causes chronotropic effects in the guinea pig atrium at concentrations much higher than those causing 6-adrenoceptor blockade. This chronotropic effect i s composed of a highly sensitive component which i s apparently mediated by 6 ^ - and 62-adrenoceptors, and a lower sensitivity component which is not blocked by propranolol or selective 6 ^ - and 6 2 -adrenoceptor antagonists, but i s blocked by bupranolol (Walter et a l . , 1984). 1.3.5. Beta-adrenoceptor agonists Adrenaline and noradrenaline are phenylethanolamine derivatives. Noradrenaline is considered to be an a- and a selective B^-adrenoceptor agonist (Vatner et a l . , 1985). Noradrenaline i s 5-10 times more active for 6 ^ - than 6 2 -adrenoceptors (Malta et a l . , 1985a). Isoprenaline, a com-pound with isopropyl substitution on the amine group, i s considered to be the prototype of nonselective 6 -adrenoceptor agonists. Substitution on the amine group of phenylethanolamine i s important for B-adrenoceptor stimu-latory effect. The greater the substituent size, the greater the B-adrenoceptor agonist activity of the compound. There are B 2-selective agonists with tert-butyl substitution, such as salbutamol and terbutaline, and those with an aromatic ring on the N-linked alkyl chain such as ritodrine, salmefamol and fenoterol. The latter compounds are believed to display increased 6-agonist potency and longer duration of action (Phillips, 1980). However, procaterol has an isopropyl substitution on the amine group as well as an ethyl group substituted at the a carbon and an amide nitrogen in a fused ring replacing the metaphenol substituent (Yoshizaki et a l . , 1976). Procaterol, a partial B 2-agonist with a selectivity for B 2 : E>\ between 100 and 1000, has been used to detect a minor population of B 2-adrenoceptors in different tissues (O'Donnell and Wanstall, 1985). The 62-agonist fenoterol has a B 2 : B^ selectivity of 20 : 1 (O'Donnell and Wanstall, 1981b). Many fl2-adrenoceptor agonists are used c l i n i c a l l y as bronchodilators while ritodrine i s used as a uterine relaxant to arrest premature labour under certain conditions (Weiner, 1985). There i s a limited availability of selective Bi-adreno-ceptor agonists. Dobutamine was classified as a B^-adreno-ceptor selective agonist (Malta et a l . , 1985a) but was also shown to have agonistic activity at a-adrenoceptors and no appreciable selectivity for either B^- or 6 2 - adrenoceptors 18 (Ruffolo et a l . , 1984). Prenalterol was suggested from whole animal and human studies to be a selective adrenoceptor agonist (Carlsson et a l . , 1977; Jennings et a l . , 1983). However, prenalterol's 6^  : 62 adrenoceptor selectivity i s dependent on tissue. It i s now believed that prenalterol i s a nonselective 6-adrenoceptor agonist and the observed cardiovascular selectivity i s due to the greater a b i l i t y of the cardiac tissue, in comparison to the vascular tissue, to respond (Malta et a l . , 1985a). Recently, McCaffery et a l . (1990) showed that prenalterol appears to act at both 6^ - and 62-adrenoceptors in humans. Other available 6i~adrenoceptor agonists include xamoterol, R0363 and OM-isoprenaline. Xamoterol i s a partial 6-adrenoceptor agonist (Nuttall and Snow, 1982; Malta et a l . , 1985b) which possesses a 100-fold selective a f f i n i t y for e^-adrenoceptors (Malta et a l . , 1985a). R0363 is also a partial agonist with 61-adrenoceptor selectivity (McPherson et a l . , 1980). It i s 2-3 times and 100-350 times less active than isoprenaline at £>1~ and 62-adrenoceptor sites, respectively (Iakovidis et a l . , 1980). Prenalterol, xamoterol and R0363 are phenoxypropanolamines where an oxymethylene group is inserted between the ring and the ethanolamine side chain. This oxymethylene group was suggested to increase 6^ -adrenoceptor activity (Raper et a l . , 1980). 6-adrenoceptor agonists which are selective for rat adipocyte l i p o l y t i c response have been developed. These include BRL 28410, BRL 35113 and BRL 35135 (Wilson et a l . , 1984). 1.3.6. B-adrenoceptor antagonists 1.3.6.1. Introduction The development of B-adrenoceptor antagonists dates back to the f i f t i e s when dichloroisoprenaline (DCI), the f i r s t 6-adrenoceptor antagonist was synthesized (Powell and Slater, 1958). Dichloroisoprenaline blocked the stimulatory effects of adrenoceptor agents on the heart as well as peripheral vasodilatation produced by sympathomimetic amines, but did not antagonize adrenoceptor mediated vasoconstriction (Moran and Perkins, 1958). The synthesis of DCI was followed by the synthesis of pronethalol which, in contrast to dichloroisoprenaline had only minor in t r i n s i c sympathomimetic activity (ISA) (Black and Stephenson, 1962). Pronethalol was shown to produce side effects in man such as lightheadedness and slight incoordination followed by nausea and vomiting (Black et a l . , 1964). It also produced lymphosarcoma and reticulum c e l l sarcoma in mice (Paget, 1963). Black et a l . (1964) later reported the synthesis of propranolol which demonstrated a better therapeutic ratio. Since the introduction of propranolol many other 6-adrenoceptor antagonists have been synthesized. 1.3.6.2. Classifications Fitzgerald (1969) originally classified B-adrenoceptor antagonists into five groups: Group 1: 6-adrenoceptor antagonists with membrane stabilizing activity and ISA and this included two subgroups depending on the degree of ISA; group 1A has high degree of ISA, agents in this group include dichloroisoprenaline; group IB, represented by pronethalol, alprenolol, KO 592 and oxprenolol, has less ISA. Group 2: B-adrenoceptor antagonists with membrane activity but no ISA; propranolol i s a prototype of this group. Group 3: 6-adrenoceptor antagonists with ISA but no membrane activity; representative drugs include INPEA (l-(4-nitrophenyl)-2-isopropylamine-ethanol. Group 4: 6-adrenoceptor antagonists with neither membrane activity nor ISA; sotalol is an example of this group. Group 5: 6-adrenoceptor antagonists having a greater activity on 6-adrenoceptors in some tissues than in others, representative drugs include practolol and butoxamine. Prichard (1978) reclassified 6-adrenoceptor antagonists into three divisions: Division I: Nonselective 6-adrenoceptor antagonists and these included four groups: Group I, with both ISA and membrane activity, such as alprenolol and oxprenolol; Group II, with membrane actions but no ISA, such as propranolol; Group III, with ISA but no membrane actions, such as pindolol and Group IV, has neither ISA nor membrane activity, such as sotalol and timolol. Division II: Cardioselective B-adrenoceptor antagonists which in turn are divided into four groups and include acebutalol in Group I, practolol in Group III and atenolol in Group IV. Division III: 6-adrenoceptor antagonists with o-adrenoceptor blocking properties, e.g., labetolol. 1.3.6.3. Chemical structure B-adrenoceptor antagonists belong to two main chemical series arylethanolamines and aryloxypropanolamines. Those which are aryloxypropanolamine derivatives are c l i n i c a l l y more important (Labrid et a l . , 1989). The following l i s t s the basic structural features of B-adrenoceptor antagonists: 1. The amino group: an amine substituent, which is a branched-chain alkyl group, i s required for 6-adrenoceptor antagonistic activity of aryloxypropanolamine (Labrid et a l . , 1989). A high degree of cardioselectivity i s obtained by the attachment of 3, 4 dimethoxy-phenylethyl side chain (Hoefle et a l . , 1975) and 4-amide substituted phenoxyethyl to the amino group, as with tolamolol (Augstein et a l . , 1973). 2. The aromatic ring: may be a benzenoid as in alprenolol, oxprenolol, practolol, atenolol and tolamolol, a bicyclic aromatic ring as in propranolol and nadolol or .heterocyclic ring as in pindolol and timolol. Para substitution by a r i g i d substituent of at least three atoms in size in the the phenoxy ring renders a l l compounds cardioselective. This group of compounds include practolol (Dunlop and Shanks, 1968), metoprolol (Ablad et a l . , 1973) and atenolol (Barret et a l . , 1973). The introduction of a combination of ortho and para substitution led to the development of very selective B^-adrenoceptor antagonists such as acebutalol (Phillips, 1980). ISA i s partly related to aromatic ring substitution with electron-withdrawing or polar groups which can be an N atom in the case of pindolol, methylamide group in the case of practolol and propylamide in the case of acebutalol (Labrid et a l . , 1989). 3. The side chain: any substitution in the alpha, beta or gamma position, or in the hydroxy 1 groups of the oxypropanolamine side chain, causes marked reduction in B-adrenoceptor activity. Extension of the side chain by one carbon, replacement of the ether oxygen by methylene, sulfur or nitrogen atom and methylation of the beta carbon w i l l lead to partial or complete loss of activity (Philip, 1980). Methylation of the a-carbon generally produces selective S>2~ adrenoceptor antagonists such as butoxamine (Levy, 1966) and ICI 118,551 ( B i l i s k i et a l . , 1980). 1.3.6.4. Selective B-adrenoceptor antagonists 1.3.6.4.1. Selective B2~adrenoceptor antagonist The f i r s t selective S>2-adrenoceptor antagonist to be synthesized was butoxamine (Burns and Lemberger, 1965). Bu-toxamine reduced the vasodilator effect of isoprenaline and reversed the vasodilator response of ethylnorepinephrine but had no effect on the chronotropic and inotropic effects of adrenaline and noradrenaline (Levy, 1966) . Subsequent S>2~ adrenoceptor antagonists developed include H 35/25 (Levi, 1967), IPS 339 (Imbs et a l . , 1977), a-methylpropranolol (Fitzgerald and O'Donnell, 1978) and ICI 118,551 (Bilski et a l . , 1980). The pA2 values for butoxamine in the trachea were 5.2-6.4, depending on the agonist, while in the atria they were 5.2-5.3. Its fl2 : 61 selectivity was 17 : 1 (O'Donnell and Wanstall, 1979b). The B 2 : selectivity for H 35/25 and a-methylpropranolol was 13.5 : 1 and 11 : 1, respectively, with pA2 values of 5.4-6.6 and 7.4-8.5, res-pectively (O'Donnell and Wanstall, 1979b). The low pA2 values for butoxamine and H 35/25 show that these compounds lack potency. Different values of 6 2 : &-± selectivity have been reported for IPS 339. Imbs et a l . (1977) reported pA2 values for IPS 339 of 6.0-9.2 and selectivity values of 155 : 1 in in vitro and 23-26 : 1 in in vivo experiments. On the other hand, O'Donnell and Walduck (1980) reported pA2 values of 7.3-8.0 and a selectivity value of only 3.3 : 1 for IPS 339. ICI 118,551 has a high degree of selectivity and specificity for B2-adrenoceptors with a selectivity value of 123 : 1 and a pA2 value of 9.3 and 7.2 in the uterus and the atrium, respectively ( B i l i s k i et a l . , 1983). Less selectivity 6 2 : of 53.7 : 1 and lower pA2 values of 8.7 and 7.0 were reported for this compound in the trachea and atria, respectively (O'Donnell and Wanstall, 1980). ICI 118,551 has no partial agonist activity but has membrane stabilizing actions ( B i l i s k i et a l . , 1983). 1.3.6.4.2. Selective fij-adrenoceptor antagonists Practolol was the f i r s t selective B^adrenoceptor ant-agonist to be synthesized. It has 1/4-1/3 the effects of propranolol in blocking the chronotropic and inotropic effects of isoprenaline in anaesthetized dogs and 1/150 the activity of propranolol in blocking adrenaline-induced re-laxation of isolated guinea pig tracheal chain (Dunlop and Shanks, 1968). Practolol i s a partial agonist with a pA2 of 6.9. Its selectivity values are 69 : 1 when measured by comparison of pA2 values from guinea pig atria and trachea (Leclerc et a l . , 1984), 8.7 : 1 when comparisons of chronotropic action and the peripheral resistance in dogs were made or 9 : 1 from binding studies (Leclerc et a l . , 1984) . Unlike practolol, atenolol does not have partial agonistic properties. It has a pA2 value of 7.3 (Barrett et a l . , 1973). Atenolol has a 6^  : 62 selectivity ratio of 5.5 : 1 by comparing the potency ratio for antagonism of a t r i a l versus vascular actions of isoprenaline ( B i l i s k i et a l . , 1983) and 4 : 1 as measured by binding studies (Leclerc et a l . , 1984). Celiprolol i s also a 61-blocker with ISA and i t has a 100-fold greater a b i l i t y to antagonize 6^ - than 62-adrenergic stimulation (Jackson et a l . , 1987). Celiprolol, however, has 62-adrenoceptor agonist activity (Taylor, 1988). Betaxolol has a pA2 value of 8.3 and a selectivity ratio of 224 : 1. Metoprolol, on the other hand has a pA2 value of 7.5 and a selectivity ratio of 32 : 1 (Boudot et a l . , 1979) while tolamolol has a pA2 value of 8.3 and Bx : 6 2 ratio of 9.3 : 1 (Adam et a l . , 1974). B i l i s k i et 25 a l . (1983) explained that the possible reasons that drug selectivity ratios vary with different studies were due to differences in tissues, agonists or methodologies used for P A 2 determinations. 1.4. Alpha adrenoceptors 1.4.1. Classification Brown and Gillespie (1956; 1957) showed that in the per-fused cat spleen, the presence of an irreversible alpha-adrenoceptor antagonist increased noradrenaline overflow with nerve stimulation. These investigators attributed this action to the blockade of postjunctional receptors resulting in higher levels of noradrenaline. The concept of a presynaptic receptor regulation of noradrenaline release was introduced by four independent groups (Farnebo and Hamberger, 1971; Kirpekar and Puig, 1971; Langer et a l . , 1971; Starke, 1971). Starke (1972) showed that, in the rabbit heart, the relative potencies of phenylephrine, oxymetazoline and naphazoline at the presynaptic receptors did not agree with those at the postsynaptic receptors. In the cat spleen, phenoxybenzamine was more potent in blocking postsynaptic rather than presynaptic receptors (Langer, 1973). This led to the classification of a-adrenoceptors into a^- and a2~subtypes based on anatomical distribution of receptors. Prejunctional adrenoceptors mediating inhibition of the release of noradrenaline were termed a 2 while postjunctional adrenoceptors mediating contraction were termed a^ (Langer, 1974). Subsequently, compounds that are selective for subtypes of adrenoceptors became available and a-adrenoceptors were classified on the basis of their d i f f e r e n t i a l selectivity for various adrenoceptor agonists and antagonists (McGrath, 1982; Timmermans and Van Zweiten, 1982). Starke (1981) defined a^- and a 2- adrenoceptors as a^- with antagonist a f f i n i t y of prazosin » corynanthine = yohimbine > rauwolscine while a 2 with a f f i n i t y of rauwolscine = yohimbine » corynanthine = prazosin. In addition to their existence in prejunctional sites, a 2-adrenoceptors were also found to exist postsynaptically in vascular smooth muscles, platelets, pancreas, the central nervous system and other tissues (Timmermans and Van Zweiten, 1982). Further subclassifications of a-adrenoceptors have been made: 1.4.1.1. a^-adrenoceptors In 1982, two different classifications of a^-adrenoceptors were proposed. One classification recognized a new subtype termed a±s which was stimulated by SGD 101/75 and blocked by phenoxybenzamine. The other subtype was sensitive to noradrenaline but not SGD 101/75 (Coates et a l . , 1982). The second classification differentiated a--adrenoceptors into a i a which was sensitive to phenylethanolamines and imidazolines and a ^ which was only sensitive to phenylethanolamines (McGrath, 1982). In 1986, ai~adrenoceptors were again classified into a ^ and a^B according their a f f i n i t i e s to phentolamine and WB 4101. The site with higher a f f i n i t y for phentolamine was termed a ^ while that with lower a f f i n i t y was termed <*IB- WB4101 labeled only a ^ subtype while prazosin had equal a f f i n i t y at both subtypes. The prazosin : phentolamine potency ratio for a n d a l B * s 3 * 5 : 1 a n d 8 0 : 1 respectively (Morrow and Greese, 1986) . In the same year, a-± adrenoceptors were also cl a s s i f i e d into C*IH and a ^ according to their high or low a f f i n i t y for prazosin, respectively (Flavahan and Vanhoute, 1986)., a^-adrenoceptors were divided into a l a which has higher a f f i n i t y for WB 4101 and allows influx of calcium through dihydropyridine sensitive channels and a ^ which has lower a f f i n i t y for WB 4101 and stimulates inositol phospholipid hydrolysis (Han et a l . , 1987). Chlorethylclonidine inactivated but did not affect a^ a (Minneman et a l . , 1988). Muramatsu et a l . (1990) showed that a^-adrenoceptors could be classifie d into three subtypes a^a, ^ I L a n d a l N B Y antagonist a f f i n i t y ; a^jj with a f f i n i t y prazosin > HV723 = WB4101 > yohimbine, a^L where prazosin = HV723 = WB4101 > yohimbine and where HV723 > WB4101 > prazosin > yohimbine. am i s sensitive while a ^ and auj are insensitive to chlorethylclonidine. 1.4.1.2. a2-adrenoceptors There i s evidence that the antagonist SK&F 104078 is more selective for post- than presynaptic a2~adrenoceptors which suggest that there may be two different subtypes of a2~adrenoceptors (Hieble et a l . , 1988). Postjunctional a2~ adrenoceptors were proposed to be classified into a2A- and a 2 B ~ a d r e n o c e P t o r s according to functional (Turner et a l . , 1984) and binding studies (Bylund et a l . , 1988). Alpha-2A has low a f f i n i t y for prazosin whereas a 2B has high a f f i n i t y for prazosin. A third, a 2C has been proposed to be present in the opossum kidney OK c e l l line (Murphy and Bylund, 1988). In the rat brain, quantitative autoradiography showed that there are two classes of receptors, a 2 s which i s rauwolscine sensitive and a 2 i which i s insensitive to rau-wolscine (Boyajian et a l . , 1987). Results from binding studies are also consistent with this classification (Boyajian and Leslie, 1987). Alpha 2B and a 2 s have been lo-calized to the caudate nucleus and this suggests that they are similar while a 2 ^ i s considered to be equivalent to a 2^ (Regan et a l . , 1988). a2K has a selective ligand oxy-metazoline while a 2 B has ARC-239 and chlorpromazine as se-lective ligands. a 2^ and a 2B have a dissociation constant ratio (prazosin to yohimbine) of 240-570 and 5.4, respec-tively (Murphy and Bylund, 1988) . a 2 c has a high a f f i n i t y for prazosin and a prazosin to yohimbine dissociation constant ratio of of 40 (Murphy and Bylund, 1988). 1.4.2. Identification and characterization 1.4.2.1. Binding studies [ 3H]-dihydroergocryptine which labels both a^- and a 2-adrenoceptor subtypes with equal a f f i n i t y was the f i r s t ligand used to study a-adrenoceptors (Miach et a l . , 1978). The proportions of ay- to a2-adrenoceptors were estimated by the analysis of biphasic displacement curves generated by selective agents (Hoffman et a l . , 1979). Subsequently, a^-adrenoceptors were identified by the antagonists, [^ Hj-WB 4101 and [ 3H]-prazosin (Morrow and Greese, 1986). adrenoceptors were identified with [ 3H]-clonidine, [3H] aminoclonidine, [3H] yohimbine, [3H] rauwolscine, and [3H] idazoxan. Binding studies have been performed in a variety of tissues including rat brain (Cheung et a l . , 1982; Morrow and Greese, 1986), vas deferens (U'Prichard and Snyder, 1979), guinea pig lung (Barnes et a l . , 1979), calf brain membranes (U'Prichard and Synder, 1977), rabbit and human bladder (Levin et a l . , 1988), rabbit uterine smooth muscle (Williams et a l . , 1976), human platelets (Cheung et a l . , 1982), human spleen, colon and kidney (Dickinson et a l . , 1986), rat t a i l artery (Cheung and Triggle, 1988), bovine retinal blood vessels (Forster et a l . , 1987) and rat mesenteric artery (Agrawal and Daniel, 1985). 1.4.2.2. Purification and isolation Different molecular masses for ai-adrenoceptor binding sites have been reported: 45,000 (Guellaen et a l . , 1982), 59,000 (Graham et a l . , 1982) and 85,000 (Venter, 1987). The proteins of lower molecular mass may be protolytic fragments of the intact 85,000 a^-adrenoceptor binding site. Target size analysis showed that a^-adrenoceptor binding site has a Mr of 160,000 indicating that they are present in the membrane in the form of dimers (Venter et a l . , 1984). The hepatic a^-adrenoceptor has a tertiary structure and is stabilized by disulfide and strong hydrophobic bonds (Parini et a l . , 1987). Several studies have shown that a 2-adrenoceptor binding sites have a Mr of 64,000 - 65,000 including binding sites purified from platelets and porcine brain (Regan et a l . , 1986; Repaske et a l . , 1987), [3H]phenoxybenzamine a f f i n i t y labeled human platelets (Regan et a l . , 1986) and [3H] parazidoclonidine photolabelled adrenal cortical a-adreno-ceptor binding sites (Jaiswal and Sharma, 1985). Other studies showed that a2-adrenoceptor binding sites have a Mr of 85,000 and i t was concluded that rat liver a^- and platelet a 2-adrenoceptors are isoreceptors and dimers with the same molecular mass 85,000 and target size 160,000 (Shreeve et a l . , 1985; Venter, 1987). 1.4.2.3. Cloning and sequencing 1.4.2.3.1. a^-adrenoceptors The cloning of the cDNA which encodes the hamster a--adrenoceptor was reported. The pharmacological properties of this receptor, namely, the source (a vas deferens derived c e l l line), low a f f i n i t y for WB4101, inhibition by chlorethylclonidine and i t s coupling to inositol phospho-l i p i d metabolism, resembled those described for the a^g— adrenoceptor subtype (Cotecchia et a l . , 1988). A second a^-adrenoceptor was cloned from the bovine brain cDNA library. This receptor showed pharmacological properties proposed for a l A but unlike the a ^ adrenoceptor, i t was sensitive to inhibition by chlorethylclonidine and was not expressed in tissues such as rat vas deferens and hippocampus where the a^-adrenoceptors have been previously found (Schwinn et a l . , 1990). Recently a third cDNA clone from rat brain has been identified with high a f f i n i t i e s for prazosin and WB4101 (Harrison et a l . , 1991). The hamster a^-adrenoceptor has an amino acid sequence of 515 residues. It has seven membrane spanning domains of 20-25 hydrophobic residues connected by three extracytoplasmic and three cytoplasmic loops with an extracellular NH2 terminus and a COOH intracellular terminus (Gottechia et a l . , 1988). The bovine brain a^-adrenoceptor has a structure similar to that of the hamster a^-adreno-ceptor. Both have 72% identity within the membrane spanning domain which i s consistent with the presence of a very similar ligand binding domain. Sequence conservation extends to those regions of the third cytoplasmic loop and carboxyl-terminal cytoplasmic t a i l which are presumed to l i e closest to the plasma membrane. This might suggest similar effector functions of these receptors (Schwinn et a l . , 1990). 1.4.2.3.2. o^-adrenoceptors Three different genes for <*2-adrenoceptors have been cloned. The f i r s t i s from human platelets and has been termed ot2c10 (Kobilka et a l . , 1987b). The second i s from human kidney and resides on chromosome 4 ((X2C4) (Regan et a l . , 1988) and the third resides on chromosome 2 ( a 2 C 2 ) (Lomasney et a l . , 1990). Kobilka et a l . (1987b) proposed that &2C10 ^ s similar to <*2h' a2 c4' w a s suggested to be equivalent to c*2B receptor (Regan et a l . , 1988), however Lorenz et a l . (1990) proposed that i t represents both c^B and c^C. In contrast, Harrison et a l . (1991) concluded that a2C4 could correspond to the a 2 c subtype in the opossum kidney c e l l OK line. The &2^2 was found to be at2B~ like (Harrison et a l . , 1991). As with any G-protein linked receptor, a 2-adrenoceptors are formed of seven hydrophobic membrane spanning domains with three extracellular and three intracellular loops. a2c10~> a2 c4~ a n c* a2 c2-adrenoceptors are formed of 450, 461 and 451 residues, respectively (Lomasney et a l . , 1990). The greatest similarity between a2 c10 a n c* 2C4 ( 7 5%) (Regan et a l . , 1988), and between a2C2 and <*2C4 and a2C2(75% & 74%) are in the transmembrane regions. The amino terminus, the carboxyl terminus and the third cytoplasmic loop represent the most divergent domains (Lomasney et a l . , 1990). Employing chimeric receptors i t was shown that the seventh membrane spanning domain of o^-adrenoceptors may contain the major determinant of ligand spec i f i c i t y (Kobilka et a l . , 1988). 1.4.3. Molecular mechanisms 1.4.3.1. a^-adrenoceptors Hokin and Sherwin (1957) were the f i r s t to report that a-adrenoceptors activate phosphatidyl inositol turnover. They showed that adrenaline stimulated phosphatidyl inositol turnover in salivary gland slices and that this response was blocked by dibenamirie and ergotamine. The effect of adrenaline on phosphatidyl inositol labeling in hepatocytes was 1000 times more sensitive to blockade by prazosin than by yohimbine. This demonstrated that the effect i s mediated via the activation of a^-adrenoceptors (Tolbert et a l . , 1980). It i s now widely accepted that a^-adrenoceptors are coupled to phosphoinositide turnover (Garcia-Sainz, 1987). The i n i t i a l step after agonist induced receptor activation involves the activation of guanine nucleotide regulatory protein (Gx). Evidence for Gx involvement came from binding studies in which G x decreased the a f f i n i t y of adreno-ceptors for agonists (Lynch et a l . , 1985). Wallace and Fain (1985) also showed that G x stimulates phosphoinositide breakdown in isolated liver membranes. Garcia Sainz (1987) suggested that G x appears to be insensitive to pertusis toxin and i s different from adenyl cyclase coupled G^ and Gs. Guanine nucleotide activates phospholipase C enzyme which in turn leads to the hydrolysis of phosphatidyl-inositol-(4,5)-biphosphate to inositol-(1,4,5)-triphosphate [Ins(1,4,5)P 3] and diacylglycerol. Ins(1 , 4 ,5)P 3 acts as a secondary messenger activating the release of calcium from endoplasmic reticulum and calciosomes thereby increasing intracellular calcium concentration. Ins(l , 4,5 ) P 3 i s metabolized to either the inactive I n s ( l , 4 ) P 2 or the potentially active Ins(1,3,4,5)P4. The latter i s proposed to f a c i l i t a t e calcium entry across the plasma membrane and promote calcium movement between various intracellular non-mitochondrial stores (Nahorski, 1990). Diacylglycerol a c t i -vates protein kinase C which may be involved in the propagation of the hormonal signal and is part of a feedback system through phosphorylating ai-adrenoceptors (Garcia-Sainz, 1985). Han et a l . (1987) provided evidence for the existence of two a!-adrenoceptor subtypes with two biochemical responses; <*ib~ involves the hydrolysis of inositol phospholipid while c t i a - involves the activation of dihydropyridine sensitive calcium channels. This i s in accordance with results .from rat aorta experiments which show that the partial agonist Sgd 101/75 produced contraction by f a c i l i t a t i n g extracellular calcium entry which i s sensitive to blockade by nifedipine while agonists with higher int r i n s i c activity f a c i l i t a t e phosphatidyl-inositol turnover (Chiu et a l . , 1987). 1.4.3.2. an-adrenoceptors Alpha-2 adrenoceptors are coupled to adenyl cyclase via (Limbird, 1988). G^  i s a heterotrimic protein with a, B and i subunits and i s activated in the presence of GTP. It i s possible that a2-adrenoceptor agonists attenuate the activity of adenyl cyclase by dissociating the a and 6 subunits of G^. The dissociated B-subunit interacts with the a-subunit of G x thereby preventing i t from stimulating adenyl cyclase (Homey and Graham, 1985). Limbird (1988) reviewed other a2-adrenoceptor mediated pathways that lead to secretion or contraction responses. One such pathway is the acceleration of Na+/H+ exchange which plays an important role in adrenaline-evoked dense granule release from human platelets. This pathway involves the increase of cellular pH which sensitizes phospholipase A 2 resulting in the activation of arachidonic acid metabolism and the generation . 35 of various cyclooxygenase derivatives which leads to inositol triphospate and diacylglycerol formation, increase in intracellular calcium, activation of protein kinase and f i n a l l y , dense granule release. Isom et a l . (1987) reported the existence of this pathway in neuroblastoma X Glioma c e l l s . North et a l . (1987) showed that the activation of ci£2-adrenoceptors leads to an increase in membrane potassium conductance in guinea pig submucous plexus, rat locus coeruleus and substantia gelatinosa. Potassium channel activation leads to hyperpolarization which depresses neurotransmitter and hormonal release. This activatation possesses properties of an inward r e c t i f i e r current which may involve GTP binding protein (Limbird, 1988). Inhibition of selected voltage-dependent calcium channels was also proposed to be one of the pathways involved in the inhibition of neurotransmitter and hormonal release (Limbird, 1988). 1.4.4. a-adrenoceptor antagonists Ergotoxin was the f i r s t a-adrenoceptor antagonist to be described (Dale, 1906). a-adrenoceptor antagonists can be divided into four groups: 6-haloethylamine alkylating agents, imidazoline analogs, piperazinyl quinazolines and indole derivatives (Hoffman and Lefkowitz, 1990). The best known haloalkylamine derivatives are dibenamine and phenoxybenzamine. Phenoxybenzamine forms a reactive ethyleniminium or aziridinium ion and is covalently conjugated with a-adrenoceptors leading to irreversible blockade of the receptor. It has a slight selectivity for a^-adrenoceptors and i t inhibits both neuronal and extraneuronal tissue uptake of catecholamines. In addition, i t has antimuscarinic, antihistaminic and antiserotoninergic actions (Hoffman and Lefkowitz, 1990). Meier and Yonkman (1949) were the f i r s t to report the adrenolytic properties of the immidazoline derivative, phentolamine. Roberts et a l . (1952) showed that phentolamine increased femoral art e r i a l flow, decreased peripheral resistance and blocked the constrictor response of adrenaline in the innervated hindlimb of the dog. Phentolamine is a nonselective a-adrenoceptor antagonist which also blocks serotonin receptors and release histamine from mast cel l s (Hoffman and Lefkowitz, 1990). McPherson and Angus (1989) reported that phentolamine, at concentrations higher than those required to block a-adrenoceptors, antagonized the vascular action of the potassium channel opener, cromakalim. The reported pA2 values for phentolamine are 7.1 and 7.9 for the rat mesenteric artery (McPherson et a l . , 1984) and thoracic aorta (Digges and Summer, 1983), respectively. Phentolamine was shown to release insulin from isolated mouse is l e t s (Schulz and Hasselblatt, 1989). The prototype for piprazinylquinazolines i s prazosin; other members of this group include terazosin, doxazosin and trimazosin. A l l derivatives of piprazinylquinazolines are selective antagonists of a^-adrenoceptors (DeJonge et a l . , 1986). Prazosin i s also an inhibitor of cyclic nucleotide phosphodiesterase (Hess, 1975) and i t causes vasodilatation but l i t t l e reflex tachycardia (Hoffman and Lefkowitz, 1990). Other selective a^-adrenoceptor antagonists include corynanthine, WB 4101, YM12617 (De Marini et a l . , 1987). The pA2 values for prazosin, WB 4101, YM12617 and corynanthine are 8.9 (Honda et a l . , 1985), 8.8 (Melchiorri et a l . , 1984), 10.1 (Honda et a l . , 1985) and 6.6 (Weitzell et a l . , 1979), respectively. The f i r s t preferentially selective a 2-adrenoceptor antagonist to be reported was yohimbine (Starke et a l . , 1975a). Rauwolscine i s one of the yohimbine diastereomers which i s more selective for a2-adrenoceptors than yohimbine (Starke, 1981). Lattimar et a l . (1984) showed that some benzoquinolizines (WY25309, WY26392 and WY26703) were a 2-selective antagonists and these compounds are more potent and selective than yohimbine. Chapleo et a l . (1983) reported a series of benzodioxan analogs with a 2-adrenoceptor antagonist selectivity of which idazoxan i s the prototype. 1.4.5. Vascular a-adrenoceptors Prejunctional a-adrenoceptors have been identified in many vascular tissues for example, the rabbit pulmonary artery (Starke et a l . , 1975b), rabbit ear artery (Drew, 1979), rabbit and cat autoperfused hindlimb (Steppeler et a l . , 1978; Pichler and Kobinger, 1978). The existence of postjunctional a^- and a 2-adrenoceptors was f i r s t demonstrated by the inabi l i t y of prazosin to completely antagonize noradrenaline-induced contractions of the isolated human palmar d i g i t a l arteries (Moulds and Jauernig, 1977) , while i t was able to antagonize noradrenaline-induced contractions of human visceral arteries (Jauernig et a l . , 1978) . This indicated that there were prazosin-resistant and prazosin-sensitive vasoconstrictor a-adrenoceptors in human vascular smooth muscle. Later, the existence of postsynaptic a^- and a 2-adrenoceptors was shown in rats (Timmermans et a l . , 1979; Drew and Whiting, 1979), rabbits (Hamilton and Reid, 1982), dogs (Langer et a l . , 1981) and cats (Timmermans, 1981). 1.5. Aim of the thesis 1.5.1. Role of B-adrenoceptors in the vasculature The B-adrenoceptors of peripheral vascular smooth muscles were originally considered to be of the 6 2-adrenoceptor subtype (Lands et a l . , 1967). There i s now growing evidence that Bi~adrenoceptors may also be present in the vascular smooth muscles of different beds such as the canine renal vasculature (Taira et a l . , 1977), rat pulmonary artery (O'Donnell and Wanstall, 1981a), cat cerebral vessels (Edvinsson and Owman, 1974), rat jugular vein (Cohen and Wiley, 1978), rat aorta (O'Donnell and Wanstall, 1984a), rat femoral and mesenteric arteries (Fujimoto et a l . , 1988) and the vasculature supplying the adipose tissue (Belfrage, 1978) . The subclassification of B-adrenoceptors in the coronary vasculature remains controversial. Most in vitro studies showed that canine (O'Donnell and Wanstall, 1984b; Nakane et a l . , 1988; Toda and Okamura, 1990), porcine (Drew and Levy, 1972; Johansson, 1973), rabbit (Delande et a l . , 1974), bovine (Purdy et a l . , 1988), rat (Nyborg and Mikkelsen, 1985), human and monkey (Toda and Okamura, 1990) and sheep (Brine et a l . , 1979) coronary arteries contain only B--adrenoceptors. Binding studies showed that porcine (Schwartz and Velly, 1983), bovine (Vatner et a l . , 1986) and canine (Nakane et a l . , 1988) coronary arteries contain both B^- and Q>2-adrenoceptors with a ratio of 6^ to &2 °f 65 : 35, 1.5-2 : 1 and 74.-77 : 23-26 respectively. In an extensive review, Feigl (1983) concluded that most in vivo studies showed that E>2-adrenoceptors predominate in the coronary vasculature. By the use of electromagnetic flow measurements, Lucchesi and Hodgeman (1971) showed that 6 -adrenoceptors in the canine circumflex coronary artery were of the B^-subtype. Recently, in vivo studies showed that both 2>i~ and ti>2-adrenoceptors are present in the canine (Jackson et a l . , 1987; Trivella et a l . , 1990) and bovine (Vatner et a l . , 1986) coronary vasculatures. The aim of the present work was to determine the functional distribution of subtypes of 6-adrenoceptors in the resistance blood vessels of pentobarbital-anaesthetized rats. The effects of atenolol and ICI 118,551, selective B^- and 62-adrenoceptor antagonists, respectively, on cardiac and vasodilator response of isoprenaline were investigated by means of the dual radiolabeled microsphere technique. The exact role of 6-adrenoceptors in mediating contraction or relaxation of the venous system i s s t i l l controversial. Beta-adrenoceptors have been shown to be present in the human saphenous veins (Coupar, 1970), rat jugular vein (Dukles and Hurlbert, 1986), rabbit f a c i a l vein (Pegram et a l . , 1976), rabbit portal vein (Sutter, 1965; Hughes and Vane, 1967) and lateral saphenous vein (Guimaraes and Osswald, 1968). They mediated venodilatation to a degree which depends on the pre-existing tone and segment of vein studied. However, at high doses isoprenaline caused contractile responses which were abolished by a-adrenoceptor blockade (Sutter, 1965; Coupar, 1970). In humans, isoprenaline injection caused venoconstriction (Eckstein and Hamilton, 1959) or venodilatation (Beck et a l . , 1970) of the forearm vein. Leenen and Reeves (1987) showed that both R^-and 62-adrenoceptors were involved in the augmentation of venous return. In anaesthetized dogs, isoprenaline increased venous return and this effect was neither abolished by hexamethonium (Kaiser et a l . , 1964) nor carotid sinus denervation (Imai et a l . , 1978). In conscious dogs with cardiac output maintained constant, isoprenaline reduced MAP and increased central venous pressure (Bennett et a l . , 1984). In sedated dogs treated with hexamethonium, the selective 62-adrenoceptor agonist terbutaline did not have any effect on MCFP, an index of body venous tone (Guyton et a l . , 1973; Pang and Tabrizchi, 1986), but reduced venous compliance (Lee et a l . , 1987). In anaesthetized open-chest dogs, a single dose of isoprenaline did not produce any change in MCFP at normal tone but i t caused venodilatation after venous tone was increased with angiotensin II (Hirakawa et a l . , 1984). Rothe et a l . (1990) demonstrated that isoprenaline has l i t t l e influence on the MCFP in anaesthetized mongrel dogs. The aim of this study was to determine the dose-response effects of isoprenaline on MCFP in conscious rats. MCFP is the equilibrium pressure which would occur throughout the circulation i f a l l the pressures were brought to an equilibrium (Guyton, 1955). MCFP was shown experimentally to be directly proportional to venous return (Guyton, 1955) and mathematically to be inversely related to venous compliance (Grodins, 1959). The mathematical bases of MCFP was formulated by Grodins (1959). Q = (Pa - pv)/R (a) Pa = BV a/C a (b) P v = BV V/C V (c) BV = BVa + BVV (d) Where Q = cardiac output during steady state; P a and P v = ar t e r i a l and venous pressures, respectively; R = systemic vascular resistance; C a and C v = arter i a l and venous compliances, respectively; BVa and BVV = arter i a l and venous blood volumes. Equations (e) and (f) are obtained by rearranging these equations: P a = BV/(Ca + Cv) + CvRQ/(Ca + Cv) (e). P v = BV/(Ca + Cv) - CaRQ/(Ca + CV) ( f ) . When the circulation i s stopped, i.e., (Q = 0) , P a = P v = BV/(Ca + C v), at that time an equilibrium pressure can be obtained throughout the circulation. This pressure i s called the MCFP. 1.5.2. Pressor responses to B-adrenoceptor antagonists Paradoxical pressor responses to B-adrenoceptor antagonists have been reported in humans in certain c l i n i c a l conditions such as insulin-induced hypoglycemia (McMurty, 1974; Lioyd-Mostyn and Oram, 1975), pheochromocytoma (Prichard and Ross, 1966) and > patients treated with methyldopa (Nies and Shand, 1973). Different conditions of mental and physical stress also led to a pressor response to B-adrenoceptor antagonist. Andren et a l . (1981) showed that an increase in noise level during the administration of propranolol was associated with a pressor response. Waal-Manning (1974) showed that propranolol produced an increase in diastolic pressure during hand grip and mental arithmetic stress. Drayer et a l . (1976) reported that propranolol produced a pressor response in 11% of patients. In another group of patients with psychosis, 50% of the patients developed hypertension after propranolol administration and most of them had increased catecholamine levels in the urine (Atsmon et a l . , 1972). In a l l these conditions there was an increase in the activity of the sympathetic nervous system (Cleophas et a l . , 1988). Zahir (1971) reported that in young hypertensive patients, propranolol pretreatment markedly attenuated the hypotensive effect of phentolamine. Phentolamine-induced orthostatic hypotension was also prevented by 6-blockade (Majid et a l . , 1974). In anaesthetized dogs, propranolol did not increase sys-temic ar t e r i a l pressure but caused an increase in the femoral perfusion pressure (Nakane and Kusakari, 1966). It also produced sustained vasoconstriction in the denervated autoperfused hind limbs of dogs (Kayaalp and Kiran, 1966). The vasoconstrictor response was s t i l l present after block-ade of a-adrenoceptors by phentolamine (Kayaalp and Turker, 1967). The f i r s t report of a pressor response in rats was in 1969, where Dasgupta reported that propranolol produced an increased MAP in urethane-anaesthetized rats. This pressor response was blocked by pretreatment with reserpine, but not by pretreatment with hexamethonium nor phenoxybenzamine. These results were confirmed by Yamamoto and Sekiya (1969) who showed that either pronethalol or propranolol was cap-able of producing a sustained rise in MAP. This pressor re-sponse was markedly reduced by adrenalectomy or. ganglionic blockade but markedly potentiated by a-adrenoceptor blockade. A pressor response to a 6-adrenoceptor antagonist was also reported by other investigators (Regoli, 1970; Sugawara et a l . , 1980; Himori et a l . , 1984; Himori and Ishimori, 1988). Propranolol administered either orally (Kato et a l . , 1976) or i.v. (Nakao e t a l . , 1975) produced a pressor re-sponse in both conscious, spontaneously hypertensive and renal hypertensive rats. In conscious normotensive rats pretreated with phentolamine, d, 1 and dl forms of pro-pranolol, as well as practolol and YB-2, produced dose-dependent pressor responses. The maximal effects were similar for different isomers of propranolol but the thres-hold dose for the pressor effect of the d-isomer was higher than that of 1-isomer (Nakao et a l . , 1975). Gomes et a l . (1978) also showed that in conscious rats pretreated with phentolamine, propranolol restored MAP to almost the control levels. Tabrizchi et a l . (1988) showed that propranolol, atenolol and ICI 118,551 produced a dose-dependent increase in MAP in phentolamine-treated conscious rats. The pressor response to propranolol was present only in rats rendered hypotensive by phentolamine but not in those treated with methacholine nor sodium nitroprusside. These results sug-gest that a pressor response to propranolol requires the presence of an a-adrenoceptor blockade (Tabrizchi and Pang, 1989). We aimed to study the conditions under which a pressor response to a B-adrenoceptor antagonist occurs. We decided to study haemodynamic changes during infusion of phento-lamine and after selective and nonselective blockade of B-> adrenoceptors by atenolol and propranolol, respectively, in both conscious rats and urethane-anaesthetized rats. We also examined whether or not pressor responses to 6-adreno-ceptor antagonists occured in phentolamine-treated rats under conditions where the renin-angiotensin system had been inhibited. This was done in order to demonstrate the importance of the latter system. Propranolol failed to produce a pressor response in pen-tobarbital-anaesthetized rats in our preliminary studies, and so we also investigated the effects of various anaes-thetic agents on the pressor response to 6-adrenoceptor an-tagonists. In order to investigate the reason for the absence of a pressor response to propranolol in pentobarbital-anaesthetized rats, we determined regional flow distribution in anaesthetized rats during phentolamine-infusion and again after the injection of propranolol in the presence of phentolamine-infusion. In another group of pentobarbital-anaesthetized rats adrenaline was also infused prior to infusion of phentolamine and 6-adrenoceptor antagonist injection in an attempt to c l a r i f y i f absence of adrenaline inhibits 6-adrenoceptor antagonist pressor responses. Lastly, we determined whether 6-adrenoceptor antagonists reverse the effect of a-adrenoceptor blockade in an in vitro vascular smooth muscle preparation where experimental conditions could be r i g i d l y controlled. 2. MATERIALS AND METHODS 2.1. Preparation of the rats 2.1.1. In vivo experiments 2.1.1.1. Measurements of MAP and HR Male Sprague-Dawley rats from Charles river (300 -400 g) were used in a l l experiments. The right femoral artery and both femoral veins of the anaesthetized rats were cannulated for the measurement of MAP by a pressure transducer (P23DB, Gould Statham, CA, USA), and for injection of drugs, respectively. A l l cannulae were f i l l e d with heparinized saline (25 I.U./ml). HR was determined electronically from the upstroke of the art e r i a l pulse pressure using a tachograph (Grass, Model 7P4G). Different anaesthetic agents were used in various studies, namely, halothane (4% in air for induction and 1.5% in air for maintenance), urethane (1 g/kg, i.p.), pentobarbital (65 mg/kg, i.p.), amobarbital (100 mg/kg i.p.), ketamine (125 mg/kg, i.p.) and chloralose (90 mg/kg, i.p.). In conscious rat experiments, halothane was used to briefly anaesthetize the rats so as to allow cannulations of the femoral arteries and veins. The cannulae were tunneled s.c. to the back of the neck, exteriorized and secured. Rats were allowed 4 h to recover from the effects of surgery and anaesthesia before further use. 2.1.1.2. Measurements of the MCFP MCFP measurements in conscious rats were determined by the method of Yamamoto et a l . (1980). Male Sprague Dawley rats were anaesthetized with halothane (4% in air for induction and 1.5% for maintenance). Catheters were placed in the femoral artery for the measurement of the MAP and HR, in the right femoral vein for the infusion of drugs and in the inferior vena cava for the measurement of the CVP. A saline-filled-balloon-tipped catheter was inserted into the right atrium via the right external jugular vein. The proper location of the balloon was tested by inflation of the balloon to stop the circulation completely. This was shown by a simultaneous decrease in MAP to less than 25 mmHg and an increase in CVP. A l l cannulae were f i l l e d with heparinized saline (25 I.U./ml) and tunneled to the back of the neck, exteriorized and secured. The rats were allowed 24 h to recover from the effects of surgery and anaesthesia before further use. 2.1.1.3. Measurements of CO and BF Rats were prepared as described for the measurement of MAP and HR. In addition, a cannula was inserted into the l e f t ventricle via the right carotid artery for the injection of microspheres and another cannula was inserted into the l e f t femoral artery for the withdrawal of blood. 2.1.2. In vitro experiments Sprague-Dawley rats (250 - 300 g) were k i l l e d by a blow on the head followed by cervical dislocation. A small ring segment of the main pulmonary artery was immediately removed, mounted over two horizontal stainless steel rods and placed inside a 20 ml organ bath f i l l e d with Kreb's solution bubbled with 95% 0 2 and 5% C0 2 at 37 °C and containing desipramine HC1 (10~5 M) and corticosterone HC1 (10~ 5 M) to prevent neuronal and tissue uptake of noradrenaline, respectively. One of the rods was attached to a force-displacement transducer (Grass FT03) for isometric recording on a Grass Polygraph (Model 79D). The preparation was adjusted and maintained at a passive force of 10 mN and equilibrated for 1 h before further use. 2.2. Experimental protocol 2.2.1. Selectivity of atenolol and ICI 118.551 Six groups of experiments (I-VI, n = 4 in each group) were performed in pentobarbital-anaesthetized rats to test the &i and 6 2 selectivity of atenolol (100 /xg/kg) and ICI 118,551 (30 /xg/kcj) , respectively. In groups I and II, dose-chronotropic response curves for dobutamine (0.5 to 128 /xg/kg) were obtained 10 min prior to and 5 min after i.v. injection of atenolol and ICI 118,551, respectively. In groups III and IV, dose-depressor response curves for salbutamol (0.05 -6.4 jug/kg) were determined 10 min prior to and 5 min after i.v. injection of atenolol and ICI 118,551, respectively. In group V, the chronotropic effect of isoprenaline (32 ng/kg/min) was investigated 10 min before and 5 min after i.v. injection of atenolol. In group VI, the vasodepressor effect of the same dose of isoprenaline was investigated 10 min before and 5 min after i.v. injection of ICI 118,551. 2.2.2. S>i and fi2-adrenoceptor stimulation on haemodvnamics  in pentobarbital-anaesthetized rats Five groups of rats (n = 8 in each group) were used to investigate the effects of vehicle, mixed 6-stimulation, fi^-stimulation, fi2-stimulation and mixed 6-blockade in groups VII - XI, respectively on MAP, HR, CO, TPR and blood flows. In groups VII and VIII, microspheres were injected into the l e f t ventricle 30 min after surgery. After 5 min, normal saline (0.9% NaCl2, 0.013 ml/min/rat) or isoprenaline (32 ng/kg/min), was infused into groups VII and VIII, respectively. Microspheres were injected a second time 10 min after the start of vehicle or isoprenaline infusion. Groups IX, X and XI were treated similar to group VIII except that ICI 118,551 (30 M9/kg) , atenolol, (100 /xg/kg) or both of these blockers, respectively, were i.v. injected immediately after the f i r s t injection of microspheres and isoprenaline infusion was started 5 min after the injection of a 6-adrenoceptor antagonist. MAP and HR recordings during the f i r s t and second injections of microspheres were used to indicate responses during control conditions and drug treatments, respectively. 50 2.2.3. Effect of isoprenaline on MCFP in conscious rats Rats were divided into six groups (n = 6 in each group) in a complete random design. In group XII, dose-response curves of isoprenaline on MAP, HR and MCFP were constructed. Individual doses for isoprenaline were infused (2.5 x 10~ 1 0 - 8 x 10~9 mol/kg /min) for 5 min; each dose was followed by a recovery period of 10 min. In group XIII, normal saline was infused at the same rate as isoprenaline and this group served as the time control for group XII. In group XIV, hexamethonium (4.6 - 7.6 x 10~7 mol/kg/min) was continuously infused and at 10 min after the start of the infusion a dose-response curve to isoprenaline was constructed. Group XV which served as a control for group XIV, was treated similarly to group XIV except that, instead of isoprenaline, normal saline was infused at the same rate as isoprenaline. In each experiment in group XIV and XV, the lowest dose of hexamethonium producing > 50% inhibition of the tachycardic response to acetylcholine (20 /ig/kg) was used. In group XVI, noradrenaline was continuously infused (7.1 x 10~8 mol/kg/min) and at 20 min after the start of the infusion, individual doses of isoprenaline were infused (5 x 10" 1 0 - 4 x 10~9 mol/kg/min). Group XVII rats, the controls for group XVI, were treated similarly to group XVI except that normal saline was infused in place of isoprenaline. 51 2.2 .4. Pressor response to 6-adrenoceptor antagonists in  phentolamine-treated rats 2.2.4.1. Haemodynamic changes in urethane-anaesthetized  rats Four groups of rats v (n = 8 in each group) were used to investigate the effects of i.v. infusions of normal saline (group XVIII), phentolamine (group XIX), propranolol in rats given phentolamine (group XX) and saline in rats given phentolamine (group XXI) on MAP, HR, CO, blood flows and vascular conductances. In groups XVIII and XIX, the f i r s t injection of radioactively-labelled microspheres was conducted 30 min after surgery and this was followed immediately by the infusion of saline (0.026 ml/min/rat) or phentolamine (300 jig/kg/min) , respectively. Ten min later, a second set of microspheres was injected. In group XX, phentolamine infusion was started 30 min after surgery and this was followed, 10 min later, by the injection of the f i r s t set of microspheres. After another 10 rain, propranolol (100 /xg/kg) was injected i.v. This was followed by the injection of a second set of microspheres 1 min after the injection of propranolol. The same protocol as in group XX was followed in group XXI except that instead of propranolol, saline (0.1 ml/rat) was injected. 2.2.4.2. Haemodynamic changes in conscious rats Four groups of rats (XXII - XXV, n = 6 in each group) were used to investigate the effects of normal saline (XXII), phentolamine (XXIII), propranolol in rats given phentolamine (XXIV) and atenolol in rats given phentolamine (XXV) on MAP, HR, CO, TPR, blood flows and vascular conductances. After injecting the f i r s t set of microspheres, normal saline (0.026 ml/min/rat) or phentolamine (300 Mg/kg/min) was infused i.v. into groups XXII and XXIII, respectively. Fifteen min after the start of saline or phentolamine infusion, a second set of microspheres was injected. In group XXIV, phentolamine was continuously infused followed 10 min later by the injection of the f i r s t set of microspheres. After another 5 min, propranolol (100 Mg/kg) was i.v. injected. This was followed by the injection of a second set of microspheres 30 s after the injection of propranolol. The same protocol as XXIV was followed in group XXV except that instead of propranolol, atenolol (100 Mg/kg) was i.v. injected. 2.2.4.3. Effects of anaesthetic agents 2.2.4.3.1. Effects of urethane. pentobarbital and halothane  on dose-response curves to propranolol. atenolol and  ICI 118.551 Rats were divided into nine groups: groups XXVI, XXVII, and XXVIII (n = 6) were anaesthetized with urethane: groups XXIX (n = 5), XXX (n = 6) and XXXI (n = 6) were anaesthetized with pentobarbital; Groups XXXII (n = 8), XXXIII (n = 6) and XXXIV (n = 6) were anaesthetized with halothane. A l l rats were continuously i.v. infused with phentolamine (300 /xg/kg/min) . After 10 min of infusion, a dose-response curve to i.v. bolus injections of a 6-adrenoceptor antagonist was constructed in each group of rats: propranolol (3 x 10~9 - 1.92 x 10~7 mol/kg) in groups XXVI, XXIX and XXXII; ICI 118,551 (2 x 10"9 -1.28 x 10"7 mol/kg) in groups XXVII, XXX and XXXIII; atenolol (3 x 10"9 - 3.84 x 10"7 mol/kg) in groups XXVIII, XXXI and XXXIV. MAP recordings were noted at 1 min after the injection of each dose of a 6-adrenoceptor antagonist. 2.2.4.3.2. Effects of pentobarbital, amobarbital, ketamine  and Chloralose on i.v. bolus of propranolol Rats were divided into four groups (n = 5-6 in each group): Groups XXXV, XXXVI, XXXVII and XXXVIII were anaes-thetized with pentobarbital, amobarbital, ketamine and chlo-ralose, respectively. A l l rats were continuously infused with phentolamine (300 /xg/kg/min) . Ten min after phento-lamine infusion, propranolol (3 x 10~7 mol/kg, i.e. 100 /xg/kg) was i.v. injected into each rat. MAP was noted 10 min after phentolamine infusion and 1 min after the injection of propranolol. 2.2.4.3.3. Effects of i.v. infusion of adrenaline on i.v.. bolus propranolol and atenolol in pentobarbital-anaes- thetized rats Two groups of pentobarbital-anaesthetized rats (n = 6 in each group) were used. Groups XXXIX and "XL were given continuous i.v. infusion of adrenaline (300 ng/kg/min) followed 10 min later by continuous i.v. infusion of phento-lamine (300 /xg/kg/min) . After another 10 min, propranolol (3 x 10"7 mol/kg) and atenolol (3 x 10"7 mol/kg, i.e. 100 /xg/kg) were i.v. injected into groups XXXIX and XL, respec-tively. MAP was noted 10 min after adrenaline and phento-lamine infusions and 1 min after the injection of a 6-adrenoceptor antagonist. 2.2.4.3.4. Haemodynamic changes in pentobarbital-anaes- thetized rats Two groups of rats were used to investigate the haemo-dynamic effects of normal saline (group XLI, n = 6) and propranolol (group XLII, n = 8). After injecting the f i r s t set of microspheres, normal saline (0.026 ml/min/rat) or phentolamine (300 /xg/kg/min) was i.v. infused into group XLI and XLII, respectively. Ten min after the start of saline or phentolamine infusion, a second set of microspheres was injected. In group XLI and XLII, 15 min after the start of infusion of saline or phentolamine, respectively, saline or propranolol (100 /xg/kg) was i.v. injected followed 30 s later by the injection of a third set of microspheres. 2.2.4.4. Effects of captopril Rats were divided in three groups (XLIII - XLV, n = 6 in each group). Captopril (5 mg/kg) was injected as an i.v. bolus into rats in a l l three groups. Ten min later, phentolamine was continuously infused (300 /xg/kg/min) . Ten min after the start of phentolamine infusion, propranolol (100 Mg/kg) , atenolol (100 Mg/kg) and ICI 118,551 (30 /xg/kg) were i.v. injected in groups XLIII, XLIV and XLV, respectively. 2.2.5. In vitro cumulative dose-response curves of 6- adrenoceptor antagonists Isolated rat pulmonary arteries were divided into three groups (XLVI, XLVII and XLVIII n = 6 in each group) . They were contracted with noradrenaline (10~6 M) and at the plateau of the contractile response, phentolamine (10~ 6 M) was added and l e f t in the bath for 50 min. The tissues were then washed and allowed 1 h to recover. Afterwards, the tissues were again contracted with noradrenaline (10~6M) and relaxed with phentolamine (10~6 M) for 20 min. Then, in the presence of noradrenaline and phentolamine, cumulative dose-response curves for propranolol (10~9 - 10~6 M), ICI 118,551 (10~9 - 10"5 M) and atenolol (10~9 to 3 X 10"5 M), were con-structed in groups XLVI, XLVII and XLVIII, respectively. The maximum force and E C 5 0 values were obtained from indi -vidual dose-response curves. 2.3. The microsphere technique 2.3.1. Method CO, blood flow and vascular conductance were determined by the reference sample method (Malick et a l . , 1976; Pang, 1983). Radioactively labeled microspheres, 5 7Co, 1 1 3 S n and 5 1 C r (15 jum diameter, Du Pont, Canada) were used. They were suspended in F i c o l l 70 (10% in chlorbutanol) (Sigma Chemical Co., St. Louis, MO, USA) and Tween 80. Ten seconds before the injection of microspheres, blood was withdrawn (Harvard infusion/withdrawal pump) from the femoral artery into a heparinized syringe at a rate of 0.35 ml/rain for 1 min. A 0.15 ml sample of a vigorously vortexed precounted microsphere suspension (containing 20,000 - 40,000 microspheres) labeled with either 5 7Co, 1 1 3 S n or 5 1 C r was then injected and flushed with (0.2 ml) saline over 10 s into the l e f t ventricle. In half of the dual-isotope studies, 5 7Co was given f i r s t and 1 1 3 S n second. In the other half of the experiments, the order of administration of isotopes was reversed. In t r i p l e isotope studies, attempts were made to alter as much as possible the sequence in which the isotopes were given. This was done to avoid a possibility of a variation in the distribution between microspheres labeled with different isotopes and to avoid variations due to different counting efficiencies for the different isotopes. At the end of the experiments, the animals were k i l l e d by an overdose of pentobarbital. Whole organs (lungs, heart, liver, stomach, intestine, caecum, colon, kidneys, spleen, testis and brain), as well as representative samples from skeletal muscle (30 - 4 0 g) and skin (30 - 40 g) were removed, weighed and loaded into vials for counting radioactivity. The samples of skin were obtained from the dorsal and ventral areas and muscle samples were taken from the chest, abdomen and back. Large organs were cut into small pieces and loaded into several vials to a level less than 3 cm from the base. When blood flow to the l e f t kidney differed more than 20% from that of the right kidney, the experiment was rejected, as i t was assumed that the mixing of the microspheres was not adequate. Blood samples, tissue samples, syringes used for the injection and flushing of the microsphere suspension and for the collection of blood, and test tubes used for holding the microsphere samples were counted for radioactivity using a Searle 1185 series dual channel automatic gamma counting system (Nuclear-Chicago, I l l i n o i s , USA). In the dual isotope experiments, correction of Co counts was made by substracting Sn spillover (5 - 8%) from Co counts. In the t r i p l e isotope experiments, there was correction for the Co counts by substracting the Sn and Cr spillovers (8% each) and there was also correction for the Cr counts by substracting the Sn spillover (11%). 2.3.2. Calculations Blood withdrawal rate (ml/min) x t o t a l i n j e c t e d cpm CO (ml/min) = cpm i n withdrawn blood MAP (mmHg) TPR (mmHg.min/ml) = CO (ml/min) Blood withdrawal rate (ml/min) x t i s s u e cpm Tissue BF (ml/min) = • cpm i n withdrawn blood blood flow (ml/min) Tissue conductance (ml/mmHg.min) = •-MAP (nunHg) Total amount of radioactivity (cpm) injected was obtained by subtracting the amount of radioactivity l e f t in the tube, injecting syringe, and flushing syringe from the amount of radioactivity originally present in the tube. Radioactivity in the blood was obtained by adding the amount of radioactivity in the blood sample, in the cannula and in the syringe used for collecting blood. 2.4. Measurements of MCFP MCFP measurements were made in conscious, unrestrained rats after temporarily stopping the circulation by means of inflating the balloon previously inserted into the right atrium. Within 5 s following inflation of the balloon with the injection of saline, MAP decreased while CVP increased to a plateau value. The difference between steady state CVP, measured within 5 s of circulatory arrest, and baseline CVP, before the inflation of the balloon i s referred here as VPP. Samar and Coleman (1978) reported that there was incomplete equilibration of arter i a l and venous pressures. To correct for this, MCFP was calculated from the following equation (Yamamoto et a l . , 1980): MCFP (mmHg) = VPP'+ 1/60(FAP-VPP). FAP represents the f i n a l a r t e r i a l pressure (mmHg) obtained within 5 s following circulatory arrest. 2.5. S t a t i s t i c a l analysis A l l results were analysed by analysis of variance (ANOVA). In some experiments, data were logarithmically transformed before s t a t i s t i c a l analysis to obtain normal distribution. Duncan's multiple-range test was used to com-pare group means. A probability of error p < 0.05 was pre-selected as the criterion for s t a t i s t i c a l significance^ 2.6. Drugs In vivo; isoprenaline HC1, atenolol, dl propranolol HC1, norepinephrine bitartrate, adrenaline (Sigma Chemical Co., St. Louis, MO, USA), phentolamine HC1 (Ciba Pharmaceuticals, N.J., USA) and hexamethonium bromide (K and K Lab., CA, USA) were dissolved in normal saline. ICI 118,551 HC1 (Imperial Chemical, Macclesfield, Cheshire, England) and dobutamine HCl ( E l i L i l l y Canada, Toronto, Ontario) were dissolved in d i s t i l l e d water. Salbutamol sulphate vials (5 mg/lOml) were obtained from Allen & Hanburys (Toronto, Montreal) and the drug solution was diluted with normal saline. The following anaesthetic agents were used: halothane (Ayerst Lab.> Montreal, Canada), a-chloralose (BDH Chemical Ltd., Poole, England), urethane (ethyl carbamate) and ketamine HCl (Sigma Chemical Co., MO, USA), sodium pentobarbital (M.T.C. Pharmaceuticals, Ontario, Canada) and amobarbital ( E l i L i l l y & Co., Ontario, Canada). In vitro: Drugs used were desipramine HCl, corticosterone (Sigma Chemical Co., St Louis, USA), norepinephrine bitartrate, propranolol, atenolol, ICI 118,551 and phentolamine HC1. With the exception of nor-adrenaline which was made up in 0.01 N HC1, a l l other stock solutions were made up in d i s t i l l e d water. Dilution of drugs were made with Kreb's solution which has the following composition (mM): NaCl, 112; KC1, 4.5; NaHC03, 26.2; KH2 P0 4, 1.2; MgCl2, 1.2; EDTA, 0.026; Glucose, 11.1 and CaCl 2, 2.5. 3. RESULTS 3.1. Selectivity of atenolol and ICI 118.551 The E D 5 0 values for dobutamine chronotropic responses and salbutamol vasodepressor responses with and without the presence of a B-adrenoceptor antagonist (I - IV) are shown in Table 1. The injection of dobutamine in group I caused a dose-dependent increase in HR, from 345 ± 10 to a maximum of 503 ± 8 beats/min. After i.v. injection of atenolol in group I, dobutamine also caused a dose-dependent increase in HR, from 348 ± 9 to a maximum of 483 ± 11 beats/min, but the second curve was shifted to the right, with a significant increase in the E D 5 0 value. In group II, dobutamine also caused a dose-dependent increase in HR, from 328 ± 27 to a maximum of 455 ± 24 beats/min in the control condition. After i.v. injection of ICI 118,551 in group II, dobutamine had a similar dose response curve, with HR increased from 342 ± 18 to a maximum of 470 ± 20 beats/min and the E D 5 0 value unchanged. In group III, salbutamol caused a dose-dependent decrease of MAP, from 98 ± 4 to 50 ± 6 mmHg. After treatment - with atenolol, the dose response curve of salbutamol was similar to that in the control condition, with MAP decreased from 95 i 2 to 48 ± 4 mmHg and the E D 5 0 value unchanged. In group IV, salbutamol also caused a dose-dependent decrease in MAP from 98 ± 3 to 40 ± 3 mmHg in the control condition. ICI 118,551 caused a parallel s h i f t to the right of the salbutamol vasodepressor curve with MAP decreased from 97 ± 1 to 40 ± 4 mmHg and a significant Table 1. Effect of atenolol (100 Mg/kg) and ICI 118,551 (30 Mg/kg) on the E D 5 0 values of the chronotropic effect of dobutamine and vasodepressor effect of salbutamol in pentobarbital-anaesthetized rats (groups I - IV, n = 4 per group). Drugs Dobutamine E D 5 0 (Mg/kg) Salbutamol E D 5 0 (Mg/kg) Control Treatment Control Treatment Atenolol 5.8 ± 0. 8 23.2 ± 3.5a 0.19 ± 0.07 0.18 ± 0.07 ICI 118,551 5.9 ± 0. 7 5.6 ± 0.7 0.20 ± 0.10 0.84 ± 0.32a Values represent mean ± S.E. a S i g n i f i c a n t l y different from control values (p < 0.05). t 63 increase in the E D 5 0 value. In group V, infusion of isoprenaline increased HR from 320 ± 12 to 427 ± 20 beats/min, whereas in group VI isoprenaline decreased MAP from 100 ± 3 to 88 ± 4 mmHg. Atenolol completely abolished this chronotropic effect in group V, while ICI 118,551 completely abolished the vasodepressor effect of isoprenaline in group VI. 3.2. Effects of B^- and B2-adrenocept6r stimulation 3.2.1. Effects on MAP. TPR. CO and HR The effects of vehicle, mixed 6-stimulation, B^-stimula-tion, B2-stimulation a n c* mixed B-blockade in groups VII -XI, respectively, on cardiovascular functions are shown in Figures 1 (MAP and TPR) and 2 (CO and HR) . Mixed B-, R^-and B2-stimulation, and mixed 6-blockade were attained by i.v. infusion of, isoprenaline alone, isoprenaline in rats pretreated with ICI 118,551, isoprenaline in rats pretreated with atenolol and isoprenaline in rats treated with both atenolol and ICI 118,551, respectively. The infusion of normal saline (group VII) did not significantly affect MAP, TPR, CO or HR. Isoprenaline (group VIII) caused a s i g n i f i -cant increase in HR (Fig 2) but did not significantly alter MAP, TPR or CO. Isoprenaline in rats given ICI 118,551 (group IX) did not significantly alter MAP, TPR or CO, but caused a significant increase in HR (Fig 2) . Isoprenaline in rats pretreated with atenolol (group X) altered neither HR nor CO, but significantly decreased MAP and TPR. 64 ~ 2.00 E E d) I E E DC Q. 0.00 VI I VIII IX X X I D) z E E < 150 100 50 0 VI I VIII IX X X I Fig. 1. Effects of normal saline (VII), isoprenaline (32 ng/kg/min, VIII), ICI 118,551 (30 Mg/kg) with iso-prenaline (IX), atenolol (100 fig/Kg) with isoprenaline (X) and, ICI 118,551 and atenolol with isoprenaline (XI) on total peripheral resistance (TPR) and mean art e r i a l pressure (MAP) in five groups (n = 8 each) of pentobarbital-anaesthetized rats. Open bars denote pretreatment and hatched bars denote post-treatment values. Values are mean ± S.E. Significantly different from control values (p < 0.05). 65 150 i VII VIII IX X X I 600 i VII VIII IX X X I Fig. 2. Effects of normal saline (VII), isoprenaline (32 ng/kg/min, VIII), ICI 118,551 (30 /xg/kg) with iso-prenaline (IX) , atenolol (100 /xg/kg) with isoprenaline (X) and, ICI 118,551 and atenolol with isoprenaline (XI) on heart rate (HR) and cardiac output (CO) in five groups (n = 8 each) of pentobarbital-anaesthetized rats. Open bars denote pretreatment and hatched bars denote post-treatment values. Values are mean ± S.E. Significantly different from control (p < 0.05). 66 Isoprenaline in rats pretreated with both 6-adrenoceptor antagonists (group XI) did not significantly affect MAP, TPR, CO or HR, although there was a tendency for TPR to increase. 3.2.2. Effects on blood flow and vascular conductances The infusion of normal saline (group VII) altered nei-ther tissue blood flow nor vascular conductance in any or-gans or tissues (Fig. 3). The infusion of isoprenaline (group VIII) caused a slight but not significant increase in coronary blood flow but a large and a significant increase in muscle blood flow (Fig. 4a) . When flow was normalized for variations in arte r i a l pressure to give conductance values, isoprenaline was found to cause a significant increase (by 50%) in coronary arte r i a l conductance and a large increase (three times control value) in skeletal muscle vascular conductance (Fig. 4b). Flows and vascular conductances in other organs and tissues were not significantly affected. The infusion of isoprenaline in rats treated with ICI 118,551 to reveal 6]_-stimulation (group IX) caused significant increases in coronary and muscle blood flow and conductances (Fig. 5a and 5b). Flows and vascular conductances in other organs and tissues were not significantly affected by this treatment. The increase in muscle vascular conductance (by 40%) in group IX was significantly less than that in group VIII. 62-stimulation by isoprenaline in rats treated 67 Fig. 3. Effects of normal saline on the distribution of blood flow (a) and vascular conductance (b) in pentobarbital-anaesthetized rats (group VII, n = 8). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), liver (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki) , spleen (Sp), 40 g of skeletal muscle (Mu), 40 g of skin (Sk), testis (Te) and brain (Br). Control (open bars); Saline (hatched bars). 68 (a) C E o o o _l CO 25 i 20 15-10-5 0 r 1 ! H 1 1 I rfi 1 I Lg Ht Li St In C o Ki Sp Mu Sk Te Br (b) 0.30 1 I 0.25 -I CD I 0.20 a 0.15 0.10 0.05 0.00 c_> < I— o Q o o f n I I I 1 I I i Lg Ht Li St In C o Ki Sp Mu Sk T e Br 69 Fig. 4. Effects of isoprenaline (32 ng/kg/min) on the distribution of blood flow (a) and vascular conductance (b) in pentobarbital-anaesthetized rats (group VIII, n = 8). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), l i v e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki) , spleen (Sp), 40 g of skeletal muscle (Mu), 40 g of skin (Sk), testis (Te) and brain (Brj. Control (open bars); isoprenaline (hatched bars). Significantly different from control (p < 0.05). (a) 25 1 70 o o o —i DO 20 1 15 10 = 5 -0 1 I 1 i 1 I 1 •I l H IfPi Lg Ht Li St In C o Ki So Mu Sk T e Br (b) 0.30-I 0.25" | 0.20 o <c O :=> Q o 0.15 0.10 0.05 0.00 -K-i I 1 i Id*. I 1 Lg Ht Li St In C o Ki Sp Mu Sk Te Br 71 Fig. 5. Effects of isoprenaline (32 ng/kg/min) on the distribution of blood flow (a) and vascular conductance (b) in pentobarbital-anaesthetized rats (group IX, n = 8) pretreated with ICI 118,551 (30 /xg/kg) . Values are mean ± S.E. Organs or tissue samples are: lungs (Lg) , heart (Ht), li v e r (Li), stomach (St), intestine (In) , colon and caecum (Co), kidneys (Ki), spleen (Sp), 40 g of skeletal muscle (Mu), 40 g of skin (Sk), testis (Te) and brain (Br). Control (open bars); isoprenaline in the presence of ICI 118,551 (hatched bars). Significantly different from control (p < 0.05). (a) 25 i 72 o _i u_ o o o _i CO 20-15; 10-5 •0 r JL i * 1 I I I . 1 ^ 4 l Lg Ht Li St In C o Ki Sp Mu Sk Te Br (b) CD O < I— <_3 Z3 Q a o 0.30 0.25 0.20 0.15 0.10 0.05 0.00 CM i ft S cm cm Lg Ht Li St In C o K i Sp Mu Sk Te Br 73 Fig. 6. Effects of isoprenaline (32 ng/kg/min) on the distribution of blood flow (a) and vascular conductance (b) in pentobarbital-anaesthetized rats (group X, n = 8) pretreated with atenolol (100 /tg/kg) . Values are mean ± S.E. Organs or tissue samples are: lungs (Lg) , heart (Ht), liver (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp), 40 g of skeletal muscle (Mu), 40 g of skin (Sk), testis (Te) and brain (Br). Control (open bars); isoprenaline in the presence of atenolol (hatched bars). Significantly different from control (p < 0.05). 74 (a) o _i u_ o o o _l CQ 25 20 15 10 5 .0 I I I k Li Lg Ht Li St In C o K i Sp Mu Sk T e Br (b) o z «c I— o Q o 0.30 0.25 0.20 0.15 0.10 0.05 0.00 _Q2L 1 I I 1 I I i 1 Lg Ht Li St In C o K i Sp Mu Sk T e Br 75 F i g . 7. E f f e c t s of isoprenaline (32 ng/kg/min) on the d i s t r i b u t i o n of blood flow (a) and vascular conductance (b) i n pentobarbital-anaesthetized r a t s (group XI, n = 8) pretreated with ICI 118,551 (30 /zg/kg) and atenolol (100 Mg/kg)• Values are mean ± S.E. Organs or t i s s u e samples are: lungs (Lg), heart (Ht), l i v e r ( L i ) , stomach (St), i n t e s t i n e (In), colon and caecum (Co) , kidneys (Ki), spleen (Sp), 40 g of s k e l e t a l muscle (Mu) , 40 g of skin (Sk) , t e s t i s (Te) and brain (Br) . Control (open bars); isoprenaline i n the presence of JCI 118,551 and atenolol (hatched bars). S i g n i f i c a n t l y d i f f e r e n t from control (p < 0.05). 76 (a) Q O O _l CO 25 i 20 15 1 10 5 O i I 4 I. I ft L I i rn nil Lg Ht Li St In C o Ki Sp Mu Sk T e Br (b) cn o z <c I— o Q Z o o 0.30-0.25 -0.20-0.15 -0.10-0.05 -0.00 ^ r*"teh I I I 1 I Lg Ht Li St In C o Ki Sp Mu Sk Te Br with atenolol (group X) caused a large increase in muscle blood flow (Fig. 6a) similar to that in group VIII which received isoprenaline only (Fig. 4a); vascular conductances in the heart and muscle were also similarly, significantly increased (Fig. 6b). Flows and conductances in other tissues and organs were also not significantly altered by isoprenaline in rats pretreated with atenolol. The infusion of isoprenaline in rats treated with both adrenoceptor blockers (group XI) caused significant decreases in blood flows and conductances in kidneys, colon and caecum. Flows and vascular conductances in other beds were not significantly affected by this treatment (Fig. 7a and 7b). 3.3. Effects of isoprenaline on MCFP in conscious rats Table 2 includes the control values of MAP, HR and MCFP for the six groups (XII - XVII). There were no significant differences in control haemodynamic values among the groups. Mean baseline CVP in a l l groups was 2.9 ± 0.6 mmHg (n = 36) prior to the inflation of the a t r i a l balloon or any drug (or vehicle) treatment. None of the treatments significantly altered values of baseline CVP. The infusion of saline in group XIII caused no changes in MAP and HR but i t produced a small and gradual decrease in MCFP with time which reached s t a t i s t i c a l significance at the last dose (Fig. 8) . In group XII, isoprenaline dose-dependent ly increased HR and decreased MAP, accompanied by a small increase in MCFP which, when compared with the predrug 78 Table 2. Control values (means ± S.E.) of mean a r t e r i a l pressure (MAP), heart rate (HR) and mean circulatory f i l l i n g pressure (MCFP) in conscious rats (groups XII - XVII, n = 6 per group). MAP HR MCFP (mmHg) (beats/min) (mmHg) Group XII 110 + 4 416 + 9 5. 5 + 0. 3 Group XIII 108 + 3 377 ± 14 5. 4 + 0. 1 Group XIV No hexamethonium 112 + 3 395 ± 11 5. 4 + 0. 1 Hexamethon ium 98 + 3 a 386 ± 14 4. 7 + 0. l a Group XV No hexamethonium 111 + 4 368 ± 14 5. 7 + 0. 2 Hexamethonium 100 + 6 a 365 ± 16 5. 0 + 0. l a Group XVI No noradrenaline 112 + 5 386 ± 11 5. 2 + 0. 1 Noradrenaline 156 + 2 a 369 ± 14 7. 9 + 0. 2 a Group XVII No noradrenaline 108 + 4 375 ± 13 5. 4 + 0. 2 Noradrena1ine 143 + 7 a 352 ± 12 7. 1 + 0. 3 a a S i g n i f i c a n t l y different from values before the administration of a drug (p < 0.05). 79 Fig. 8. Dose-response curves for the effects (represented as change from control values) of isoprenaline (group XII) or saline (group XIII) on mean art e r i a l pressure (MAP), heart rate (HR) and mean circulatory f i l l i n g pressure (MCFP) in conscious, intact rats^ Each point represents the mean ± S.E. (n = 6 each). Significantly different from the normal saline group (p < 0.05). • Isoprenaline A Saline 80 20 O -20 -40 Change in M A P (mmHg) -60 Mr- i- -* * *--10 -9 -8 140 120-100-80 60 40 20 0 -20 Change in HR (beats/min) _1 i *• A -10 -9 -8 Change in M C F P (mmHg) 0 i -1 H -2 -10 -9 -8 L o g ( mol /kg/min ) control value prior to the infusion of isoprenaline, reached s t a t i s t i c a l significance at the fourth infused dose and when, compared with the corresponding MCFP value in the time-control group (XIII), reached s t a t i s t i c a l significance at a l l doses (Fig. 8) . In both groups XIV and XV, hexa-methonium caused similar decreases in MAP and MCFP but i t had no significant effect on HR (Table 2) . Saline infusion in group XV affected neither MAP nor HR but caused a small and gradual but insignificant decline in MCFP (Fig. 9). Isoprenaline in group XIV increased HR and decreased MAP but i t had no significant effect on MCFP when compared with either the predrug control value within the same group or the corresponding readings in the saline group (XV) (Fig. 9) . In groups XVI and XVII, noradrenaline caused similar increases in MAP and MCFP and small but insignificant reductions in HR (Table 2). The infusion of saline in group XVII did not significantly affect MAP. There was a tendency for HR to gradually increase and MCFP to decrease with the passage of time but these changes are not s t a t i s t i c a l l y significant (Fig. 10) . Isoprenaline decreased MAP and MCFP and increased HR when compared with the corresponding predrug control values within the same group or with the corresponding MAP, MCFP and HR readings in the time-control group. Isoprenaline, however, did not decrease MCFP back to the control level prior to the infusion of noradrenaline (Fig. 10). 3.4. Pressor response to B-adrenoceptor antagonists 82 Fig. 9. Dose-response curves for the effects (represented as change from control values) of isoprenaline (group XIV) or saline (group XV) on mean art e r i a l pressure (MAP), heart rate (HR) and mean circulatory f i l l i n g pressure (MCFP) in conscious, hexamethonium-treated rats. Each point represents the mean ± S.E. (n = 6 each) . Significantly different from the normal saline group (p < 0.05) • Isoprenaline A Saline -60 • ' — 1 -10 -9 -8 Change in HR (beats/min) -10 -9 -8 Change in MCFP (mmHg) L o g ( mol /kg/min ) 84 Fig. 10. Dose-response curves for the effects (represented as change from control values) of isoprenaline (group XVI) or saline (group XVII) on mean arte r i a l pressure (MAP), heart rate (HR) and mean circulatory f i l l i n g pressure (MCFP) in conscious, noradrenaline-treated rats. Each point represents the mean ± S.E. (n = 6 each) . Significantly different from the normal saline group (p < 0.05). • Isoprenaline * Saline 20 O -20 -40 -60 Change in M A P (mmHg) I L 1-1 -* 85 -9.50 -9.00 -8.50 -8.00 140 -120-100 80 60 40 20 0 -9.50 Change in HR (beats/min) I * •9.00 -8.50 -8.00 1 O --1 -Change in M C F P (mmHg) I ! -9.50 -9.00 -8.50 -8.00 L o g ( mol /kg/min ) 3.4.1. Haemodynamic changes in urethane-anaesthetized rats 3.4.1.1. Effects on MAP. TPR. CO and HR The effects of saline (group XVIII), phentolamine (XIX), propranolol in phentolamine-treated rats (group XX) and saline (group XXI) in phentolamine-treated rats on cardio-vascular functions are shown in figures 11 (MAP and TPR) and 12 (CO and HR). Groups XVIII and XXI are time controls for groups XIX and XX, respectively. MAP and HR readings were taken at the time of injection of the microspheres. The i n -fusion of normal saline in group XVIII did not produce any significant effects on MAP and HR. There was a tendency for CO to increase and TPR to decrease in the 10 min period be-tween the injections of the two sets of microspheres, but these changes were not s t a t i s t i c a l l y significant. The i n -fusion of phentolamine in group XIX significantly decreased MAP by reducing TPR but did not alter HR. CO was slightly but riot significantly decreased by phentolamine. The in-fusion of phentolamine in groups XX and XXI caused similar decreases in MAP as in group XIX, from 84 ± 2 to 50 ± 2 and from 94 ± 4 to 57 ± 2 mmHg, respectively. The injection of propranolol in rats given phentolamine in group XX s i g n i f i -cantly increased MAP to a level (90 mmHg) which i s slightly, but not significantly, higher than control MAP (84 mmHg) prior to the infusion of phentolamine, and i t raised TPR but altered neither HR nor CO. The pressor response to propra-nolol reached a peak within 1 min of injection of the drug and was sustained at approximately 90% of the peak response 87 1.50i 150 i 0) I 100-Fig. 11. Effects of normal saline (XVIII), phentolamine (300 /ig/kg/min, XIX), propranolol (100 /xg/kg) in the presence of phentolamine (XX), and saline in the presence of phentolamine (XXI), on total peripheral resistance (TPR) and mean arte r i a l pressure (MAP) in four groups (n = 8 each) of urethane-anaesthetized rats. Open bars denote pretreatment and hatched bars ^denote post-treatment values. Values are mean ± S.E. Significantly different from pretreatment (p < 0.05). 88 150i XVIII X IX X X XXI 600-c € 400 • c/) Fig. 12. Effects of normal saline (XVIII), phentolamine (300 /xg/kg/min, XIX) , propranolol (100 /xg/kg ) in the presence of phentolamine (XX), and saline in the presence of phentolamine (XXI), on heart rate (HR) and cardiac output (CO) in four groups (n = 8 each) of urethane-anaesthetized rats. Open bars denote pretreatment and hatched bars denote post-treatment values. Values are mean ± S.E. Significantly different from pretreatment (p < 0.05). 10 min later, at the time of termination of the experiments. The injection of saline in rats given phentolamine in group XXI altered neither MAP nor HR. There was again a tendency for TPR to decrease and CO to increase with the passage of time but these changes were not s t a t i s t i c a l l y significant. 3.4.1.2. Effects on blood flows and vascular conductances: The infusion of saline in group XVIII neither affected blood flow nor vascular conductance in any organs or tissues (Fig. 13). The infusion of phentolamine in group XIX significantly decreased flows in the l i v e r , stomach, colon and caecum, and kidneys but did not affect flows in any other organs or tissues (Fig. 14a). Phentolamine significantly increased vascular conductances in the muscle and skin beds but did not affect conductance in other beds (Fig. 14b). In group XX rats previously treated with phentolamine, propranolol reduced muscle flow but increased flows to the lungs, heart, liver, intestine, colon and caecum, kidneys and spleen. Flows in the other organs or tissues were not affected (Fig. 15a). When flow was norma-lized for MAP (conductance), propranolol caused significant decreases in arte r i a l conductances in the muscle, skin and kidneys but did not affect conductances in other beds (Fig. 15b) . The injection of saline in group XXI did not produce any significant change in flow or ar t e r i a l conductance in any tissue or organ (Fig. 16). 90 Fig. 13. Effects of normal saline infusion on the distribution of blood flow (a) and vascular conductance (b) in urethane-anaesthetized rats (group XVIII, n = 8) . Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), l i v e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki) , spleen (Sp) , 40 g of skeletal muscle (Mu) , 40 g of skin (Sk) and brain (Br). Control (open bars); normal saline (hatched bars). (b) CD g < Q O O 0.30 0.25 0,20 0.15 -0.10-0.05 -0.00 r=fl 4 Lg Ht Li St In C o K i Sp Mu Sk Br 92 Fig. 14. Effects of phentolamine infusion (300 /xg/kg/min) on the distribution of blood flow (a) and vascular conductance (b) in urethane-anaesthetized rats (group XIX, n = 8) . Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), l i v e r (Li) , stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp), 40 g of skeletal muscle (Mu) , 40 g of skin (Sk) and brain (Br^. Control (open bars) ; phentolamine (hatched bars). Significantly different from control (p < 0.05). 93 (a) 3 O O O —J CO 25-20-15-10-5 O -X-1 I I 1 J 3 B . Lg Ht St In C o Ki Sp Mu Sk Br (b) CD 3: <_> <c (— o ZD o o o 0.30-0.25 -0.20 0.15 i 0.10 0.05 1 0.00 P r 1 ! Lg Ht Li St In C o K i Sp Mu Sk Br 94 Fig. 15. Effects of propranolol (100 jug/kg) on the dis-tribution of blood flow (a) and vascular conductance (b) in phentolamine-treated (3 00 jig/kg/min) , urethane-anaes-thetized rats (group XX, n = 8). Values are mean ± S.E. Organs or tissue samples are: Lungs (Lg), heart (Ht), liv e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp), 40 g of skeletal muscle (Mu) , 40 g of skin (Sk) and brain (Br) . Phento-lamine treatment (open bars) ; propranolol iij the presence of phentolamine treatment (hatched bars). Significantly different from phentolamine treatment (p < 0.05). (a) o _1 u. o o o _l 00 25 -20-15 10 5 1 0 i _ L l J r l ™ • — ^ i Lg Ht St In C o K i Sp Mu Sk Br (b) < I— ZD Q Z o o 0.30 0.25 0.20-0.1 5 -0.10 0.05 1 0.00 1 1 1 -x-I Lg Ht Li St In C o Ki Sp Mu Sk Br 96 Fig. 16. Effets of normal saline on the distribution of blood flow (a) and vascular conductance (b) in phento-lamine-treated (300 iig/kg/min) , urethane-anaesthetized rats (group XXI, n = 8). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg) , heart (Ht) , liver. (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp),40 g of skeletal muscle (Mu) , 40 g of skin (Sk) and brain (Br) . Phentolamine treatment (open bars); saline in the presence of phentolamine (hatched bars). (a) o o o _l en 25 • 20-15 -10-5 0 r i Lg Ht Li St In C o Ki Sp Mu Sk Br (b) zz. < I— o Q o 0.3Oi 0.25 0.20 0.15 0.10 0.05 0.00 i 3 * r 1 * 1 " t ' - w ' I I _1 Lg Ht Li St In C o Ki Sp Mu Sk Br 3.4.2. Haemodynamic changes in conscious rats 3.4.2.1. Effects on MAP, TPR. CO and HR Figure 17 shows the changes of MAP and TPR, while figure 18 shows the effects on CO and HR after the administrations of normal saline, phentolamine, propranolol in phentolamine-treated rats and atenolol in phentolamine-treated rats in groups XXII, XXIII, XXIV and XXV, respectively. The i n -fusion of normal saline in group XXII did not significantly affect MAP, TPR, CO or HR. The infusion of phentolamine in group XXIII significantly increased HR and decreased MAP by reducing TPR since CO was not altered. Phentolamine in groups XXIV and XXV caused similar decreases in MAP as that in group XXIII, from 110 ± 4 to 73 ± 3 mmHg and from 111 ± 2 to 71 ± 3 mmHg, respectively. HR was also increased from 365 ± 16 to 450 ± 15 and from 368 ± 12 to 471 ± 8 beats/min in group XXIV and XXV, respectively. The subsequent injec-tion of propranolol (group XXIV) and atenolol (group XXV) in rats given phentolamine significantly increased MAP back to control levels (114 ± 3 and 112 ± 5 mmHg, respectively) . The pressor effects of propranolol and atenolol were accom-panied by an increase in TPR, no change in CO and reduced HR. 3.4.2.2. Effects on blood flows and vascular conductances: The infusion of normal saline in group XXII affected neither blood flow nor vascular conductance in any organ or tissue (Fig. 19). The infusion of phentolamine in 99 Fig. 17. Effects of normal saline (XXII), phentolamine (300 jug/kg/min, XXIII) , propranolol (100 /zg/kg) in the presence of phentolamine (XXIV) and, atenolol (100 /zg/kg) in the presence of phentolamine (XXV) , on total peripheral resistance (TPR) and mean arter i a l pressure (MAP) in four groups (n = 6 each) of conscious rats. Pretreatment (open bars); post-treatment (hatched bars). Values are mean ± S.E. Significantly d i f f e r -ent from pretreatment (P < 0.05). 100 150 c E _i E 100 m. O O 50 X X I I XXI I I X X I V X X V 600 400-c E •—. *-> ro <D ~ 200 n i X X I I XXI I I X X I V X X V Fig. 18. Effects of normal saline (XXII), phentolamine (300 jug/kg/min, XXIII) , propranolol (100 M9/kg) in the presence of phentolamine (XXIV) and, atenolol (100 /xg/kg) in the presence of phentolamine (XXV) , on heart rate (HR) and cardiac output (CO) in four groups (n = 6 each) of conscious rats. Pretreatment (open bars); gost-treatment (hatched bars). Values are mean ± S.E. Significantly different from pretreatment (p < 0.05). 101 Fig. 19. Effects of normal saline infusion on the distribution of blood flow (a) and vascular conductance (b) in conscious rats (group XXII, n = 6) . Values are mean ± S.E. Organs or tissue samples are: lungs (Lg) , heart (Ht), l i v e r (Li), stomach (St), intestine (In) , colon and caecum (Co), kidneys (Ki), spleen (Sp), 30 g of skeletal muscle (Mu), 30 g of skin (Sk), testis (Te) and brain (Br). Control (open bars); normal saline (hatched bars). 102 (b) E E o < I— o Q z: o o 0.30-0.25-0.20-0.15 -0.10-0.05 -0.00 fl i i Lg Ht Li St In C o Ki Sp Mu Sk Te Br group XXIII significantly increased flows in the lungs, heart and skeletal muscle, but decreased flows in the stomach, kidneys and spleen (Fig. 20a). Phentolamine significantly increased vascular conductances in the lungs, heart and skeletal muscle but i t did not affect conductances in other vascular beds (Fig. 20b). In group XXIV rats previously treated with phentolamine, propranolol reduced muscle flow, increased flow to the lungs but did not affect flows in other organs or tissues (Fig. 21a). Propranolol caused significant decreases in vascular conductances in the heart, intestine, kidneys, skeletal muscle and skin (Fig. 21b). The injection of atenolol in group XXV decreased blood flow to the skeletal muscle and increased blood flows to the lungs, intestine, caecum and colon, spleen, testis and brain (Fig. 22a). Atenolol reduced vascular conductances in the intestine, kidneys, skeletal muscle and skin (Fig. 22b). 3.4.3. Effects of anaesthetic agents 3.4.3.1. Effects of urethane. pentobarbital and halothane  on dose-response curves to B-blockers Baseline MAP (pooled values) in rats anaesthetized with pentobarbital i s significantly higher than MAP in rats anaesthetized with urethane or halothane (Table 3). Phentolamine reduced MAP in a l l nine groups (XXVI - XXXIV) of rats (Table 3) . An i . v. bolus of propranolol, IGI 118 ,551 or atenolol dose-dependently increased MAP in rats 104 Fig. 20. Effects of phentolamine infusion (300 /zg/kg/min) on the distribution of blood flow (a) and vascular conductance (b) in conscious rats (group XXIII, n = 6) . Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), li v e r (Li), stomach (St), intestine (In), colon and caecum (Co) , kidneys (Ki), spleen (Sp), 30 g of skeletal muscle (Mu) , 30 g of skin (Sk) , testis (Te) and brain (Br), gontrol (open bars); phentolamine (hatched bars). Significantly different from control (p < 0.05). « 105 (a) O O 25' 20-15 10" 5 " 0 X 1 i i. I 1 Lg Ht Li St In C o Ki Sp Mu Sk Te Br (b) 0.30 1 Lg Ht Li St In C o K i Sp Mu Sk Te Br Fig. 21. Effects of propranolol (100 /zg/kg) on the distribution of blood flow (a) and vascular conductance (b) in phentolamine-treated (300 /zg/kg/min) conscious rats (group XXIV, n = 6). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht) , li v e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp), 30 g of skeletal muscle (Mu) , 30 g of skin (Sk) , testis (Te) and brain (Br). Phentolamine-treated (open bars); pro-pranolol after phentolamine treatment (hatched bars). Significantly different from phentolamine treatment (p < 0.05). 107 (a) Q O O 25-20-15-10-5 0 I I i I Lg Ht Li St In C o K i Sp Mu Sk T e Br (b) C E B E o < I— <_> Q O o 0.30-0.25-0.20-0.15-0.10 0.05 i 0.00 1 I I i i i Lg Ht Li St In C o K i Sp Mu Sk Te Br 108 Fig. 22. Effects of atenolol (100 /xg/kg) on the distribution of blood flow (a) and vascular conductance (b) in phentolamine-treated (300 /xg/kg/min) , conscious rats (group XXV, n = 6) . Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht) , l i v e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp), 30 g of skeletal muscle (Mu), 30 g of skin (Sk), testis (Te) and brain (Br). Phentolamine-treated (open bars); atenolol after phentolamine treatment (hatched bars). Signifcantly different from phentolamine treatment (p < 0.05). (a) o o o _i CO 25 i 20 15 10 5i O I 1 f I i Lg Ht Li St In C o Ki Sp Mu Sk Te Br (b) e o < I— o o o o 0.30i 0.25 0.20 0.15 0.10 0.05 0.00 I I .1 nj I Lg Ht Li St In C o K i Sp Mu Sk Te Br 110 Table 3. Mean a r t e r i a l pressure (means ± S.E.) prior to and 10 min after the infusion of phentolamine (300 ixcr/kg/min) in rats anaesthetized with urethane (Groups XXVI - XXVIII), pentobarbital (Groups XXIX - XXXI) or halothane (Groups XXXII - XXXIV). n Control Phentolamine Urethane: Group XXVI 6 92 + 7 56 + 6 a Group XXVII 6 84 + 2 48 + 6 a Group XXVIII 6 92 + 7 47 + 2 a Pooled 18 89 + 3 51 + 3 a Pentobarbital: Group XXIX 5 105 + 3 66 + 6 a Group XXX 6 103 + 7 77 + 4 a Group XXXI 6 100 + 3 h 80 + 2 a Pooled 17 102 + 2 b 75 + 3 a Halothane: Group XXXII 8 98 + 1 69 + 3 a Group XXXIII 6 84 + 1 47 + 2 a Group XXXIV 6 83 + 3 61 + 2 a Pooled 20 89 + 2 63 ± 2 a a S i g n i f i c a n t l y different from control values (p < 0.05). b S i g n i f i c a n t l y different from pooled values in rats anaesthetized with urethane or halothane (p <0.05). anaesthetized with urethane but not pentobarbital (Fig. 23, 24 and 25) . In rats anaesthetized with halothane, ICI 118,551, but neither propranolol nor atenolol, caused a small dose-dependent increase in MAP. The maximal increase in MAP in response to the highest dose of ICI 118,551 under the influence of halothane (group XXXIII) was approximately 25% of the corresponding MAP value under the influence of urethane (group XXVII) even when baseline MAPs prior to and after the infusion of phentolamine were similar in the two groups (Table 3). 3.4.3.2. Effects of pentobarbital, amobarbital. ketamine  and chloralose on i.v. bolus of propranolol In groups rats anaesthetized with pentobarbital (XXXV), amobarbital (XXXVI) and chloralose (XXXVIII), phentolamine reduced MAP while propranolol did not produce any significant effect on MAP (Fig. 26). In ketamine anaesthetized rats (XXXVII) , MAP was higher than in rats anaesthetized with pentobarbital, amobarbital and chloralose and phentolamine caused a greater reduction in MAP. The injection of propranolol partially restored MAP to a level which i s lower than baseline MAP (Fig. 26). 3.4.3.3. Effect of adrenaline infusion on i.v. bolus  propranolol and atenolol in pentobarbital-anaesthetized rats 112 O) X E E CL < 0 D) C ITS O -20 ^ U r e -thane 60 -40 " 20 • 0 • -9 • P e n t o - * H a l o -barbital thane •8 -7 •6 Propranolol ( log mol/kg ) Fig. 23. Dose-response curves for propranolol on mean arte r i a l pressure (MAP) in groups of urethane (XXVI), pentobarbital (XXIX) and halothane (XXXII) anaesthetized rats pretreated with phentolamine (300 /xg/kg/min) . Each point represents the mean ± S.E. (n = 5-8 each). 113 I E E < (D c ITS U A U r e -thane 60 1 40 20" o --20 • P e n t o - * H a l o -barbital thane -9 -8 -7 -6 ICI 1 18,551 ( log mol/Kg ) Fig. 24. Dose-response curves for ICI 118,551 on mean arte r i a l pressure (MAP) in groups of urethane (XXVII), pentobarbital (XXX) and halothane (XXXIII) anaesthetized rats pretreated with phentolamine (300 ixg/kg/min) • Each point represents the mean ± S.E. (n = 6 each). 114 I e < U) C (0 JZ U A U r e -thane 60 40 " 20" 0 --20 •9 • P e n t o - A H a l o -barbital thane •8 7 ~6 Atenolol ( log mol/kg ) Fig. 25. Dose-response curves for atenolol on mean ar t e r i a l pressure (MAP) in groups of urethane (XXVIII), pentobarbital (XXXI) and halothane (XXXIV) anaesthetized rats pretreated with phentolamine (300 /xg/kg/min) . Each point represents the mean ± S.E. (n = 6 each). 115 150n Pentobarbital Amobarbital K e t a m i n e Chloralose Fig. 26. Mean arter i a l pressure (MAP) in groups of rats anaesthetized with pentobarbital, amobarbital, ketamine or chloralose (n = 5-6 each) during control conditions (open bars), 10 min after the start of a continuous infu-sion of phentolamine (300 /zg/kg/min) (hatched bars) and 1 min after the injection of propranolol (100 /zg/kg) during the infusion of phentolamine (cross-hatched bars}. S i g -nificantly different from control (p < 0.05); " S i g n i f i -cantly different from phentolamine treatment (p < 0.05). 116 Fig. 27. Mean ar t e r i a l pressure (MAP) in pentobarbital-anaesthetized rats (n = 6 each) during control conditions (open bars) , 10 min after the start of a continuous adrenaline (300 ng/kg/min) infusion (hatched bars), 10 min after the start of phentolamine (300 /xg/kg/min) infu-sion in the presence of adrenaline (cross-hatched bars) and 1 min after the injection of propranolol (100 /xg/kg, group XXXIX) or atenolol (100 /xg/kg, group XL) during the infusions of adrenaline and phentolamine (closed bars). aSignificantly different from control (p < 0.05); Sig-nificantly different from adrenaline (p < 0.05); S i g -nificantly different from phentolamine (p < 0.05). In groups XXXIX and XL, the infusion of adrenaline caused a small increase in MAP which was significant in group XL but not in XXXIX. The infusion of phentolamine reduced MAP in both groups. An i.v. bolus of propranolol and atenolol partially restored MAP to levels lower than MAP attained after the infusion of adrenaline (Fig. 27). 3.4.3.4 Haemodynamic changes in pentobarbital-anaesthetized  rats The infusion of saline (XLI) showed that time alone did not cause any changes in MAP, TPR (Fig. 28), CO, HR (Fig. 29) blood flow or vascular conductances (Fig. 30, 31). In group XLII, the infusion of phentolamine reduced MAP and TPR (Fig. 28), CO and HR (Fig. 29), however, only the reduction in MAP reached s t a t i s t i c a l significance. Propranolol injection in the presence of phentolamine did not have any significant effects on MAP, TPR, CO or HR (Fig. 28, 29) . Phentolamine infusion increased blood flow to the lungs and reduced flows to the skin and, caecum and colon. When BF was normalized by MAP, vascular conductance was increased only in the lungs. Subsequent propranolol injection did not have any significant effects on blood flow or vascular conductances in any of the organs or tissues (Fig. 32, 33). 3.4.4. Effects of captopril The control MAPs in the three groups, XLIII, XLIV and 118 2.00i c 1.50-E X L I X L I I Fig. 28. Total peripheral resistance (TPR) and mean arterial pressure (MAP) in two groups of rats. Group (XLI, n = 6) during control conditions, 10 min after the start of saline infusion and 1 min after saline injection in the presence of saline infusion. Group (XLII, n = 8) during control conditions, 10 min after phentolamine infusion (300 /^g/kg/min) and 1 min after propranlol (100 /(^ g/kg) injection in the presence of phentolamine infusion. . Control (open bars), after f i r s t treatment (hatched bars) and after second treatment (cross-hatched bars). aSignificantly different from control (p < 0.05). 119 Fig. 29. Cardiac output (CO) and heart rate (HR) in two groups of rats. Group (XLI, n = 6) during control conditions, 10 min after the start of saline infusion and 1 min after saline injection in the presence of saline infusion. Group (XLII, n = 8) during control conditions, 10 min after phentolamine infusion (300 jiig/kg/min) and 1 min after propranolol (100 jug/kg) injection in the presence of phentolamine infusion. Control (open bars), after f i r s t treatment (hatched bars) and after second treatment (cross-hatched bars). 120 Fig. 30. Distribution of blood flow in pentobarbital-anaesthetized rats (group XLI, n = 6) , during control conditions (closed bars), 10 min after the start of normal saline infusion (hatched bars) and 1 min after normal saline injection during the continuous infusion of normal saline (cross-hatched bars). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), l i v e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp), 30 g of skeletal muscle (Mu) , 30 g of skin (Sk) , testis (Te) and brain (Br) . 122 Fig. 31. Vascular conductance in pentobarbital-anaesthetized rats (group XLI, n = 6) , during control conditions (closed bars), 10 min after the start of normal saline infusion (hatched bars) and 1 min after normal saline injection during the continuous infusion of normal saline (cross-hatched bars). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), l i v e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp), 30 g of skeletal muscle (Mu) , 30 g of skin (Sk) , testis (Te) and brain (Br), 123 £ d) X E E LU U z < h-o D Q Z O O 0.00 Lg Ht D) I E E E LU O Z < h-u D Q Z 0 U 0.05 i 0.00 Ki Sp Mu Sk T e Br 124 Fig. 32. Distribution of blood flow in pentobarbital-anaesthetized rats (group XLII, n = 8), during control conditions (closed bars), 10 min after the start of phentolamine (300 jug/kg/min) infusion (hatched bars) and 1 min after propranolol (100 /xg/kg) injection during the continuous infusion of phentolamine (cross-hatched bars). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), l i v e r (Li), stomach (St), intestine (In), colon and caecum (Co), kidneys (Ki), spleen (Sp) , 30 g of skeletal muscle (Mu) , 30 g of skin (Sk), testis (Te) and brain (Br). aSignificantly different from control (p < 0.05). 126 Fig. 33. Vascular conductance in pentobarbital-anaes-thetized rats (group XLII, n = 8), during control condi-tions (closed bars), 10 min after the start of phento-lamine (300 /zg/kg/min) infusion (hatched bars) and 1 min after propranolol (100 /zg/kg) injection during the con-tinuous infusion of phentolamine (cross-hatched bars). Values are mean ± S.E. Organs or tissue samples are: lungs (Lg), heart (Ht), l i v e r (Li), stomach (St), intes-tine (In) , colon and caecum (Co), kidneys (Ki), spleen (Sp) , 30 g of skeletal muscle (Mu) , 30 g of skin (Sk) , testis (Te) and brain (Br). aSignificantly different from control (p < 0.05). 127 £ X E E LLI u Z < h-u D Q Z O o 0 .30 i 0 .00 L g Ht £ I £ £ 1 LU u Z < u D Q Z 0 u Ki Sp Mu Sk M i l M l~l T e Br XLV were, 106 ± 4, 105 ± 1 and 107 ± 2 mmHg, respectively. Captopril reduced MAP in a l l three groups, however, only the decrease in the second group reached s t a t i s t i c a l s i g n i f i -cance. Phentolamine reduced MAP in the three groups while subsequent injections of propranolol, atenolol and ICI 118,551 increased MAP; however, MAP in the three groups were not restored back to control levels prior to the infu-sion of phentolamine (Fig. 34). The pressor response to atenolol lasted for only 1 min after which MAP f e l l back to the phentolamine baseline value while those to propranolol and ICI 118,551 were sustained for the 20 min observation time before the termination of the experiment. 3.5. In vitro cumulative dose response curves of 6-adreno- ceptor antagonists In the three groups (XLVI, XLVII and XLVIII) of isolated — 6 • rat pulmonary arteries, noradrenaline (10 °M) caused an i n -crease in force which was subsequently reduced by phento-lamine and maintained for 50 min. After a f u l l recovery of the response, noradrenaline again caused a similar increase in force which was similarly reduced by phentolamine. The subsequent addition of propranolol, ICI 118,551 and atenolol dose-dependently increased force (Fig. 36) with E C 5 0 values of 4.1 ± 0.3 X 10"8, 1.2 ± 0.3 x 10"7 and 3.8 ± 1 X 10"7 M, respectively. Maximum forces developed in response to the 6-adrenoceptor antagonists were not significantly different from force produced by noradrenaline (Fig. 35). 129 150n XLIII X L I V X L V Fig. 34. Mean art e r i a l pressure (MAP) in groups of con-scious rats (n = 6 each) during control conditions (open bars), 10 min after the injection of captopril (5 mg/kg) (closed bars), 10 min after the start of phentolamine i n -fusion (300 jug/kg/min) (hatched bars) and 1 min after the injection of propranolol (100 /zg/kg) , atenolol (100 jug/kg) or ICI 118,551 (30 /xg/kg) during the infusion of phentolamine (cross-hatched bars) i n groups XLIII, XLIV and XLV, respectively. aSignificantly different from control (p < 0.05); "Significantly different from capto-p r i l (p < 0.05); c s i g n i f i c a n t l y different from phento-lamine after captopril injection (p < 0.05). 130 12 i Propranolo l ICI 118 .551 A t e n o l o l Fig. 35. Maximum developed force in isolated rat pul-monary artery (n = 6 each) in response to noradrenaline (10~^ M) (open bars), in the presence of phentolamine (10~ 6 M)(cross-hatched bars) and both phentolamine and 6-blockers (hatched bars). a S i g n i f i c a n t l y different from control (p < 0.05); "Significantly different from phento-lamine and noradrenaline (p < 0.05). 131 • P r o - O A teno lo l A ICI pranolol 1 1 8 , 5 5 1 4 i -9 -8 -7 -6 -5 -4 LOG DOSE ( M ) Fig. 36. Cumulative dose-response curves for propra-nolol, ICI 118,551 and atenolol on the isolated rat pul-monary artery in the presence of noradrenaline (10~° M) and phentolamine (10~ 6 M). 4 DISCUSSION 4.1. Selectivity of atenolol and ICI 118.551 ICI 118,551 (30 Mg/kg) shifted the dose-response curve for the vasodepressor effect to salbutamol but had no effect on the chronotropic response to dobutamine. Atenolol (100 ixg/kg) , on the other hand, shifted the dose-response curve for the chronotropic effect of dobutamine and had no effect on the vasodepressor action of salbutamol. Furthermore, ICI 118,551 completely abolished the vasodepressor and atenolol completely abolished the chronotropic effect of isoprenaline, indicating the effectiveness of blockade of B>2~ and B1-adrenoceptors by ICI 118,551 and atenolol, respectively. 4.2. Role of fij- and 62~a<3renoceptors * n ^ n e vasculature Infusion of the saline did not affect haemodynamics, indicating the reproducibility of the dual-isotope microsphere technique used in these experimental conditions. Infusion of the low dose of isoprenaline significantly increased HR and tended to decrease MAP and TPR and increase CO; however the latter changes were small and not s t a t i s t i c a l l y significant. Although CO and TPR were not significantly affected, distribution of regional blood flow was altered, with flow markedly increased in the muscle bed and slightly but not significantly increased in the coronary bed. Vascular conductances in both the muscle and coronary beds were significantly elevated suggesting the relative importance of 6-adrenoceptors in the skeletal muscle and coronary vasculature. Vascular conductance was not significantly changed in the lungs, liver, gastrointestinal tract, testis and brain suggesting the relative lack of 6 -adrenoceptors in the vascular beds in these sites. There was a tendency for conductance in the kidneys and skin to decrease, perhaps as a result of a compensatory increase in vasomotor influence to maintain MAP. When isoprenaline was infused into rats pretreated with ICI 118,551 to obtund 6i~adrenoceptor stimulation, HR was increased to an extent similar to that in rats that received isoprenaline only and MAP, CO and TPR also were not affected. Blood flows and conductances in coronary and muscle beds were increased. The increase in muscle vascular conductance was considerably less than that in rats that received isoprenaline only, suggesting that B^-adrenoceptor stimulation played a minor role in the vasodilator effect of isoprenaline in the muscle bed. Since B^-adrenoceptor stimulation caused both chronotropic and coronary dilator effects, i t i s not clear whether the increase in coronary conductance was entirely secondary to increased metabolic requirements or, in addition, there was a direct vasodilator effect on e^-adrenoceptors in the coronary vasculature. Flow and conductances in other vascular beds were not affected by 6^-adrenoceptor stimulation. The infusion of isoprenaline in in the presence of ateolol to obtund 62-adrenoceptor stimulation decreased MAP 134 and TPR but did not affect HR and CO. Since the previous infusion of isoprenaline alone at the same rate did not significantly reduce MAP and TPR, this suggests that blockade of the cardiostimulatory effect of isoprenaline by atenolol unmasked the depressor effect of isoprenaline. In the atenolol-treated rats that received isoprenaline, there was increased blood flow and conductance in the muscle beds, whereas only conductance was increased in the coronary bed; the magnitudes of these increases were similar to those in rats that received isoprenaline only. Flow and conductance in other organs and tissues were not significantly affected. Therefore, our results suggest that only fi2-adrenoceptors play a major role in the vasodilator effect of isoprenaline. The infusion of isoprenaline in rats that received both ICI 118,551 and atenolol did not significantly affect MAP, TPR, CO and HR. There was, however, a tendency for CO to decrease and TPR to increase. The slight increase in vasomotor tone after nonselective blockade of 6-adrenoceptors i s possibly due to the antagonism of the effects of endogenously-released catecholamines on 6-adrenoceptors. There were reductions in blood flows and vascular conductances in the kidneys, colon and caecum. Increase in flow and conductances in the coronary or skeletal muscle beds were no longer evident. This confirms that the slight increase in skeletal muscle blood flow after the infusion of isoprenaline in the presence of ICI 118,551 described previously was most probably due to the activation of B^-adrenoceptors and not due to an unblocked residual S>2~ adrenoceptor stimulation. To summarize, the vascular beds most sensitive to the actions of isoprenaline are those of the coronary and skeletal muscle. The vasodilator effects of isoprenaline in these two beds are mediated via the activation of both &i~ and B>2-adrenoceptors. Other studies have shown the presence of B^-adrenoceptors in the vascular smooth muscles of different beds such as the canine renal vasculature (Taira et a l . , 1977), rat pulmonary artery (O'Donnell and Wanstall, 1981a), cat cerebral vessels (Edvinsson and Owman, 1974), rat jugular vein (Cohen and Wiley, 1978), rat aorta (O'Donnell and Wanstall, 1984a), rat femoral and mesenteric arteries (Fujimoto et a l . , 1988) and the vasculature supplying the adipose tissue (Belfrage, 1978). In anaesthetized cats treated with phentolamine, vasodi-l a t a t o r responses to noradrenaline as well as to the selec-tive B-adrenoceptor agonists oxymethyleneisoprenaline and RO 363 were blocked by atenolol while vasodilator responses to adrenaline were blocked by butoxamine. This shows that in the cat, activation of either B^- or 62-adrenoceptors mediates vasodilatation (McPherson et a l . , 1981). In phentolamine-treated conscious dogs, the administration of noradrenaline or endogenously-released noradrenaline e l i c i t e d peripheral vasodilatation which was blocked by 136 atenolol suggesting that B^-adrenoceptors mediate vasodilatation under these conditions (Vatner et a l . , 1985). In the coronary vasculature, most in vitro experiments show the presence of 6 ^ - but not 62-adrenoceptors (O'Donnell and Wanstall, 1984b; Toda and Okamura, 1990; Baron et a l . , 1972; Drew and Levy, 1972; Nakane et a l . , 1988; Johansson, 1973; Delande et a l . , 1974; Purdy et a l . , 1988). Since large coronary arteries were used in a l l these studies, the results may not be indicative of responses in resistance blood vessels. Nyborg and Mickkelson (1985) studied B-adrenoceptor subtypes in isolated rat intramyocardial resistance arteries (i.d. = 200 itm) and they concluded that B^-adrenoceptors were present in these vessels. Binding studies (Schwartz and Velly, 1983; Vatner et a l . , 1986; Nakane et a l . , 1988) suggested the presence of both subtypes of 6-adrenoceptive sites in the coronary vasculature. Most in vivo studies suggested the presence of B 2 -adrenoceptors in the coronary vasculature (Feigl, 1983). However, there is' a technical problem with the use of beating hearts for these studies since they were incapable of demonstrating the presence of coronary B^-adrenoceptors, because B^^-adrenoceptor stimulation increases myocardial work thereby causing metabolic coronary vasodilatation. Lucchesi and Hodgeman (1971) used both isoprenaline and calcium chloride to increase coronary blood flow and they attributed the difference between the increases in coronary blood flow induced by the two agents to be the result of direct stimulation of coronary 6-adrenoceptors by isoprenaline. Since the selective 6i-adrenoceptor blocker practolol abolished the isoprenaline effect, the authors concluded that 62-adrenoceptors were present only in the coronary vasculature. However, Jackson et a l . (1987), using the same method in which calcium chloride i s compared to a 6-adrenoceptor agonist (but with a more selective 6 i~blocker celiprolol) showed that the isoprenaline-mediated increase in coronary blood flow was attenuated but not completely blocked by celiprolol while that of noradrenaline was completely blocked by ce l i p r o l o l . Although, celiprorol i s known to have 6 2-agonistic effect (Taylor, 1988), the authors used a dose (0.3 mg/kg) which was shown to have no effect on zinoterol (selective 62-agonist)-induced increase in hindlimb blood flow indicating that this dose of cel i p r o l o l has no effect on 62-adrenoceptors. The authors proposed the presence of both 6 ^ - and 62-adrenoceptors in the coronary vasculature. The use of calcium chloride as an inotropic agent was c r i t i c i z e d as i t has direct vasoconstrictor in addition to inotropic effects (Mark et a l . , 1972). Arrested heart preparations were used to separate the increase in coronary blood flow due to metabolic effects from that due to direct stimulation of coronary 6-adrenoceptors. However, there i s a drawback of this preparation due to i t s unphysiological condition. It was concluded from the result of one study using potassium chloride arrested hearts that 62-adrenoceptors predominate in the coronary vasculature (Gross and Feigl, 1975). Tri v e l l a et a l . (1990), using a canine nonbeating atrioventricular-blocked cardiac preparation to determine coronary blood flow during prolonged asystoles after stopping cardiac pacing, concluded that both B>x~ a n d ^ 2 " adrenoceptors are present in the coronary vasculature. Discrepancies in experimental findings between different laboratories may have resulted from the use of different species of animals and different methodologies, with in vitro and in vivo results indicative of responses in large arteries and small resistance vessels, respectively. Because small vessels are important determinants in blood flow distribution i t may not be valid to use results from in vitro studies which use large arteries to indicate responses in resistance blood vessels. In addition, in vitro results may depend on whether or not care was taken to preserve the intimal surface of the vasculature during preparation, as evidence shows that the a r t e r i a l endothelium may contribute to 6-adrenoceptor-mediated relaxation response (Rubanyi and Vanhoutte, 1985). 4.3. Role of B-adrenoceptors in the venous system We examined the effect of isoprenaline under different experimental conditions, namely, basal conditions, after ganglionic blockade with hexamethonium, and after the elevation of vascular tone with the infusion of noradrenaline. In the three control groups, there was a consistent but small gradual decline of MCFP. This has been observed previously (Waite et a l . , 1988; D'Oyley et a l . , 1989). In the intact rat, isoprenaline decreased MAP, and increased both HR and MCFP. The increase in MCFP was more prominent when the readings were compared with those of the time control group. To determine i f the venoconstrictor effect was mediated by a direct effect, or via reflex sympathetic activation, isoprenaline was also given to rats previously treated with hexamethonium so as to attenuate autonomic nerve activity. We selected to use a dose of hexamethonium which inhibited the tachycardic response to acetylcholine by >50%. This regimen was chosen to allow for a degree of venous tone in order that further venodilatation could be seen. In accordance with published results (D'Oyley and Pang, 1990), hexamethonium reduced both MAP and MCFP but had no significant effect on HR. Under these conditions, isoprenaline again dose-dependently increased HR and reduced MAP but failed to have any significant effect on MCFP. This indicated that the constrictor effect of isoprenaline on venous tone in the intact rat was indeed a consequence of reflex activation of the sympathetic nervous system. There was, however, a small but insignificant increase in MCFP at the highest isoprenaline dose level possibly as a result of incomplete ganglionic blockade. The infusion of noradrenaline significantly increased MAP and MCFP while slightly but insignificantly reduced HR. It has been shown that the infusion of noradrenaline into rats pretreated with propranolol caused dose-dependent increases in MAP as well as MCFP and reduced HR (Pang and Tabrizchi, 1986). This suggests that the lack of a significant bradycardic response in the present study was due to the opposing influence of direct /^-adrenoceptor stimulation. Under the condition of elevated vascular tone, the infusion of isoprenaline increased HR but reduced both MAP and MCFP. In summary, in intact rats isoprenaline increases MCFP by an indirect effect, i.e., hypotension-induced venoconstriction. The direct effect of isoprenaline i s dependent on the i n i t i a l venous tone so that after ganglionic:blockade, isoprenaline had no venodilator effect while in conditions of high venous tone isoprenaline produced venodilatation. In in vitro venous preparations such as the rabbit portal vein (Sutter, 1965; Hughes and Vane, 1967), rat jugular vein (Duckies and Hurlbert, 1986) and canine saphenous vein (Guimaraes and Osswald, 1969), isoprenaline, at low doses, caused relaxation. Therefore, our in vivo results showing direct venodilator responses to isoprenaline are in accordance with the results of low doses of isoprenaline in in vitro studies. Our results are also in accordance with studies on perfused vascular beds where the intraarterial injection of isoprenaline caused small dilator responses in the veins of the paw and muscle of the foreleg of dog (Abboud et a l . , 1965). The venodilator effects were enhanced when isoprenaline was injected in the presence of venous constriction induced by the intraarterial infusion of noradrenaline. Webb-Peploe and Shepherd (1969), using dog perfused lateral saphenous veins, showed that the effect of isoprenaline i s proportional to the i n i t i a l degree of vein wall tension. This conclusion was based on the findings that after sympathectomy, isoprenaline had no dilator effect, while during venoconstriction produced by either e l e c t r i c a l stimulation of the lumbar sympathetic chain or infusion of venoconstrictor drugs as serotonin, potassium chloride, noradrenaline and adrenaline, isoprenaline produced venodilatation. Our results are not in accordance with the experiments in dogs which showed that isoprenaline increases venous return by stimulating 6-adrenoceptors. Kaiser et a l . (1964), using an in vivo preparation consisting of complete cardiopulmonary bypass surgery and a bubble oxygenator, showed that isoprenaline caused a displacement of blood from the vasculature of a dog to the oxygenator which denotes venoconstriction. Since the venoconstriction was blocked by the nonselective 6-adrenoceptor antagonist nethalide, but not by hexamethonium, the authors concluded that 6-adrenoceptors mediated venoconstriction. An alternative explanation for the 6-adrenoceptor increase in venous return was proposed by Green (1977), who suggested that isoprenaline dilated the hepatic outflow vessels thereby reducing the splanchnic venous time constant and the effective splanchnic back pressure resulting in the release of splanchnic blood volume for redistribution to other areas of the systemic circulation. Our results are also not in agreement with those of Imai et a l . (1978) who used an open-loop method in dogs with cardiac output held constant. In these experiments, isoprenaline increased the venous return; the increase was not blocked by the interference of sinoaortic baroreceptor reflex but was completely abolished by propranolol. They concluded that fi-adrenoceptors mediated the increase in venous return by decreasing venous resistance. The drawback of experiments using the open loop reservoir system was reviewed by Greenway (1982) who suggested that drug induced-changes in the reservoir volume (unstressed volume) could be due to changes in a r t e r i a l or venous resistances, heart rate and contractility in addition to changes in venous compliance. Therefore, although i t is possible to determine that there i s a change in reservoir volume, i t i s not possible to determine which factor caused the effect. He also warned that extracorpreal circuits may modify drugs effects. Our results are also not in accordance with the results of another study in conscious dogs with cardiac output maintained constant via the production of atrioventicular block and alteration of ventricular rate to compensate for changes in stroke volume and in which isoprenaline was found to reduce MAP and increase central venous pressure (Bennett et a l . , 1984). However, since autonomic function was not interfered with, changes in central venous pressure could be a result of reflex mechanisms. In ganglionic blocked sedated dogs, terbutaline, a selective B2"-agonist, did not affect MCFP but i t reduced venous compliance (Lee et a l . , 1984). The authors concluded that 62-adrenoceptors mediate venoconstriction. Hirakawa et a l . (1984) showed that in anaesthetized and open-chest dogs, a single dose of isoprenaline did not produce any changes in MCFP under normal conditions but caused venodilatation after venous tone was increased with infusion of angiotensin II. Our observations also i l l u s t r a t e the a b i l i t y of isoprenaline to dilate veins in conditions of elevated vascular tone. Rothe et a l . (1990) also showed that in anaesthetized dogs, isoprenaline had l i t t l e effect on MCFP. The lack of reflex venoconstrictor effect of isoprenaline at normal tone may be a consequence of modulation by pentobarbital of autonomic reflex mechanisms. Pentobarbital has been shown to decrease plasma catecholamine levels, rectal temperature, MAP and HR in dogs suggesting that the drug suppresses' a c t i v i t i e s of the sympathetic nervous system (Baum et a l . , 1985). When given i.p. into conscious rats, pentobarbital lowered MCFP (Samar and Coleman, 1978), suggesting that i t reduced sympathetic tone to the venous system. In whole animal experiments, isoprenaline may cause variable venous effects, depending on the methodologies used for the estimation of venous responses, experimental conditions, presence or absence of anaesthetic agents and species of animals. Therefore, interpretations of results are d i f f i c u l t due to the complexity of cardiovascular control mechanisms in the intact animals. In humans, when isoprenaline was injected locally in the forearm vein at a dose which did not produce systemic effects, there was venodilatation (Beck et a l . , 1970). When infused i.v. at doses which produce systemic effects, isoprenaline constricted the human forearm vein (Eckstein and Hamilton, 1959) suggesting that the venoconstrictor response of isoprenaline was l i k e l y mediated by reflex mechanisms rather than direct stimulation of 6-adrenoceptors on venous smooth muscle. Venous return changes have been inferred from changes in left-ventricular end-diastolic dimension (LVED) determined by echocardiography. By this method, i t was shown that isoprenaline, adrenaline and terbutaline directly while hydralazine indirectly (by increasing endogenous sympathetic nerve activity) increased venous return by stimulation of B-adrenoceptors in the veins. This study concluded that adrenaline mediates ncreases in venous return mainly by stimulating &2~ adrenoceptors and to a lesser extent by stimulating 6^ -adrenoceptors. In contrast, endogenous sympathetic activity appears to increase venous return primarily via activation of 6i-adrenoceptors (Leenen and Reeves, 1987). However, two other possible mechanisms were given to account for this increase. First, 6-adrenoceptor stimulation may increase LV compliance and this may increase LVED. Secondly, 6-adrenoceptor stimulation may change respiration and intrathoracic pressures thereby increasing venous return. As reviewed by Greenway (1982), venous return (i.e, cardiac output) i s affected by venous compliance as well as other factors which include cardiac contractility, HR, a r t e r i a l and venous resistances. Therefore, i t is not valid to use changes in venous return to reflect changes in venous compliance. 4.4. Pressor response to 6-adrenoceptor antagonists in  phentolamine-treated rats 4.4.1. Haemodynamic changes In urethane-anaesthetized rats, baseline blood flow to the skeletal muscle was markedly greater than the corresponding readings in conscious rats, halothane-anaesthetized (Tabrizchi and Pang, 1987) and pentobarbital-anaesthetized rats. This suggests that urethane anaesthesia enhances blood flow to the skeletal muscle bed. Urethane anaesthesia has been shown to block a 2-adrenoceptors (Armstrong et a l . , 1982). Since i t has been shown that either a^- or (*2-adrenoceptor blockade in the halothane-anaesthetized rats increased the percent distribution of CO to the skeletal muscle bed (Tabrizchi and Pang, 1987), i t might be reasoned that urethane increased flow in the skeletal muscle bed via the blockade of postjunctional <*2~ adrenoceptors. The infusion of phentolamine in urethane-anaesthetized rats decreased MAP primarily by reducing TPR. CO was slightly but not significantly decreased while HR was not altered by phentolamine. Blood flow was decreased in the li v e r , stomach, colon and caecum, and kidney. This reduction of blood flow was probably a consequence of decreased perfusion pressure caused by phentolamine since conductance was not decreased in any of the named organs. Conductance was significantly increased by phentolamine in skeletal muscle and skin beds suggesting that a-adrenoceptors are important in mediating a resting vasoconstriction in these beds. In phentolamine-treated rats, propranolol increased MAP by increasing TPR. Blood flow to the lungs, heart, liv e r , intestine, colon and caecum, kidneys and spleen was increased while flow to the skeletal muscle was decreased. Since MAP was also increased, the increase of flow in some beds may reflect passive changes secondary to the increase in perfusion pressure. Normalization of flows to conductances shows decreases in skeletal muscle, skin and kidneys and no changes in other vascular beds. The decreases in muscle and skin conductances produced by propranolol are in accordance with the reversal of the vasodilator effect of phentolamine in these beds. The decrease in conductance in the kidney bed suggests that changes in addition to the reversal of a-adrenoceptor blockade also may have taken place. In conscious rats, phentolamine decreased MAP by reducing TPR. HR was increased probably via hypotension-induced reflex changes in autonomic nerve a c t i v i t i e s . Blood flows were decreased in the stomach, kidneys and spleen. The reduction of blood flow in these vascular beds was probably a consequence of decreased perfusion pressure since vascular conductance was not decreased in any organ or tissue. Blood flows and vascular conductances were i n -creased in the lungs, heart and skeletal muscle. This indi -cates that a-adrenoceptors are most important in these vas-cular beds although the increase in the coronary blood flow was also due to the increase in metabolic demands as a result of the reflex increase in heart rate caused by phentolamine. The increase in flows and conductances to the lungs and skeletal muscle are in accordance with the effects of phentolamine on the distribution of CO in halothane-anaes-thetized rats (Tabrizchi and Pang, 1987). It i s important to note that the number of microspheres found in the lungs i s the sum of spheres trapped in the bronchial circulation and those reaching the venous side of the circulation through the arteriovenous anastomoses. Bronchial blood flow i s very small and large changes in the bronchial flow should not affect greatly the number of microspheres in the lungs (Hof and Hof, 1989). Therefore our results suggest that phentolamine vasodilates the arteriovenous anastomoses vessels which are in accordance with results in the dog leg showing that phentolamine increased arteriovenous flow in that bed (Spence et a l . , 1972). In phentolamine-treated conscious rats, propranolol increased MAP by increasing TPR since CO was not altered. HR was reduced as a result of the blockade of B^-adrenoceptors. Blood flow to the lungs was increased while flow to the skeletal muscle was decreased. The increase in blood flow to the lungs was related to passive changes secondary to the increase in MAP since vascular conductance in the lungs was not altered. Conductances were decreased in the heart, intestine, kidneys, skeletal muscle and skin beds. In phentolamine-treated rats, atenolol produced a pressor response which was of similar magnitude to that produced by propranolol. MAP was also increased by the elevation of TPR since CO was not altered. HR was reduced due to the blockade of 6^-adrenoceptors. Blood flow to the lungs, intestine, colon and caecum, spleen, testis and brain was increased while skeletal muscle blood flow was reduced. The increase in blood flow was due to passive changes secondary to the increase in MAP since conductances in these beds were not altered. Vascular conductance was reduced by atenolol in the intestine, kidneys, skeletal muscle and skin beds. It is important to note that the dose of atenolol used in the present study is selective for the blockade of B^-adrenoceptors since i t has been shown that atenolol (100 Mg/kg) did not shift the E D 5 0 value of salbutamol for lowering MAP in conscious rats (Tabrizchi et a l . , 1988). Therefore, selective blockade of ^-adrenoceptors by atenolol and nonselective blockade by propranolol produced pressor responses of similar magnitudes and constrictions of intestine, kidneys, skeletal muscle and skin vascular beds. The failure of both atenolol and propranolol to decrease lung flow and conductance i s most lik e l y due to the lack of control of the arteriovenous anastomoses by 6-adrenoceptors (Spence et a l . , 1972). Coronary conductance was reduced by propranolol but not by atenolol. Our results suggest that 62-adrenoceptors are important in the mediation of vasodilatation in the coronary vasculature and confirm our previous results on the effects of isoprenaline on coronary conductance. They are in agreement with those of in vivo studies as reviewed by Feigl (1983). In contrast to our results on coronary flow, Vatner and Hintze (1983) reported that propranolol and atenolol produced similar degrees of coronary vasoconstriction in phentolamine-treated dogs. The use of different species of animals, doses of drugs and experimental conditions may account for the different observations on coronary flow with selective 6 ^ - and nonselective 6-blockade. It has been speculated that the pressor effect caused by a 6-adrenoceptor antagonist was due to the antagonism of 6 2 -adrenoceptor-mediated vasodilatation (Kayaalp and Turker, 1979; Yamamoto and Sekiya, 1969; Himori et a l . , 1984; 150 Himori and Ishimori, 1988). However, previous studies in our laboratory have shown that the injection of very small doses of either atenolol or ICI 118,551 into the conscious rat caused similar pressor response (Tabrizchi et a l . , 1988) . This suggests that the blockade of vasodilator B>2~ adrenoceptors i s not the mechanism of this pressor response. Our haemodynamic studies in conscious rat confirm that the pressor response to 6-adrenoceptor antagonists i s indeed not due to the antagonism of i^-adrenoceptor-mediated vasodilatation since atenolol and propranolol produced similar haemodynamic effects. In urethane-anaesthetized rats, propranolol reversed the vasodilator effect of phentolamine in the skin and skeletal muscle and in addition, i t vasoconstricted the kidneys. In conscious rats, either propranolol or atenolol reversed the vasodilator effect of phentolamine mainly in the skeletal muscle but in addition, both drugs vasoconstricted the kidneys, intestine and skin. This shows that the skeletal muscle vasculature is the most-affected bed. This may also explain the observation that in urethane-anaesthetized rats where the skeletal muscle blood flow is high, propranolol produced a pressor response with or without the infusion of phentolamine (Yamamoto and Sekiya, 1979;. Regoli, 1970) . However, in conscious rats, pretreatment with phentolamine is needed to produce a pressor response (Tabrizchi et a l . , 1988). Tabrizchi and Pang (1989) also showed that propranolol did not produce a pressor response in conscious rats pretreated with either sodium nitroprusside or the cholinergic agonist methacholine suggesting that prior a-adrenoceptor blockade i s needed for the pressor response to a 6-adrenoceptor antagonist. However, i t i s not clear why conductance was decreased in the kidneys, skin and intestine with the 6-adrenoceptor antagonists although phentolamine did not increase conductance in these beds. It is l i k e l y that during the infusion of phentolamine, other endogenous vasopressor agents are released to oppose the vasodilator effect of phentolamine. Phentolamine was shown to increase the secretion of catecholamines (Tabrizchi and Pang, 1987) and renin (Keeton and Campbell, 1981). Activation of the renin-angiotensin system may oppose the vasodilatatory effects of phentolamine (Gardiner and Bennett, 1988). It has been shown that angiotensin II exerts the most prominent vasoconstrictor effects in the kidneys and skin (Pang, 1983). It i s possible that phentolamine did vasodilate the kidneys and skin bed, however, these effects were concealed by the vasoconstrictor effects of the endogenously-released angiotensin II. Therefore, i t i s possible that the vasoconstrictor effect of the 6-adrenoceptor antagonists in the kidneys and skin beds also were due to antagonism of vasodilator effects of phentolamine (presumably due to unopposed vasoconstrictor action of angiotension II in these beds). To explore the possible role of the renin-angiotensin system in the pressor response to 6-adrenoceptor antagonists, captopril was injected in three groups of rats before the start of phentolamine infusion and the subsequent injection of 6-adrenoceptor antagonists propranolol, ICI 118,551 and atenolol. Since MAP was reduced more by phentolamine in the presence of captopril than in i t s absence in a l l the groups, i t suggests that the renin-angiotensin system was involved with opposing the direct vasodilator effect of phentolamine. In the absence of the renin-angiotensin system a l l three 6-adrenoceptor antagonists produced pressor responses but MAPs were not restored to the i n i t i a l prephentolamine control value. This confirms the importance of the renin-angiotensin system in the production of the pressor response. 4.4.2. Effects of anaesthetic agents In urethane-anaesthetized rats pretreated with phentolamine, i.v. bolus doses of propranolol, atenolol or ICI 118,551 each produced a dose-dependent increase in MAP which restored MAP to control values. However, in halothane-anaesthetized rats pretreated with phentolamine, the injection of propranolol or atenolol did not produce a pressor response. On the other hand, ICI 118,551 caused a small dose-dependent increase in MAP. . The maximum rise was only 25% of that in conscious rats (Tabrizchi et a l . , 1988; Tabrizchi and Pang, 1989) and urethane-anaesthetized rats in the present study. It has been shown that phentolamine decreases MAP in halothane-anaesthestized rats by reducing cardiac output and not total peripheral resistance (Tabrizchi and Pang, 1987). It i s therefore conceivable that the lack of pressor response with propranolol and atenolol in phentolamine-treated halothane-anaesthetized rats i s related to the possible further reduction of cardiac output via the blockade of A^-adrenoceptors. In addition, halothane has been reported to attenuate the pressor re-sponse to both phenylephrine and azepexole in dogs (Kenny et a l . , 1990) and to block the pressor response to N G-nitro-L-arginine in rats (Wang et a l . , 1991). Halothane also re-duced the amplitude of oscillations produced by nor-adrenaline in the rat isolated mesenteric vein and this was attributed to the inhibition of calcium release from the sarcoplasmic reticulum (Marijic et a l . , 1990). This anaes-thetic agent also reduced phenylephrine-induced contraction in isolated rat aorta (Sprague et a l . , 1974) and serotonin-and acetylcholine-induced contraction in endothelium free porcine coronary artery and this was explained to be due to the inhibitory effect of halothane on agonist induced-inositol phosphate formation (Ozhan et a l . , 1990). Su and Zhang (1990) showed that halothane decreased tension development in the intact aortic ring due to combined effects of a depression of Ca* -induced activation of the contractile proteins and a decrease of sarcoplasmic reticulum C a 2 + accumulation leading to reduced C a 2 + release for muscle contraction. Therefore, i t is possible that non-specific inhibition o f Ca 2 +-release i s responsible for the the inhibition of pressor response to 6-adrenoceptor antagonists. In pentobarbital-anaesthetized, phentolamine-treated rats, a l l 6-adrenoceptor antagonists failed to produce a pressor response. Pentobarbital has been shown to decrease plasma catecholamine levels in rats (Farnebo et a l . , 1979) and dogs (Zimpfer et a l . , 1982; Baum et a l . , 1985). Holmes and Schneider (1973) reported that pentobarbital reduced acetylcholine-induced catecholamine release in the isolated bovine chromaffin vesicles. The mechanism may involve the interruption of a link between receptor activation and catecholamine release. In order to examine whether or not catecholamines affect the pressor response to 6-adrenoceptor antagonists, adrenaline was infused into two additional groups of rats. The infusion of adrenaline in rats pretreated with phentolamine caused markedly greater reductions in MAP. The subsequent injections of both propranolol and atenolol partially restored MAP. Since propranolol and atenolol caused similar pressor responses, i t i s unlikely that the partial reversal involved only the blockade of vasodilatory 62-adrenoceptors. These results are in agreement with those of Tabrizchi and Pang (1990) which show that adrenaline i s required for the partial restoration of the pressor response to a 6-adrenoceptor antagonist in phentolamine-treated rats. The haemodynamic changes in pentobarbital-anaesthetized rats showed that phentolamine increased blood flow only to the lungs while blood flow was reduced in the caecum, colon and skin. Vascular conductance was increased only in the lungs which indicates that phentolamine i s an important vasodilator of arteriovenous anastomoses. In contrast to the situation in conscious rats, blood flow to the skeletal muscle was not altered by phentolamine. Propranolol did not produce changes in MAP, flow or conductance in any vascular bed. This again shows that i t i s important to have increased blood flow to the skeletal muscle by a-adrenoceptor blockade in order to produce a pressor response to a B-adrenoceptor antagonist. Further experiments were carried out to examine i f other barbiturate anaesthetic agents similarly suppressed the pressor response to propranolol. The results show that propranolol also failed to produce a pressor response in rats anaesthetized with amobarbital. Under the influence of ketamine, propranolol partially reversed the hypotensive effect of phentolamine. The reason for the partial reversal by B-adrenoceptor antagonists of phentolaminerinduced hypotension in ketamine-anaesthetized rats i s not clear at the present time. However, ketamine i s known to stimulate the cardiovascular system by centrally-mediated sympathomimetic effects, and to inhibit intraneuronal and extraneuronal catecholamine uptake (Riou et a l . 1989) . This may explain why MAP was higher in animals anaesthetized with ketamine than with other anaesthetic agents. It has been shown in in vivo studies that, in the absence of autonomic control, ketamine has direct myocardial depressant properties in dogs (Schwartz and Horwitz, 1975). It i s therefore possible that following a- and B-adrenoceptor blockade, the direct myocardial depressant effect of ketamine attenuated the pressor effect of propranolol. In in vitro studies, ketamine, in doses relevant to those used in surgical induction, inhibited the development of spontaneous mechanical activity and lowered baseline tension of rat aortae and portal veins. In addition, i t attenuated agonist-induced contractions of rat aortic strips (Altura et a l . , 1980). The relaxant effect of ketamine in the rabbit ear artery was proposed to be due to a decrease of Ca 2 +-influx through the plasma membrane or an interference with the process of signal transduction between receptor occupation on the plasma membrane and C a 2 + release from intracellular stores via the inhibition of hydrolysis of phosphatidylinositol 4,5-biphosphate ( P I P 2 ) (Kanamura et a l . , 1989).. Therefore, i t i s possible that ketamine attenuated the pressor response to B-adrenoceptor antagonists by i t s direct relaxant effect. In chloralose-anaesthesia, pressor response to a B-adrenoceptor antagonist was completely abolished. Chloralose had a profound negative inotropic effect on the dog heart-lung preparation (Bass and Buckley, 1966). Charney et a l . (1970) showed that chloralose anaesthesia in dogs was associated with a transient increase in cardiac output which was abolished by a- and B-adrenoceptor blockade. The intrinsic depressant effect of chloralose, like that of ketamine, may have been normally masked by the sympathomimetic effects of the anaesthetic. However, following a- and 6-adrenoceptor blockade, the myocardial depressant effect of chloralose may have been unmasked resulting in the abolition of the pressor response to a 6 -adrenoceptor antagonist. Our results show that anaesthetic agents variably affect the response to a 6-adrenoceptor antagonist. The injection of a 6-adrenoceptor antagonist caused a pressor response in phentolamine-treated conscious rats and urethane-anaesthetized rats. Pressor responses to propranolol were attenuated in phentolamine-treated rats anaesthetized with ketamine or halothane and were abolished in rats anaesthetized with barbiturates, halothane and chloralose. The reasons for the differential effects of anaesthetic agents on the response to B-adrenoceptor antagonists remain obscure. 4.5. Reversal of a-adrenoceptor blockade by B-adrenoceptor  antagonists in the isolated rat pulmonary artery The rat pulmonary artery which contains both a- and 6 -adrenoceptors was used to test the hypothesis that a 6 -adrenoceptor. antagonist reverses the effect of a-adrenoceptor blockade. It was shown by Fleish and Hooker (1976) that 6-adrenoceptors mediate relaxation in the rat pulmonary artery. The predominant 6-adrenoceptors in this preparation were reported to be of the 62-subtype although a small population of B^-subtype was also present (O'Donnell and Wanstall, 1981a). In preliminary experiments, noradrenaline produced a maximum contraction of 12.1 ± 1.3 mN indicating that this tissue was sensitive to the drug. Dose-response curves for noradrenaline were repeated four times with no significant changes in either the E C 5 0 value or maximum response. This shows that the preparation was stable over the study period. It i s to be noted that the pulmonary artery i s not a systemic artery. However, the aim of the study' was only to show i f there i s an interaction between 6-blockers and a-blockers at the level of the receptor. The responses from our in vitro preparation may not be representative of the results i n in vivo preparations of the interaction of 6-blockers and a-blockers. In in vivo the interaction between a- and 6-blockers l i k e l y occurs in small resistance vessels. Noradrenaline caused a dose-dependent increase in force. Phentolamine partially blocked the effect of noradrenaline and this antagonism was not affected by time indicating that under the experimental conditions, the effect of phentolamine was not overcome by noradrenaline. Propranolol, atenolol and ICI 118,551 were shown to restore completely and in a dose-dependent manner the effect of noradrenaline which was previously antagonized by phentolamine. Since a l l three 6-adrenoceptor antagonists were effective in reversing the effect of phentolamine, i t is most lik e l y that the vasoconstrictor effect of the 6-159 adrenoceptor antagonists was primarily due to the reversal of a-adrenoceptor blockade. This confirms that the pressor responses of B-adrenoceptor antagonists are not due to the blockade of vasodilatory 62-adrenoceptors. 4.6. Interaction between a- and B-adrenoceptor antagonists The mechanism of the pressor response to 6-adrenoceptor antagonists i s unknown, however, several hypotheses were proposed: f i r s t , 6-adrenoceptor antagonists may raise MAP and vasoconstrict tissue vasculature by antagonizing 6 2 -adrenoceptor mediated vasodilation (Kayaalp and Turker, 1967; Yamamoto and Sekiya, 1969; Himori et a l . , 1984; Himori and Ishimori, 1984). Our results are not in agreement with this hypothesis although we cannot exclude the possibility that the blockade of 62-adrenoceptors may contribute to the pressor response. Second, 6-adrenoceptor antagonist pressor effect i s attributed to a centrally mediated release of adrenal catecholamines (Kayaalp and Kiran, 1967; Sugawara et a l . , 1980). However, Tabrizchi et a l . (1989) showed that 6-adrenoceptor antagonists do not increase the levels of plasma catecholamines in conscious rats. Third, 6-adrenoceptor antagonists may reverse the effects of a-adrenoceptor blockade by displacing a-adrenoceptor antagonists by an unknown mechanism (Olivers et a l . , 1965; Regoli, 1970). Fourth, 6-adrenoceptor antagonists, via the blockade of 6-adrenoceptors, may allow more adrenaline to react with a-adrenoceptors which are not 160 blocked (Prichard and Ross, 1966; Regoli, 1970). Our results are in accordance with the last two hypotheses that a possible interaction of a- and 6-adrenoceptor antagonists occurs and this may result in subsequent stimulation of the a-adrenoceptors. The mechanism of the interaction i s not quite clear. Moreover in in vivo experiments, additional factors such as the renin-angiotensin system may also be involved in the pressor response to 6-adrenoceptor antagonists. 4.7. Conclusions 1. 6 2-adrenoceptor stimulation by a small dose of isoprenaline decreased TPR and MAP but 62-adrenoceptor stimulation affected neither TPR nor MAP. Isoprenaline initiated coronary and skeletal muscle vasodilatation via the activation of 6 ^ - and 62-adrenoceptors. Vascular conductances in other vascular beds were not affected. 2. Isoprenaline increased venous tone in intact, conscious rats. It had no effect on venous tone in rats pretreated with hexamethonium and i t decreased venous tone in rats infused with noradrenaline to produce a high venous tone. Therefore, 6-adrenoceptor stimulation mediated direct venodilatation but a high venous tone is needed. 3 . 6-adrenoceptor antagonists produced pressor responses primarily by reversing the effect of phentolamine in the skeletal muscle bed in conscious rats and urethane-anaesthetized rats. In addition, 6-adrenoceptor anta-gonists vasoconstricted skin, kidneys and intestinal vascular beds. The renin-angiotensin system had to be intact in order for complete reversal of phentolamine-induced hypotension by 6-adrenoceptor antagonists. Anaesthetic agents had varying effects on the pressor effects of 6-adrenoceptor antagonists in phentolamine-treated rats. This ranged from the absence of influence with urethane, attenuated pressor response with halothane and ketamine and absence-of a pressor response with halothane, pentobarbital, amobarbital and chloralose. 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