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Possible role of adenosine 5’-triphosphate in the cardiovascular system of the rat Poon, Christina I. 1994

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POSSIBLE ROLE OFADENOSINE 5’-TRIPHOSPHATE IN THE CARDIOVASCULAR SYSTEM OF THE RAT by CHRISTINA I. POON B.Sc., University of British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology & Therapeutics, Faculty of Medicine We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER, 1994 © Christina I. Poon  In presenting this thesis in partial fulfillment 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 and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head my of department or his by her or representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature  Department of  4-  The University of British Columbia Vancouver, Canada Date___________  II  ABSTRACT  The experiments described in this thesis were designed to characterize a possible role for adenosine 5’-triphosphate (ATP) in the cardiovascular system of the conscious rat. This study assessed the role of ATP in the control of mean arterial pressure (MAP), heart rate (HR), and mean circulatory filling pressure (MCFP) by examining the effects of receptor antagonists (of cx-adrenoceptors, P - and P 1 -purinoceptors, and autonomic ganglia), chemi 2 cal sympathectomy (by reserpine or guanethidine), and ATP per se. Furthermore, we compared the contribution of endogenous ATP and noradrenaline (NA) in basal vascular tone with that during drug-induced vasodilatation and concomittant elevation of sympa thetic nerve activity. Phentolamine (non-selective c-adrenoceptor antagonist) was found to be a more effective arterial than venous vasodilator in both basal conditions and during drug (hydralazine or nifedipine)-induced vasodilatation and reflex venoconstriction. While MCFP was not significantly decreased by phentolamine either under basal conditions or during hydralazine treatment, phentolamine did decrease MCFP in the presence of nifedipine. Following suramin treatment, the phentolamine-induced depressor effect was significantly potentiated whereas MCFP remained unchanged. Under basal conditions, mecamylamine very effectively reduced both MAP and MCFP whereas in the presence of hydralazine induced vasodilatation and elevated venomotor tone, ganglion blockade reduced MCFP but not MAP. Blockade of P -purinoceptors by suramin produced a dose-dependent increase in 2 MAP and decrease in HR neither of which was affected by hydralazine, nifedipine, mecamylamine, reserpine, or guanethidine. Suramin failed to reduce MCFP in the pres ence of hydralazine, nifedipine, or guanethidine. In contrast, mecamylamine treatment revealed a significant dose-dependent decrease in MCFP by suramin, while reserpine treatment revealed a slight but significant decline in MCFP.  III  l.v. infusion of ATP produced profound depressor and bradycardic effects. The ATP induced depressor effect was unaffected by mecamylamine and suramin whereas block ade of Pi-purinoceptors by 8-phenyltheophylline clearly and significantly attenuated this y-purinoceptors by cibacron blue only slightly and insignificantly 2 response. Blockade of P attenuated the depressor effect of ATP. ATP-induced bradycardia was not affected by mecamylamine or cibacron blue whereas 8-phenyltheophylline completely abolished this response and even revealed a slight, but insignificant, increase in HR in response toATP. Suramin slightly but insignificantly enhanced the ATP-induced bradycardia. ATP produced a slight but insignificant depression of MCFP which was unaltered in the presence of suramin, and slightly but insignificantly enhanced both during mecamylamine-induced ganglion blockade and following 8-phenyltheophylline treatment. Cibacron blue, in con trast, revealed a slight but insignificant ATP-induced increase in MCFP.  iv  TABLE OF CONTENTS Page ABSTRACT  LISTOFTABLES  ii Viii  LIST OF FIGURES  ix  ABBREVIATIONS  xi  ACKNOWLEDGEMENTS  xiii  1.  INTRODUCTION  1  1 .1. 1 .1 .1. 1.1.2. 1 .1.3. 1.1.4.  Historical Aspects of the Concept of Sympathetic Noradrenaline ATP Cotransmission and the Extracellular Effects of ATP in the Cardiovascular System NA as the primary sympathetic neurotransmitter Development of the cotransmission hypothesis Development of the NA-ATP cotransmission hypothesis Other extracellular effects of ATP in the cardiovascular system  1 1 2 2 3  1.2. 1.2.1. 1.2.2. 1.2.3. 1.2.4.  Classification and Mechanisms of Action of Cardiovascular Purinoceptors4 - and P 1 P -purinoceptors 2 4 -purinoceptorsubtypes 1 P 5 -purinoceptor subtypes 2 P 6 Presynaptic purinoceptors 10  1.3. 1.3.1. 1.3.2. 1.3.3. 1 .3.4.  Sources of Extracellular ATP in the Cardiovascular System ATP from nerves ATP from blood cells ATP from the adrenal medulla ATP from the vessel wall and other tissues  11 11 14 15 15  1.4.  Metabolism of Extracellular ATP  17  1.5. 1.5.1.  Regulation of Vascular Tone by ATP Dual regulation of blood vessel tone byATP: vasoconstriction and vasodilatation Pharmacological tools used to characterize P -purinoceptors 2 vasoconstriction of vascular x-purinoceptor-mediated 2 P smooth muscle  18  1.5.2. 1.5.3.  18 20 21  V  1.5.3.1.  1.5.3.3. 1.5.4. 1.5.4.1. 1.5.4.2. 1.5.4.3.  Pharmacological dissection of the mechanical response to sympathetic perivascular nerve stimulation Pharmacological dissection of the electrical response to sympathetic perivascular nerve stimulation Influence of the parameters of nerve stimulation y-purinoceptor-mediated vasodilatation of vascular smooth muscle 2 P y-purinoceptors and sympathetic cotransmission 2 P y-purinoceptors and NANC transmission 2 P y-purinoceptors and endothelium-dependent vasodilatation 2 P  1.6. 1.6.1. 1.6.1.1. 1.6.1.2. 1.6.1.3. 1.6.1.4. 1.6.1.5. 1.6.1.6. 1.6.1.7. 1.6.2.  Actions of ATP in Vascular Beds and in Whole Animals Vascularbeds Coronary vascular bed Hepaticvascularbed Mesenteric vascular bed Pancreatic vascular bed Intestinal vascular bed Renal vascular bed Hindlimbvascularbed Whole animals  29 29 30 31 32 32 33 33 34 34  1.7.  Aims of the Thesis  38  2.  METHODS & MATERIALS  40  2.1. 2.1 .2. 2.1.3. 2.1.3.1. 2.1.3.2.  40 40 40 40  2.1.4.  Methods Animals Surgical preparation of animals for use in MCFP studies Standard preparation Modifications to the standard preparation: experiments involving suramin Measurement and calculation of MCFP  2.2. 2.2.1. 2.2.2. 2.2.3.  Experimental Design, Statistical Analysis, and Presentation of Results Experimental design Statistical analysis Presentation of results  42 42 43 43  2.3. 2.3.1. 2.3.2.  Experimental Protocol Phentolamine and mecamylamine Suramin I  44 44 45  1.5.3.2.  22 23 25 27 27 27 28  42 42  vi 2.3.3. 2.3.4. 2.3.5.  Phentolamine Suramin II ATP  46 46 47  2.4.  Drugs  48  3.  RESULTS  49  3.1.  Controls: Time and Volume Effects of Vehicle in the Absence and Presence of Various Drug Treatments  49  3.2.  Resistance of MCFP to cL-Adrenoceptor Antagonism in Rats with Normal and Reflexly-lncreased Venous Tone Effect of phentolamine in the absence and presence of hydralazine Effect of phentolamine in the absence and presence of nifedipine Effect of mecamylamine in the absence and presence of hydralazine  61 61 61 62  Possible Role of Purinergic Neurotransmission Reflexly-Increased Venous Tone Effect of suramin in the absence and presence Effect of suramin in the absence and presence Effect of suramin in the absence and presence  69 69 69 70  3.2.1. 3.2.2. 3.2.3. 3.3. 3.3.1. 3.3.2. 3.3.3.  in Basal and of hydralazine of nifedipine of mecamylamine  3.4.1.  Does Suramin Reveal an cx-Adrenoceptor Antagonist-Sensitive Component of Venous Tone? Effect of phentolamine in the absence and presence of suramin  75 75  3.5. 3.5.1. 3.5.2.  Does Sympathetic Cotransmission Contribute to Venous Tone? Effect of suramin in the absence and presence of reserpine Effect of suramin in the absence and presence of guanethidine  78 78 78  3.6. 3.6.1. 3.6.2. 3.6.3. 3.6.4.  Characterization of the Cardiovascular Effects of Exogenous ATP Effect of ATP in the absence and presence of mecamylamine Effect of ATP in the absence and presence of suramin Effect of ATP in the absence and presence of cibacron blue Effect of ATP in the absence and presence of 8-phenyltheophylline  82 82 82 83 83  4.  DISCUSSION  89  4.1.  Resistance of MCFP to o-Adrenoceptor Antagonism in Rats with Normal and Reflexly-lncreased Venous Tone  90  4.2.  Possible Role of Purinergic Neurotransmission in Basal and Reflexly-l ncreased Venous Tone  93  3.4.  VII  4.3.  Does Suramin Reveal an o-Adrenoceptor Antagonist-Sensitive Component of Venous Tone?  96  4.4.  Sympathetic Cotransmission and Venous Tone  98  4.5.  Cardiovascular Effects of Exogenous ATP  101  4.6.  Summary and Conclusions  104  5.  REFERENCES  107  VIII  LIST OF TABLES Table  Page  1.  Pretreatment and post-treatment control values of HR, MAP and MCFP of vehicle-treated time controls.  50  2.  Pretreatment and post-treatment control values of HR, MAP and MCFP of rats treated with hydralazine or nifedipine followed by phentolamine or mecamylamine.  64  3.  Pretreatment and post-treatment control values of HR, MAP and MCFP of rats treated with hydralazine, nifedipine or mecamylamine followed by suramin.  71  4.  Pretreatment and post-treatment control values of HR, MAP and MCFP of rats treated with suramin followed by phentolamine.  76  5.  Pretreatment and post-treatment control values of HR, MAP and MCFP of rats treated with reserpine or guanethidine followed by suramin.  79  6.  Pretreatment and post-treatment control values of HR, MAP and MCFP of rats treated with cibacron blue, mecamylamine, 8phenyltheophylline or suramin followed by ATP.  84  ix LIST OF FIGURES  Figure  Page  1.1.  Dose-response curves of the effects of saline on HR, MAP and MCFP in rats treated with saline, hydralazine or suramin.  51  1.2.  Dose-response curves of the effects of saline on HR, MAP and MCFP in rats treated with vehicle or nifedipine.  52  1.3.  Dose-response curves of the effects of saline on HR, MAP and MCFP in rats treated with saline or hydralazine.  53  1.4.1.  Dose-response curves of the effects of saline on HR in rats treated with saline, hydralazine, mecamylamine or guanethidine.  54  1.4.2.  Dose-response curves of the effects of saline on MAP in rats treated with saline, hydralazine, mecamylamine or guanethidine.  55  1.4.3.  Dose-response curves of the effects of saline on MCFP in rats treated with saline, hydralazine, mecamylamine or guanethidine.  56  1 .5.  Dose-response curves of the effects of saline on HR, MAP and MCFP in rats treated with vehicle or reserpine.  57  1.6.1.  Dose-response curves of the effects of saline on HR in rats treated with saline, cibacron blue, mecamylamine, 8-phenyltheophylline or suramin.  58  1.6.2.  Dose-response curves of the effects of saline on MAP in rats treated with saline, cibacron blue, mecamylamine, 8-phenyltheophylline or suramin.  59  1.6.3.  Dose-response curves of the effects of saline on MCFP in rats treated with saline, cibacron blue, mecamylamine, 8-phenyltheophylline or suramin.  60  2.1.  Dose-response curves of the effects of phentolamine or vehicle on HR, MAP and MCFP in rats continuously infused with saline or hydralazine.  65  2.2.  Dose-response curves of the effects of phentolamine or vehicle on HR, MAP and MCFP in rats continuously infused with vehicle or nifedipine.  66  x  Figure  Page  2.3.  Dose-response curves of the effects of mecamylamine or vehicle on HR, MAP and MCFP in rats continuously infused with saline or hydralazine.  67  2.4.  Dose-response curves of the effects of mecamylamine on changes in HR and MAP produced by a test dose of methoxamine in rats.  68  3.1.  Dose-response curves of the effects of suramin or saline on HR, MAP and MCFP in rats continuously infused with saline or hydralazine.  72  3.2.  Dose-response curves of the effects of suramin or saline on HR, MAP and MCFP in rats continuously infused with vehicle or nifedipine.  73  3.3.  Dose-response curves of the effects of suramin or saline on HR, MAP and MCFP in rats continuously infused with saline or mecamylamine.  74  4.1.  Dose-response curves of the effects of phentolamine or saline on HR, MAP and MCFP in rats treated with saline or suramin.  77  5.1.  Dose-response curves of the effects of suramin or vehicle on HR, MAP and MCFP in rats treated with vehicle or reserpine.  80  5.2.  Dose-response curves of the effects of suramin or saline on HR, MAP and MCFP in rats continuously infused with saline or guanethidine.  81  6.1.  Dose-response curves of the effects of ATP or saline on HR, MAP and MCFP in rats treated with saline or mecamylamine.  85  6.2.  Dose-response curves of the effects of ATP or saline on HR, MAP and MCFP in rats continuously infused with saline or suramin.  86  6.3.  Dose-response curves of the effects of ATP or saline on HR, MAP and MCFP in rats treated with saline or cibacron blue.  87  6.4.  Dose-response curves of the effects of ATP or saline on HR, MAP and MCFP in rats treated with vehicle or 8-phenyitheophylline.  88  xi ABBREVIATIONS ADP  adenosine 5’-diphosphate  AMP  adenosine 5’-monophosphate  ATP  adenosine 5’-triphosphate  DDW EDRF EJC EJP EtOH FAP GABA HR l.a. i.p. i.v. 3 1P iu L-NAME LDV MAP MCFP MTX  double distilled water endothelium-derived relaxing factor excitatory junction current excitatory junction potential ethanol final arterial pressure ‘y-aminobutyric acid heart rate intra-arterial intraperitoneal intravenous inositol-1 ,4,5-triphosphate international unit N-nitro-L-arginine methyl ester large dense-cored vesicle mean arterial pressure mean circulatory filling pressure methoxamine  n  number of observations  NA  noradrenaline  NANC  non-adrenergic, non-cholinergic  NO  nitric oxide  NPY  neuropeptide tyrosine  p PE  probability (significance level in a statistical test)  2 PGI  prostacyclin -log of the dissociation constant standard deviation (of observed sample) standard error (of estimate mean value)  pKB s.d. s.e.mean  polyethylene  XII  SDV UTP VPP w/v  small dense-cored vesicle uridine triphosphate venous plateau pressure weight by volume approximately equals  XIII  ACKNOWLEDGEMENTS I would like to express my sincere gratitude to all those who have made my graduate studies experience a positive one. Dr. Catherine Pang, whom I have known for 4 years, first as a summer student then as a graduate student, is thanked for her invalu able scientific insight and financial support, without which this thesis would not have been possible. Kenneth Poon is thanked for his invaluable experimental assistance, data and statistical analysis, and manuscript preparation. I would also like to thank Su Lin Lim and Dr. Reza Tabrizchi for their technical assistance. Dr. Michael Walker and Dr. Hudson (of SYSTAT, Inc.) are thanked for their statistical assistance. I also wish to acknowledge the secretarial services of Elaine Jan and Margaret Wong who helped prepare my endless list of applications and letters of reference. Finally, I am grateful to the Heart & Stroke Foundation of B.C. and the Yukon and the Medical Research Council of Canada for their very generous financial support.  1  1.  INTRODUCTION  1.1.  Historical Aspects of the Concept of Sympathetic Noradrenaline-ATP Cotransmission and the Extracellular Effects ofATP h the Cardiovascular System  1 .1 .1.  NA as the primary sympathetic neurotransmitter  The concept of chemical neurotransmission was first explicitly proposed at the turn of the century when Elliot (1904) suggested that “sympathetic axons cannot excite the peripheral tissue except in the presence, and perhaps through the agency, of the adrena line or its immediate precursor...”, thus anticipating the modern view of sympathetic neuro transmission in which noradrenaline (NA), the immediate precursor of adrenaline, is re leased from sympathetic nerves and acts at specific receptors on smooth muscle near the sites of release. The view of Elliot, which was based on the similarities of the effects of sympathetic nerve stimulation and applied adrenaline, was later confirmed by Loewi (1921) who demonstrated in the perfused frog heart that tachycardia induced by sympathetic nerve stimulation was mediated by a chemical substance called “Acceleransreizstoff”, which he later identified as adrenaline (Loewi 1936). As suggested by Dale (1934), post ganglionic nerves were therefore termed “adrenergic”. It was not until 1946, however, that Euler positively identified NA as the primary transmitter in sympathetic mammalian nerves. The notion that neurons utilize a single transmitter substance is commonly referred to as Dale’s Principle. Although a misinterpretation of Dale’s original writings (see Eccles 1986), the popularization of “Dale’s Principle”, to a large extent by Eccles (1957), has allowed this idea to dominate thinking until the early 1980s. Nevertheless, departures from this principle can be traced back as far as 1955 when Koelle posed the following question:  “...  is it not likely that the terms cholinergic and adrenergic, originally proposed  by Dale... might refer to the predominant rather than the exclusive types of transmitting agents of the nerve fibers, and that these... might liberate mixtures of chemical transmit  Introduction  2 ters?”. The concept introduced by this question is currently known as cotransmission and has been a subject of rapidly growing interest since the early 1970s. 1 .1.2.  Development of the cotransmission hypothesis  The 1976 paper by Burnstock entitled “Do some nerve cells release more than one transmitter?” was pivotal to the early development of the cotransmission hypothesis. In this paper, it was very tentatively suggested that Dale’s Principle may not have universal applicability and that, in certain instances, one nerve cell may release more than one transmitter. Today, cotransmission is a generally accepted principle and the current view is that it may apply to most neurons and thus represent the rule rather than the exception (Hokfelt etal. 1986; Kupfermann 1991). In their “mature” state, many neurons contain one or several members of the following three classes of putative messengers: (i) “Classic” transmitters, i.e. the monoamines noradrenaline, adrenaline, dopamine or 5hydroxytryptamine, or acetylcholine, or amino acids, such as -y-aminobutyric acid (GABA), glycine or glutamate, (ii) a nucleotide, presumably often adenosine 5’-triphosphate (ATP), and (iii) neuropeptides, a rapidly expanding class of transmitter substances which includes neuropeptide tyrosine (NPY) amongst more than 50 different compounds (Hokfelt et a!. 1986; Stjãrne 1989; Kupfermann 1991). 1.1.3.  Development of the NA-ATP cotransmission hypothesis Some of the most compelling early evidence for sympathetic NA-ATP cotransmission  derived from studies of adrenal chromaffin cells which were shown not only to contain very large amounts of ATP, but also to release ATP by exocytosis together with a variety of proteins, collectively referred to as chromogranins. Soon after, similar evidence was ob tained from neural tissue of the sympathetic nervous system and some investigators be gan to propose that ATP coreleased with NA may have a physiological role. At the time, recent developments identifying ATP as the principal transmitter of “atropine-resistant” non-adrenergic, non-cholinergic (NANC) nerves of the gut, bladder, and portal vein (Burn stock 1972) gave support to such a role for cotransmitter ATP. Introduction  3 Studies of the vas deferens during the early and mid-i 970s, most notably by Burnstock and co-workers (reviewed in Burnstock 1986, i990d), provided the clearest evi dence for NA-ATP cotransmission. The response of the vas deferens to sympathetic nerve stimulation consists of a fast (twitch) contraction, followed by a slower, more sus tained contraction. A number of investigators suggested that the slow response was me diated by NA while the rapid twitch response utilized a different transmitter. The slow phase of the contraction is mimicked by exogenous NA; prazosin and other cL-adrenoceptor antagonists block the response; and cocaine potentiates the response (via blockade of NA reuptake). Furthermore, it was demonstrated that adrenergic neuron blocking agents selectively eliminate the slow contraction while leaving the twitch response relatively in tact. The rapid twitch contraction, on the other hand, was proposed to be mediated by ATP, since exogenous ATP was highly effective in mimicking the nerve-induced response. Although in blood vessels the contributions of ATP and NA to the mechanical response to sympathetic nerve stimulation are not as clearly separated as they are in the vas deferens, analogous studies using vascular preparations appeared to indicate that perivascular nerves also utilize ATP and NA as cotransmitters.  It was these early studies of NA-ATP  cotransmission, especially on the vas deferens, that laid the ground work for the ongoing investigation into the involvement of cotransmission in the cardiovascular system. i .1.4.  Other extracellular effects of ATP in the cardiovascular system  Apart from its action as an excitatory cotransmitter, ATP released intravascularly from non-neuronal stores can cause vasodilatation via its action as a local modulator of vascular tone (Burnstock 1 990b). Some of the earliest observations of the inhibitory ef fects of purines on the cardiovascular system date from the i 920s when Drury and Szent Gyorgyi demonstrated the potent effects of purine-containing extracts from cardiac mus cle, brain, kidney and spleen on the heart and blood vessels. These observations subse quently led to the development by Berne (1963) of a general hypothesis of adenosine function in the cardiovascular system. Although early studies implicated adenosine as the Introduction  4 primary vasodilatatory purine, recognizing the very rapid enzymatic breakdown of ATP to adenosine in the circulation, more recently it has become apparent that ATP, per Se, is also involved where adenosine had previously been thought to be the sole mediator (Winbury et al. 1953; Eikens & Wilcken 1973; Toda eta!. 1982; Hopwood & Burnstock 1987; Hopwood et al. 1989). In 1981, De Mey & Vanhoutte were the first to describe the mechanism responsible for the potent vasodilatatory actions of ATP  —  they demonstrated  that ATP, by acting on vascular endothelial cells, induces the release of endothelium derived relaxing factor (EDRF) which subsequently causes relaxation of vascular smooth muscle. Thus, ATP is often referred to as having a dual function in the regulation of vascular tone: (i) as an intravascular mediator of endothelium-dependent vasodilatation via purinoceptors located on vascular endothelial cells, and (ii) as an excitatory cotrans mitter with noradrenaline from sympathetic perivascular nerves causing vasoconstriction via purinoceptors located on smooth muscle (Burnstock & Kennedy 1986; Ralevic & Burnstock 1991).  Classification and Mechanisms of Action of Cardiovascular Purinoceptors  1.2.  Although this thesis is concerned, in particular, with the actions of ATP, it is appropri ate to discuss the receptors for both ATP and adenosine in light of the fact that extracellular ATP is susceptible to rapid enzymatic breakdown to adenosine both in the circulation and at sites of release by perivascular nerves (Gordon 1986). The interplay between ATP and adenosine in the cardiovascular system is complex: the physiological actions of one can be synergistic, antagonistic, or even modulatory with respect to the actions of the other. 1.2.1.  -purinoceptors 2 Ei- and P  Although the widespread and potent extracellular actions of purine nucleosides and nucleotides have been recognized for over 60 years, it was not until 1978 that an attempt was made to characterize and name the receptors mediating such actions. This classifi - and P 1 -purinocep2 cation system (Burnstock 1978) consisted of two major subtypes, P Introduction  5 tors, and was based on a review of the extensive literature concerning the actions of purine nucleosides and nucleotides on a wide variety of tissues. The four criteria used in this classification were: (i) the relative potencies of ATP, ADP, AMP, and adenosine; (ii) the selective actions of antagonists, particularly methylxanthines; (iii) the activation of adenylyl cyclase by adenosine but not byATP; (iv) the induction of prostaglandin synthesis byATP but not by adenosine. Thus the following classification was proposed: The Pj-purinocep tors are associated with an agonist potency order of adenosine  >  AMP  >  ADP  >  ATP;  methylxanthines such as theophylline, aminophylline, and caffeine are selective competi tive antagonists with respect to these receptors; and occupation of these receptors leads to inhibition or activation of an adenylyl cyclase system with resultant changes in levels of -purinoceptors are associ 2 intracellular adenosine 3’,5’-monophosphate (cAMP). The P ated with an agonist potency order of ATP >ADP >AMP  >  adenosine; these receptors are  not antagonized by methylxanthines and do not act via an adenylyl cyclase system. Since -purinoceptors forms a 2 its proposal, it has become apparent that neither the P 1 nor the P -  homogeneous group and that each can be separated into at least two subtypes. 1.2.2.  -purinoceptor subtypes 1 E 1 and A -subtypes (Van Calker et al. 1979) 2 1 -purinoceptors were subdivided into A P -  - and A 1 -subtypes appear to be 2 and R - and Ra-subtypes (Londos et al. 1980). The A 1 /A nomenclature is 1 A analogous to the R- and Ra-subtypes, respectively; however, the 2 more widely used. The classification is based on the relative potency series of adenine analogues and according to whether adenylyl cyclase activity is increased or decreased in 1 decreases while A 2 increases activity). This classification the presence of adenosine (A A A2a, A2b, , of P -purinoceptors has recently been expanded to include four subtypes, 1 1 and A 3 (Fredholm et al. 1994), all of which are G-protein-coupled and proposed to have the general structure that would place them in the rhodopsin-like group of the superfamily -purinoceptors act via G-proteins, not all 1 of G-protein-coupled receptors. Although all P couple exclusively to adenylyl cyclase, but instead may couple to ion channels or Introduction  6 phospholipases (Fredholm etal. 1994). The existence of Aia, Aib, and A 4 subtypes has also been tentatively proposed (Tucker and Linden 1993). Until recently, the myocardial adenosine receptor has been referred to as eitherA 1 or 3 in the literature. With the cloning of a P A 1 receptor distinct from established A 1 and A 2 subtypes (now referred to as A ) (Zhou etat 1992), this is no longer the case (Carruthers 3 & Fozard 1993).  Instead, it has been suggested that there exist two different A 1 -like  receptors, or a single receptor coupled to two different effectors, one of which mediates “direct” (cAMP-independent, atrial-specific, coupled to K channels) and the other, “antiadrenergic” (cAMP-dependent, atrial- and ventricular-specific) effects in the heart -purinoceptor 1 (Tucker & Linden 1993). In the vasculature, it has been proposed that three P subtypes are responsible for mediating the vasodilator effects of adenosine: a high affinity A2a receptor on vascular smooth muscle cells, a low affinity A2b receptor on endothelial 4 receptor also on vascular smooth cells (Bruns et a!. 1987; Nees et a!. 1987), and an A muscle (Tucker & Linden 1993). The A b-purinoceptor induces accumulation of cAMP in 2 the endothelium which may, in turn, open gap junctions with adjacent smooth muscle cells to stimulate chemical (EDRF and cyclic nucleotide) and electrical communication (Greenfield  et a!. 1 990a,b). The smooth muscle A2a receptor acts via stimulation of adenylyl cyclase, while the putative A -purinoceptor on smooth muscle has been shown to activate KATp 4 channels (Daut eta!. 1990). 1.2.3.  -purinoceptor subtypes 2 E -purinoceptor were first proposed by Burnstock and Kennedy 2 Subtypes of the P  y-purinoceptor subtypes were postulated primarily on the basis of 2 (1985). P2X- and P rank order of potency of agonists in a variety of different biological systems (highly selec -purinoceptors are not available). Generally, 2X and P 2 2 y activa tive antagonists for P tion was correlated with contraction and relaxation, respectively. Gordon (1986) extended 21 and P P this classification scheme to include -z-purinoceptors, which are believed to 2  mediate ADP-induced aggregation of platelets (Humphries et a!. 1993) and the tetrabasic Introduction  7 acid ATP -induced histamine release from rat mast cells (Dahlqvist & Diamant 1974), 4 respectively. The P z-purinoceptor is also found on macrophages and appears to repre 2 sent the opening of a fairly nonselective type of pore (Steinberg & Silverstein 1987). It has recently become apparent that many responses to purine nucleotides do not fall into the y 2 above classification, including those that are insensitive to both 2-methylthio-ATP (P agonist) and o3-methylene ATP (P2X agonist), yet are sensitive to ATP (Demolle et a!. y responses can 2 1988; Wilkinson et al. 1993). In some cases these non-P2x and non-P also be elicited by uridine triphosphate (UTP) with a similar agonist potency to ATP, which has led to the definition of the so-called “P2U” or “nucleotide” or “pyrimidine” receptor (Seifert & Schultz 1989; O’Connor eta!. 1991; Dubyak 1991). Pyrimidine receptors are coupled to phospholipase C and mediate their action via the formation of inositol-1 ,4,52 from intracellular stores. These recep triphosphate (lP ) and subsequent release of Ca 3 tors have been identified on both vascular smooth muscle (Kaithof et a!. 1993) and endothelial cells (Wilkinson eta!. 1993) where they mediate vasoconstriction and endothe hum-dependent relaxation, respectively. Finally, there also appears to be a receptor for diadenosinetetraphosphate, designated a P2D subtype (Hilderman et a!. 1991; Castro et a!. 1992). -purinoceptors responsible for mediating the “dual functions” of ATP in 2 The two P y subtypes. Although 2 the regulation of vascular tone are the excitatory 2X and inhibitory P there is mounting evidence that nucleotide receptors may also be involved, the functional significance and mechanism of action of these receptors are not as well characterized. Most evidence suggests that the vascular smooth muscle P x-purinoceptor represents an 2 2 (Benhem & Tsien 1987; Bean 1992; intrinsic ion channel permeable to Na, K, and Ca Kalthof et a!. 1993), although there have been some reports (e.g. von der Weid et a!. 1993) of ATP-induced chloride currents in aortic and other vascular preparations. These depolarizing currents are directly activated by nanomolar to micromolar concentrations of extracellularATP without the apparent involvement of G-proteins or soluble second mes Introduction  8 sengers. Thus, ATP can be added to the relatively small group of physiological agonists (acetyicholine, glutamate, GABA, glycine, and 5-hydroxytryptamine) for ligand-gated ion channels. In vascular smooth muscle, activation of P x-purinoceptors by ATP increases 2 mainly the Na but also the K and Ca 2 conductances and generates an inward current, the excitatory junction current (EJC), and a transient depolarization, the excitatory junc tion potential (EJP), which may or may not summate to fire a muscle action potential. The -channels, increases the 2 membrane depolarization activates L-type voltage-gated Ca influx of Ca , and triggers a muscle contraction (Benham 1989). In some arterial smooth 2 muscles, it has been claimed that the increase in intracellular Ca 2 is the direct result of influx through the ATP-gated channel, without a requirement for depolarization (Benham & Tsien 1987; Benham 1990). Thus, although the major cation entering through the chan nels is Na, with Ca 2 composing less than 10% of the total current, localized increases in intracellular Ca 2 concentration can be obtained. It has been suggested that these in 2 creases might directly activate contractile proteins or perhaps upregulate other Ca dependent enzymes modulating the contractile process and provide an enhanced source of Ca 2 for uptake into internal Ca 2 stores (Benham 1990). Bo and Burnstock (1993) H]cf3-methylene ATP (P2X3 used autoradiographic localization to show that specific [ purinoceptor radioligand) binding sites were associated only with the smooth muscle of several different vessels in rat, guinea-pig, and rabbit. They also found that, in general, the medium- and small-sized arteries had higher densities of P x-purinoceptor than the 2 elastic and large muscular arteries, while the veins, except for the portal vein, were sparsely labeled. y-purinoceptors results in the release of nitric oxide (NO) 2 Activation of endothelial P 2 are (Kelm et a!. 1988) and prostacyclin (PG 12) (Pearson et a!. 1983). Both NO and PGI vasodilators, although the latter is effective in only some vessels.  In vitro, NO acts  synergistically with PGI 2 to inhibit platelet aggregation (Radomski eta!. 1987). The obser vations that PGI 2 release is rapid in onset, transient, and followed by a period of Introduction  9 refractoriness (Toothill et al. 1988), whereas NO release is also rapid, but sustained for many minutes (Martin eta!. 1985a) have been interpreted as indicating two different cellu lar signalling pathways for PGI 2 and NO (Boeynaems & Pearson 1990). The initial event y-purinoceptor activation appears to be inositol phospholipid hydrolysis 2 resulting from P by a G-protein-coupled phospholipase C, with subsequent accumulation of lP 3 and re lease of intracellular Ca 2 stores (Forsberg et al. 1987; Boyer et a!. 1990; Harden et al. 1990). The increase in intracellular Ca 2 activates phospholipase A 2 and results in PGI 2 2 in order to main synthesis. NO release, in contrast, requires influx of extracellular Ca tain elevated intracellular Ca 2 concentration (Luckhoff et a!. 1988). The mechanism by y2 which extracellular Ca 2 enters the cell is not clear. The recent cloning of the P purinoceptor (Webb et a!. 1993) indicates that this receptor has significant homology with other G-protein-coupled receptors, yet may constitute a distinct family within the supertamily of G-protein-coupled receptors (Barnard eta!. 1994). y-purino2 There is growing evidence that, in some vessels, there is a population of P ceptors located on the vascular smooth muscle which mediates endothelium-independent vasodilatation (rabbit mesenteric artery, Mathieson & Burnstock 1985; rabbit cerebral ar tery, Conde et al. 1991; rabbit coronary artery, Corr & Burnstock 1991; rabbit portal vein, Brizzolara eta!. 1993). The mechanism responsible for this “direct” vasodilatation byATP may involve the G-protein-coupled opening of K channels, as demonstrated for ATP induced relaxation of intestinal smooth muscle (Hoyle & Burnstock 1989). Shirahase et y-mediated endothelium-dependent vasoconstriction in 2 a!. (1991) have demonstrated P canine basilar arteries and identified the endothelium-derived contracting factor as -purinoceptors have also been located on cardiac muscle (Burnstock 2 . P 2 thromboxaneA 1980; Pelleg et a!. 1990) and are believed to induce both a positive inotropy and an in y-purinoceptors as has been dem 2 crease in inositol-lipid metabolism via activation of P onstrated in atrial and ventricular preparations (Legssyer et al. 1988; Scamps et a!. 1990; Mantelli eta!. 1993). Introduction  10 1.2.4.  Presynaptic purinoceptors  There is substantial evidence that ATP and adenosine can reduce the release of NA from perivascular sympathetic nerves; however, until recently, it was believed that these effects were mediated by a single population of Pi-purinoceptors, thus requiring the me tabolism of ATP to adenosine (Katsuragi & Su 1982; Su 1983). It is now known that both adenosine and ATP per se are capable of prejunctional modulation of neurotransmitter release (Shinozuka eta!. 1988; Forsyth eta!. 1991; Fuder et al. 1993); however, the iden tity of the receptor(s) mediating these effects is currently a controversial matter. Some investigators have suggested that a novel “P -purinoceptor”, at which adenosine and ATP 3 are equipotent, is responsible for reducing NA release (Shinozuka et al. 1988; Westtall et -purinoceptor by either P 3 1 a!. 1 990a,b; Forsyth eta!. 1991). Stimulation of the putative P  -  - and P 1 -receptor antagonists 2 or P -agonists has been shown to be reduced by both P 2 (Westfall et al. 1990a,b). There are other investigators, however, who have proposed the 1 -purinoceptor (A 1 existence of two distinct presynaptic purinoceptors: a classical P subtype), activated mainly but not exclusively by the nucleoside adenosine, and a G y-like”-purinoceptor activated only by nucleotides (von Kugelgen et a!. 2 protein-linked “P 1989a; Kurz eta!. 1993; von Kugelgen eta!. 1993). Von Kugelgen et al. (1993) have -autoreceptor to inhibition of Ca 2 2 entry speculated that the G-protein may couple the P -adrenoceptors, 2 or enhancement of K efflux. In contrast to NA acting at presynaptic o which reduces both its own release and that of its cotransmitter ATP (Bulloch & Starke 1990; MacDonald et a!. 1992; Msghina et a!. 1992; Bao 1993), ATP acting at P -purinoc2 eptors on sympathetic axon terminals has been shown to reduce only the release of NA (White & MacDonald 1990; Westfall etal. 1990a,b; von Kugelgen eta!. 1993; Allgaier etal. 1994), although the possibility of autoinhibition by ATP of release of ATP itself has not been totally eliminated. In contrast, Evans and Surprenant (1992) have presented evi dence indicating thatATP affects neitherATP nor NA release. Interestingly, several groups (Miyahara & Suzuki 1987; Shinozuka eta!. 1992; Ishii eta!. 1 993a,b; Ishii et a!. 1994) have Introduction  11 demonstrated enhanced release of NA following activation by ATP of P - or, possibly, P 2 3 purinoceptors on perivascular sympathetic nerve terminals.  1.3.  Sources of ExtracellularATP in the Cardiovascular System ATP is well-known as a ubiquitous intracellular constituent and is synthesized in cells  by glycolysis and by mitochondrial oxidative phosphorylation. Because of its ubiquitous occurrence in all cell types (at cytosolic concentrations of 2-3 mM), total tissue contents of ATP are unlikely to reveal possible extracellular functions. Knowledge of both the release and source of ATP is therefore a much more important criterion to be satisfied if one wishes to establish specific functions for extracellularATP in a particular system. Further more, the receptor subtypes mediating ATP-induced responses in the cardiovascular sys tem are often determined by the cellular source of ATP. Plasmatic ATP, which is believed to be the primary mediator of endothelium-dependent vascular responses, may be re leased from a number of different cellular sources. Neuronally-released ATP, in contrast, is believed to act directly by activation of purinoceptors located on the vascular smooth muscle. Unlike ATP derived from neurons, platelets, and the adrenal medulla, which is released via exocytosis, other cell-derived or extra-neuronal ATP (e.g. from vascular endothelial and smooth muscle cells) is believed to be released in a non-secretory fashion (Gordon 1986). These latter cells lack storage granules containing ATP, and the ATP is apparently released from the cytoplasm (Pearson & Gordon 1979). 1.3.1.  ATP from nerves  ATP has been found to occur together with NA in both small dense-cored vesicle (SDV) and large dense-cored vesicle (LDV) fractions of the homogenates of sympatheti cally innervated tissues (Lagercrantz 1971; Klein 1982; Lagercrantz & Fried 1982). The ATP:NA ratio in large LDVs is three to five times higher than in SDVs. As the vesicles are transported down the axon to the nerve terminal, there is an increase in the content of NA but not of ATP, as the molar ratio of NA:ATP increases from 4:1 to as much as 50:1 (Klein Introduction  12 1982; Lagercrantz & Fried 1982). Thus, some have suggested that the low proportion of ATP in the mature vesicles argues against a cotransmitter role for ATP in light of the finding that exogenous ATP and NA are equipotent as smooth muscle spasmogens (Fredholm eta!. 1982). When attempts have been made to measure release of transmitter ATP from vascu lar sympathetic nerves, results have proved especially controversial. Numerous studies using “overflow” methods have demonstrated the release of radiolabeled purines during stimulation of sympathetic nerves in blood vessels (Su 1983); however, such results have been criticized because most of the radiolabel appears as adenosine rather than ATP. Although it has been assumed that released ATP is rapidly degraded to adenosine by ectonucleotidases, it is possible that adenosine itself is released during the sympathetic nerve stimulation. (Fredhoim eta!. 1982; Pons et al. 1980; White & MacDonald 1990). Further complications arise from the inability of overflow methods to distinguish between “transmitter ATP”, presumably originating from sympathetic vesicles, and “non-transmitter ATP” released from the cytosol of neuronal and non-neuronal cells in response to field stimulation (Shinozuka etal. 1991; Msghina eta!. 1992; Ishii etal. 1993b). The magnitude of this problem is illustrated by the finding (in rabbit aorta) that less than 3% of the field stimulation-induced overflow of ATP was derived from sympathetic nerves, with the re mainder originating from smooth muscle (7%) and endothelium (90%) (Sedaa et al. 1990). Although the release of ATP, and possibly adenosine, from both neuronal and non-neuro nal stores far exceeds that of NA (by factors of 350 and 2000 in the rabbit aorta and pulmonary artery, respectively) (Sedaa etaL 1990; Mohri eta!. 1993), the ratio of NAto ATP released from nerves per se is approximately 1  —  which is estimated to provide a  physiologically active concentration of ATP once released (Sedaa etal. 1990). It has been demonstrated that both the endothelial and smooth muscle “overflow” ATP is released via cj-adrenoceptor stimulation (Katsuragi & Su 1982, Buxton eta!. 1990; Sedaa eta!. 1990; Starke eta!. 1992; Shinozuka etal. 1992, lshii etal. 1993b). Interestingly, nicotine, long Introduction  13 known to induce exocytotic NA release via activation of nicotinic receptors on sympathetic nerve terminals (Loffelholz 1970), releases ATP in addition to NA in vascular preparations (Bultmann etal. 1991a). One of the currently debated issues with respect to storage and release of cotrans mitter ATP concerns the possible “dissociation” of nerve impulse-induced release of ATP and NA as reflected by the variability in ratios of released NA and ATP. In other words, it is unclear as to whether ATP released by nerve stimulation originates from purely purinergic nerves or from sympathetic nerves and, if it comes from sympathetic nerves, whether from vesicles containing both NA and ATP, or ATP alone. According to the conventional view of sympathetic cotransmission, NA and ATP are stored together in vesicles and nerve im pulses release them by exocytosis in the same proportions as they are stored (Stjãrne 1989). Numerous observations of “non-parallel” modulation of nerve-released NA and ATP under various stimulation parameters or pharmacological treatments (Kennedy et aL 1986b; White & MacDonald 1990; Bulloch & Starke 1990; Sjöblom-Widfeldt & Nilsson 1990; Starke et al. 1992; Evans & Cunnane 1992; Driessen et al. 1993) have cast doubt as to whether NA and ATP are stored in the same transmitter vesicle and released via exocytosis as a mixed multimolecular packet. For example, Sjöblom-.Widfeldt and Nilsson 2 con (1990) demonstrated in small mesenteric arteries of the rat that extracellular Ca centration differentially affected the adrenergic and purinergic components of the contrac tile response to sympathetic nerve stimulation, suggesting the possibility that NA and ATP are regulated separately. Although both components of the response were abolished by chemical sympathectomy with 6-hydroxydopamine, these authors suggested that this did not necessarily indicate that only one type of adrenergic nerve was responsible for the release of NA and ATP since it is possible, though highly speculative, that subpopulations of vesicles, or even of adrenergic nerves, with varying ATP:NA ratios may exist (Sjãblom Widfeldt 1990). An alternative, and perhaps more likely, explanation for the apparent differential regulation of NA and ATP is that transmitter release, instead of occurring via Introduction  14 complete (all-or-none) exocytosis which would require NA and ATP to be release in a fixed ratio, may occur via a non-exocytotic ion-exchange mechanism capable of partial and selective release of vesicle contents (Uvnàs etal. 1989). Evidence in support of corelease has been presented by Msghina and coworkers (Msghina et al. 1992; Msghina & Stjàrne 1993) who have demonstrated (in rat tail artery) “parallel” modulation of NA and ATP release by both presynaptic x2-autoreceptors and tetanic stimulation. Similarly, Sjöblom Widfeldt eta!. (1990; see also Sjöblom-Widfeldt 1990) showed that both adrenergic and purinergic components of the neurogenic contractile response in rat mesenteric arteries are potentiated to the same extent by tetanic stimulation. 1.3.2.  ATP from blood cells Erythrocytes, leukocytes, neutrophils, and platelets contain significant amounts of  ATP which can be released following exposure to damaging stimuli. For example, release of ATP from human erythrocytes in response to hypoxia and other haemodynamic stresses has been demonstrated (Forrester 1990). Blood platelets, in particular, are a rich source of plasmatic ATP (Detwiler & Feinman 1973) as they contain ATP, together with 5hydroxytryptamine, in “dense granules” which have been compared to storage vesicles in some neurons (Sneddon 1973). Platelets contain approximately 40 nmol of ATP and ADP per mg of protein (Da Prada et a!. 1981). The plasma concentration of ATP following platelet activation by thrombin in vitro can reach concentrations up to 20 tM (Ingerman et a!. 1979; Born & Kratzer 1984) while that in vivo, where platelet degranulation occurs following localized platelet aggregation, can approach the high micromolar range since the concentration within storage granules is of the order of 1 M (Ugurbil 1981; Meyers et a!. 1982). In addition to thrombin, adrenaline, collagen, and ADP are known to induce ATP release (reviewed in Colman 1990). Degranulation may also occur when platelets inter act, perhaps transiently, with the vascular wall (George 1985) and this could result in a smaller, more localized release of nucleotides.  Introduction  15 1.3.3.  ATP from the adrenal medulla  Cells of the adrenal medulla, like neurons and platelets, contain ATP in storage gran ules and discharge them, together with catecholamines, via degranulation (Winkler et aL 1987). ATP represents approximately 15% of the dry weight of adrenal granules (Hillarp 1959) and is stored with catecholamines in the ratio of about 4 moles amine:1 mole ATP (Winkler & Westhead 1980). Although the exact functions of this released ATP have not yet been established, it is possible that the adrenal medulla may contribute significantly to local release of ATP into the plasma (White 1988). 1 .3.4.  ATP from the vessel wall and other tissues  As mentioned previously, there are certain cells which release ATP from a cytosolic pool rather than from vesicle-bound stores. These ATP sources include skeletal (Abood eta!. 1962) and cardiac muscle cells (Paddle & Burnstock 1974; Forrester 1990), and vascular smooth muscle and endothelial cells (Pearson & Gordon 1979; Buxton et al. 1990). Given its molecular mass and anionic nature, there is generally little permeation of ATP (or MgATP) across the plasma membrane. The mechanism of release from cytosolic stores is unclear, although Forrester (1990) has demonstrated that hypoxia-induced re lease of cytosolic ATP from cardiac myocytes and human erythrocytes is substantially attenuated by inhibitors of either anion transporters or nucleoside transporters. Recently, Abraham et al. (1993) described very interesting observations which suggest that the multidrug resistance (mdrl) gene product (or P glycoprotein) can function as a channel for ATP. This raises the possibility that other proteins belonging to the “ABC” transporter superfamily may also act as conduits forATP release in response to physiological or patho logical stimuli. Using cultured endothelial and smooth muscle cells, Pearson and Gordon (1979) demonstrated that ATP release was not accompanied by any evidence of cell damage, as indicated by the lack of extracellular lactate dehydrogenase activity and absence of stain ing of cells with trypan blue, and therefore was not the result of a nonspecific increase in Introduction  16 membrane permeability such as induced by cell lysis.  Furthermore, the absence of  compartmentalization of endothelial ATP excluded the possibility of a secretory process involving degranulation. The selective release of ATP from cultured endothelium appears to be a cellular response to potentially damaging stimuli. Mechanisms have not yet been identified; however, effective stimuli include neutral proteinases such as trypsin and thrombin, which require an active catalytic site of the enzyme (Lollar & Owen 1981), and cationic proteins or polymers (LeRoy etal. 1984). In nonpathological states, it is believed that endothelial ATP is liberated by NA released during increased sympathetic nerve ac tivity since exogenous NA has been shown to stimulate release of ATP from cultured cardiac endothelial cells via a prazosin-sensitive, yohimbine-insensitive, c -adrenoceptor mediated, mechanism (Buxton et a!. 1990). Recently, Yang et a!. (1994) presented evi dence supporting the ability of ATP to stimulate ATP release from cardiac endothelial cells via an action at P y-purinoceptors. 2 1 The release of ATP from vascular smooth muscle has been shown to be an cx. adrenoceptor-mediated process (Katsuragi & Su 1982, Sedaa eta!. 1990; Starke eta!. 1992; Shinozuka et a!. 1992, lshii et a!. 1 993b) and, as described previously, contributes to the ATP “overflow” observed following electrical nerve stimulation. An ATP-evoked ATP release system, analogous to that found in cardiac endothelial cells (Yang eta!. 1994), has been demonstrated in vas deferens and ileal longitudinal smooth muscles (Katsuragi eta!. 1991; von Kugelgen & Starke 1991 b), raising the possibility that such a system may also operate in vascular smooth muscle. The functional significance of ATP released postsynaptically from vascular smooth muscle and endothelium may rest in its ability to modulate NA release by an action at presynaptic nerve terminals (reduced NA release, Shinozuka eta!. 1991; enhanced NA release, lshii eta!. 1993b).  Introduction  17  1.4.  Metabolism of ExtracellularATP The enzymes responsible for removal of ATP from the extracellular space are  ectonucleotidases. Ectonucleotidases, though primarily located in large numbers on the luminal surface of vascular endothelial cells, are widely distributed amongst other tissues and isolated cells such as skeletal, cardiac, and smooth muscle, blood platelets, and leukocytes (Ryan 1982; Pearson & Gordon 1985; Zimmerman etal. 1992). The efficiency of metabolism of ATP in the circulation has been known since 1950 when Binet and Burstein demonstrated that a bolus of ATP is virtually all removed by a single passage through the pulmonary vasculature. Sollevi et al. (1984) demonstrated that ATP intravenously admin istered to anaesthetized dogs is virtually all removed during its passage through the lung circulation. In addition, Ryan and Smith (1971) demonstrated that the mean transit time of ATP and its metabolites was indistinguishable from that of a vascular space marker (blue dextran or albumin) in the perfused rat lung, and that the half-life of nucleotides in the perfused lung is  —  0.2 s or less. In comparison, the half-life for ATP incubated in cell-free  plasma or whole blood at 37 °C is 20-30 mm and 10 mm, respectively (Jorgensen 1956; Trams etal. 1980). The heart is an additional site of efficient ATP metabolism  —  99% of a  bolus of ATP can be removed on a single passage through the coronary vasculature (Pad dle & Burnstock 1974). The metabolic pathway involves sequential dephosphorylation, with ATP being con verted to ADP, then to AMP, then to adenosine, which is taken up by active transport and rephosphorylated or further metabolized to inosine and hypoxanthine. Unlike endothelial cells, vascular smooth muscle cells lack the high-affinity adenosine transport system (Pearson et al. 1978) and convert very little of the adenosine produced extracellularly to metabolites (Slakey et al. 1990). The three separate enzymes catabolizing ATP —*  AMP  —>  —>  ADP  adenosine are ATPase, ADPase, and monophosphatase (5’-nucleotidase),  respectively (Pearson & Gordon 1985). Non-specific phosphatases do not contribute to nucleotide metabolism at the endothelial surface (Pearson et al. 1980). Introduction  18 Of particular importance to the metabolism of ATP released either intravascularly as a local modulator, or abluminally as a cotransmitter are the ectonucleotidases located on the vascular endothelium and smooth muscle, respectively. Slakey et al. (1990) have suggested that regulation of the time course of adenine nucleotide hydrolysis in the endothelium and vascular smooth muscle differs in light of the fact that the site of action and extent of phosphorylation can profoundly modify the physiologic effect of these nude otides. They demonstrated, that for smooth muscle cells, the rate of production of adeno sine is regulated predominantly by delivery of substrates to the cell surface, while for endothelial cells “feed-forward” inhibition leads to a pronounced lag in production of ad enosine. This lag, it was speculated, serves to insure that proaggregatory ADP remains in the extracellular milieu long enough to allow thrombus formation, afterwhich antiaggregatory adenosine is produced. CotransmitterATF in contrast, is rapidly metabolized to adenos 1 (or P ) receptors on sympa 3 me which subsequently acts at presynaptic autoinhibitory P 2 1 and P thetic nerve terminals (Katsuragi & Su 1982). In effector cells expressing both P receptors, the production of adenosine from ATP may serve to either attenuate or potentiate the biological responses triggered by the initial release of ATP (Dubyak & El-Moatassim 1993).  1.5.  Regulation of Vascular Tone byATP  1.5.1.  Dual regulation of blood vessel tone byATP: vasoconstriction and vasodilatation  The effect 0tATP on blood vessels depends on a number of factors, the most impor tant of which are: (i) the source of ATP, (ii) the integrity of the endothelium, and (iii) the activity of ectonucleotidases. Numerous studies have demonstrated that ATP induces vasodilatation in endothelium-intact blood vessels but vasoconstriction once the endothe hum has been removed (Kennedy et al, 1985; Machaly eta!. 1988; Read eta!. 1993), or that at low basal tone ATP causes vasoconstriction while at high tone vasodilatation is observed (Furchgott 1966; Kennedy & Burnstock 1985b; White eta!. 1985; Mathieson Introduction  19 and Burnstock 1985; Burnstock & Warland 1987a; Ralevic eta!. 1991). Other studies have shown the asymmetry in vascular responses to intraluminal and abluminal adminis tration of ATP (Cohen et a!. 1984; Chen & Suzuki 1991; Kaul et a!. 1992). Such studies have shown that intraluminal administration produces endothelium-dependent vasodilata tion while abluminal administration tends to produce vasoconstriction. Kaul and coworkers (1992) demonstrated in the perfused rabbit carotid arteries that ADP administered intraluminally and abluminally elicited endothelium-dependent vasodilatation in vessels preconstricted with phenylephrine and c,3-methyIene-ATP (a potent P x-purinoceptor 2 agonist), respectively, but not when applied abluminally to phenylephrine-constricted ar teries in the presence of an ADPase inhibitor. These results are representative of the generally accepted finding that the response of a vessel to ADP, ATP, and their analogues y-purinoceptors. Thus, 2 is a balance between opposing effects mediated by 2X- and P ATP generated by endothelial cells, platelets, and erythrocytes will act predominantly at y-purinoceptors located on the endothelium, promoting the release of EDRF and pro 2 P ducing vasodilatation, whereas ATP released from sympathetic nerve endings at the abluminal surlace will act primarily at P x-purinoceptors on smooth muscle to promote 2 constriction, perhaps with a synergistic interaction with noradrenaline (Kennedy & Burnstock 1986a; Ralevic and Burnstock 1990; Corr& Burnstock 1991; Witt eta!. 1991; Bultmann  etal. 1991a). Thus, in the presence of endothelial dysfunction, unopposed stimulation of x-purinoceptors on smooth muscle by intravascularly-derived ADP and ATP may pro 2 P duce vasoconstriction. In addition, the observations of Kaul eta!. (1992) suggest that abluminal application may result in preferential activation of smooth muscle P x-purinoc2 y-purinoceptor 2 eptors since occupation of these by o,f3-methylene-ATP unmasked a P vasodilatation. These investigators also showed that the so-called “diffusion barrier” (i.e. the smooth muscle and endothelial ectonucleotidases encountered by purine nucleotides in transit from the abluminal to intraluminal side, or vice versa), though somewhat reduc ing the endothelium-dependent vasodilatation produced by abluminal ADP in the pres Introduction  20 ence of oc,J3-methylene-ATP, may not be as great a factor in the response to abluminal nucleotides as the preferential activation of smooth muscle P x-purinoceptors and con 2 comitant “masking” of the opposing P y-mediated effect. 2 1 .5.2.  -purinoceptors 2 Pharmacological tools used to characterize P -purinoceptors has 2 Unfortunately, a lack of selective competitive antagonists for P  hampered the characterization of these receptors in the cardiovascular system by neces sitating a reliance on “relative agonist potency orders” (see Kennedy 1990; Fedan & Lamport 1990). As a result, investigators have resorted to using a procedure, first introduced by Kasakov and Burnstock (1982), which utilizes the slowly degradable P x-purinoceptor 2 agonist c3-methylene-ATP as a selective desensitization agent for P x-purinoceptors. 2 The use of cçf3-methylene-ATP as an antagonist remains the most widely used method for y-mediated responses; 2 characterizing P x-purinoceptors and for distinguishing 2X- from P 2 however, its long-lasting and selective effects in vitro (except in immature rat basilar ar tery, Byrne & Large 1986) have been very difficult to reproduce in vivo (Flavahan et a!. 1985; Bulloch & McGrath 1 988b; Taylor & Parsons 1989; Schlicker et a!. 1989; Daziel et a!. 1990).  A further complication of the use of cc,3-methylene-ATP in vivo is its  cardiodepressant effect (Delbro eta!. 1985; Flavahan eta!. 1985). Reactive blue 2 (also known as cibacron blue), an anthraquinone sulfonic acid dye, was first introduced by Kerr and Krantis (1979) as a new class of P -purinoceptor antagonist and was later shown to 2 y-purinoceptors (Burnstock & Warland 2 be a noncompetitive antagonist selective for P 1987a; Hopwood etal. 1989; Taylor et al. 1989), although competitive activity has also been demonstrated (Houston eta!. 1987). Although reactive blue 2 appears to display a y-purinoceptors at low concentrations, nonspecific effects such 2 degree of selectivity for P as antagonism of acetylcholine or adenosine (Burnstock & Warland 1 987b; Taylor et a!. 1989) are not uncommon at higher concentrations, thus limiting the usefulness of the compound.  Introduction  21 -purinoceptor antago 2 The most recent and promising candidate demonstrating P nism is the trypanocidal drug suramin which, interestingly, has also been utilized in the treatment of cancer and acquired immunodeficiency syndrome (reviewed in Voogd et a!. 1993). Dunn and Blakely (1988) initially introduced suramin as a potential P x-purinoceptor 2 antagonist in the mouse vas deferens; however, it has since been demonstrated in a number of tissues that suramin inhibits not only 2X- but also P y-mediated effects (Den 2 Hertog eta!. 1989; Hoyle eta!. 1990; von Kugelgen & Starke 1991 c). Leff and coworkers (1990; 1991) conducted a quantitative pharmacological analysis of suramin’s actions and concluded that, indeed, suramin is a genuine competitive antagonist at P x-receptors 2 although its affinity for these receptors is low (pKB  =  5.17) and it requires a relatively long  time to achieve equilibrium in vitro. Similar studies have yet to be carried out for antago nism of P y-purinoceptors by suramin. Reports demonstrating that suramin competitively 2 antagonizes responses to UTP acting on vascular smooth muscle nucleotide (P2U) recep tors (Kalthof et a!. 1993) and to ADP acting on platelet P -r-purinoceptors (Hourani et a!. 2 1992) seem to indicate that suramin cannot distinguish between the proposed subtypes of -purinoceptors. In the rabbit saphenous artery, suramin has been shown to inhibit re 2 P sponses not only to ATP and c,f3-methylene-ATP but also to histamine and 5hydroxytryptamine, thus raising doubts about its selectivity (Nally & Muir 1992; see also Schlicker eta!. 1989). 1.5.3.  x-purinoceptor-mediated vasoconstriction of vascular smooth muscle 2 E In many in vitro, and relatively fewer in vivo, preparations, the mechanical and elec  trical responses to perivascular nerve stimulation are partially resistant to oe-adrenoceptor antagonists (Glick eta!. 1967; Holman & Surprenant 1980; Suzuki & Kou 1983; Hirst & Neild 1980; Hirst & Lew 1987). This knowledge, together with the observation that sympa thetic nerve stimulation is associated with the release of purines (Su 1975, 1983), eventu ally led to the proposal that sympathetic NA-ATP cotransmission operates in blood ves sels (reviewed in Westfall eta!. 1990c; Burnstock 1990c,d; von Kugelgen & Starke 1991a). Introduction  22 1.5.3.1. Pharmacological dissection of the mechanical response to sympathetic perivascular nerve stimulation The sympathetic nerve-mediated electrical and mechanical responses have been pharmacologically dissected into two distinct components: the adrenergic component, which is sensitive to block by o-adrenoceptor antagonists such as prazosin, and the purinergic component, which is susceptible to desensitization by c3-methylene-ATP or antagonism by suramin. Studies conducted using the rabbit saphenous artery exemplify the experi mental approach taken in a number of other vessels (Burnstock & Warland 1 987b; Warland & Burnstock 1987). In this particular vessel, only 28% of the vasoconstrictor response to sympathetic nerve stimulation is blocked by prazosin while the remainder is abolished by oL,f3-methylene-ATP. Furthermore, all contractions can be eliminated by either blocking the nerve action potential with tetrodotoxin or destroying sympathetic nerves with guanethidine or 6-hydroxydopamine, thus indicating a sympathetic nerve origin for both NA and ATP and suggesting corelease (Burnstock & Warland 1987b; Warland & Burnstock 1987).  Reserpine treatment, which depletes sympathetic nerves of their  catecholamine content, tailed to abolish neurogenic contractions despite a 95.7% reduc tion in NA content of the tissue. The response remaining after reserpine treatment, how ever, could be eliminated by desensitization with cL,f3-methylene-ATP but not prazosin (Warland & Burnstock 1987). A very similar approach has also been used to identify ATP as a sympathetic cotransmitter in a number of other vessels including the rabbit mesenteric (von Kugelgen & Starke 1985), hepatic (Brizzolara & Burnstock 1990), jejunal (Evans & Cunnane 1992), and saphenous arteries (Nally & Muir 1992), the guinea-pig submucosal arterioles (Evans & Surprenant 1992), the dog basilar (Muramatsu et al. 1981) and mesenteric arteries (Muramatsu 1987), and the rat mesenteric arteries (Sjöblom-Widfeldt et a!. 1990).  It should also be noted that chemical sympathectomy, with either 6-  hydroxydopamine or guanethidine, has also been used to ascertain whether oc-adrenoceptor antagonist-resistant nerve-mediated responses are due to ATP released trom sympathetic Introduction  23 nerves as a cotransmitter orATP released as the primary transmitter from NANC nerves. For example, in the rabbit portal vein, in which NANC neurotransmission is well-character ized, chemical sympathectomy fails to abolish the response to perivascular nerve stimula tion, nerve-mediated release of tritiated purines, and quinacrine fluorescence (which al lows histochemical localization of ATP) (Burnstock et a!. 1979; Burnstock et al. 1984). 1 .5.3.2. Pharmacological dissection of the electrical response to sympathetic perivascular nerve stimulation Electrophysiological studies have demonstrated that not only is there an adrenoceptor antagonist-resistant component of the contractile response to sympathetic nerve stimula tion, but there is also an analogous component to the smooth muscle electrical response (Cheung 1982; Kurlyama 1983; Sneddon & Burnstock 1985; Allcorn eta!. 1985; Kennedy  eta!. 1 986b; Sjöblom-Widfeldt 1990; Nally & Muir 1992). This response is the EJP and is generally characterized as a short-latency, rapid membrane depolarization brief duration  (-  (—  10 mV) of  1 s) (reviewed in Hirst & Edwards 1989). Similar EJPs have been re  corded from many systemic arteries after sympathetic stimulation and are typically fol lowed by a slow depolarization of  2 mV that lasts for many seconds and is associated  with an c-adrenoceptor antagonist-sensitive contraction. With brief stimuli, the EJP is not associated with a muscle contraction. However, increasing stimulus strength results in 2 channels, consequently introducing an associ summation of EJPs which activates Ca ated component in the contractile response. Although the slow c-adrenoceptor-mediated depolarization is larger under these conditions, it is not sufficient to initiate an action po tential. With a further increase in stimulus strength, the EJP still triggers an action poten tial and associated contraction, but the o-adrenoceptor-mediated depolarization is now sufficiently large to trigger an action potential as well, thereby increasing the associated tension response.  Here, the c-adrenoceptor-mediated constriction precedes the c  adrenoceptor-mediated depolarization, suggesting that a membrane potential change is 2 has been released from not required for tension development, presumably because Ca Introduction  24 intracellular stores. The majority of blood vessels produce EJPs; however, not all are followed by a neurogenic oL-adrenoceptor-mediated depolarization and it is not uncom mon for low concentrations of NA to produce large contractions in the absence of any detectable membrane change (Hirst & Edwards 1989). Nevertheless, it is very unusual for neurona!Iy-released NA to evoke constriction without causing a membrane potential change at some stage (Hirst & Edwards 1989). The suggestion that the EJP is mediated by ATP released from sympathetic nerves as a cotransmitter has met with some controversy. Although it has been demonstrated that the EJP can be mimicked by exogenous ATP (Suzuki 1985), is abolished by chemical sympathectomy (guanethidine or 6-hydroxydopamine) but not reserpine (Cheung 1982; Sneddon & Burnstock 1985; Kennedy et al. 1986b; Cunnane & Evans 1989), and is blocked by suramin or desensitization with oj3-methylene-ATP (Allcorn et all 985; Sneddon & Burnstock 1985; Kennedy etal. 1986b; Ramme eta!. 1987; Nally & Muir 1992), some have proposed that NA may, in fact, be responsible for the EJP in spite of its resistance to o-adrenoceptor antagonists. This alternate hypothesis assumes that NA activates spe cialized receptors (‘y-adrenoceptors) which are restricted to regions near sympathetic nerve varicosities (Hirst & Neild 1981; Luff et a!. 1987). This is based on the observation that very large concentrations of noradrenaline (> 1 mM), in the absence (Hirst & Neild 1980; Hirst et a!. 1982) or presence (Laher et al. 1986; Bevan et a!. 1987) of c-adrenoceptor blockade, mimic the initial, rapid electrical and mechanical responses observed during sympathetic nerve stimulation. In addition, it has been suggested that postjunctional x adrenoceptors are found only in extra-junctional areas and that antagonism of neuronal responses by prazosin is due to non-specific depression of smooth muscle function rather than oci-receptor blockade (Hirst & Neild 1981; Neild & Zelcer 1982). However, they adrenoceptor hypothesis is difficult to reconcile with the observation that reserpine pretreatment, which greatly diminishes the release of NA, abolishes the second, slow onset depolarization but has no effect on the initial generation of EJPs (Suzuki eta!. 1984). Introduction  25 More recently, Hirst and Jobling (1989) showed that cx,j3-methylene-ATP eliminates EJPs caused by perivascular nerve stimulation but has no effect on arterial responses suppos edly mediated by ‘y-adrenoceptors, thus supporting the notion that ATP is responsible for the generation of EJPs.  Interestingly, this same study found that neither EJP5 nor -y  adrenoceptor responses could be elicited in veins even though veins responded to ap -purinoceptors 2 plied ATP with a contractile response, suggesting that the distribution of P has no correlation with the ability of sympathetic nerves to initiate an EJP. 1.5.3.3. Influence of the parameters of nerve stimulation There are two types of inconsistency in the literature which appear to indicate a large degree of variability in the role of ATP as a cotransmitter and cast in doubt the possible physiological role for NA-ATP cotransmission. First, in some vessels, the EJPs but not neurogenic contractions are resistant to o-adrenoceptor blockade (e.g. rabbit ear artery: Allcorn et a!. 1985), while in others both the EJP and contractile response are totally abolished by c3-methylene-ATP (Ramme eta!. 1987). Second, some investigators have reported cL-ad renoceptor antagonist-resistant contractile responses to nerve stimulation  (e.g. rabbit ear artery: Kennedy et a!. 1986) whereas, in the same tissue, others have demonstrated contractions which are virtually abolished by cL-adrenoceptor blockade (e.g. rabbit ear artery: Allcorn eta!. 1985). Most commonly, there are reports of elimination of both the EJP and a significant proportion of the contractile response by c,f3-methyleneATP (e.g. Sjöblom-Widfeldt 1990). It is becoming more and more apparent that the source of these discrepancies rests in the variations in stimulation parameters used to elicit neu rotransmission and contraction. It appears that contractions evoked by short trains of low frequency stimuli are predominantly purinergic, whereas longer periods of stimulation and! or higher frequencies are usually required to reveal a substantial noradrenergic compo nent of contraction (Kennedy eta!. 1 986b; Sjöblom-Widteldt eta!. 1990; Sjöblom-V’Jidfeldt & Nilsson 1990; Evans & Cunnane 1992). Thus, the stimulation frequency-dependence of the proportions of ATP and NA contributing to the contractile response is probably re Introduction  26 sponsible for observations in the rabbit saphenous (Burnstock & Warland 1987) and ileocolic arteries (Bulloch & Starke 1990)  —  short periods of electrical stimulation elicited monophasic,  cç3-methylene-ATP-sensitive vasoconstriction, whereas longer periods produced biphasic vasoconstriction of which the first phase was oc,3-methylene-ATP-sensitive while the sec ond phase was prazosin-sensitive. Based on the observation that NA, in addition to ATP, -adrenoceptor 2 is released during low frequency nerve stimulation, and that yohimbine (0L antagonist) enhanced c,f3-methylene-ATP-sensitive EJPs, Ramme etal. (1987) concluded that NA released by low frequency stimulation and/or short trains of stimuli does not reach the threshold concentration required to elicit contraction through postjunctional o -ad renoceptors. Therefore, 2 adrenoceptors, but is sufficient to activate prej unctional o stimulation at higher frequencies for longer durations must release sufficient NA to acti -adrenoceptors to elicit a contraction. 1 vate enough postjunctional o Although this hypothesis also explains findings in the dog basilar artery (Muramatsu et al. 1981) and rabbit saphenous artery (Burnstock & Warland 1987b), Evans and Surprenant (1992) were unable to demonstrate an x-adrenoceptor-sensitive component of the contractile response of guinea-pig submucosal arterioles even at stimulation parameters known to elicit substantial noradrenergic contractions in all arteries studied to date. In this particular study, the fact that reserpine had virtually no effect on neurogenic contractions eliminated a possibility of y-adrenoceptor involvement, thus suggesting that ATP is the sole mediator of vasoconstriction in this vessel while NA modulates transmitter -receptors. The estimation that submuscosal arterioles contrib 2 release via presynaptic a ute up to 40% of the total mesenteric splanchnic resistance (Parks & Jacobson 1987) and contribute significantly to the maintenance of systemic blood pressure, together with the findings of Evans and Surprenant (1 992), suggest an important physiological role forATP. Burnstock (1 990d) has speculated upon the physiological significance of the dependence of relative rates of cotransmitter release on stimulation parameters (i.e. functional activity of the nerve terminal) in proposing that NA may be the most important component of Introduction  27 sympathetic cotransmission during activities such as gentle exercise, while ATP might be the more important component during stress when short burst frequencies occur in sym pathetic nerves. Indeed, the in vivo activity of sympathetic nerves is highly irregular in both humans and animals (Delius etal. 1972; see also Nilsson etal. 1985). 1 .5.4.  y-purinoceptor-mediated vasodilatation of vascular smooth muscle 2 E  y-purinoceptors located on the vascular 2 ATP-induced vasodilatation can occur via P y-purinoceptors 2 smooth muscle or endothelium (see section 1.2.3.). Smooth muscle P are activated by ATP released from either sympathetic or NANC nerves, whereas intraluminally-released ATP (e.g. ATP from platelets and endothelium) is believed to act on endothelial P y-purinoceptors. 2 1 .5.4.1. P y-purinoceptors and sympathetic cotransmission 2 y-purinoceptors have been identified on vascular smooth 2 Blood vessels in which P y-purinoceptors, in 2 muscle cells, though few in comparison to those with endothelial P clude rabbit mesenteric (Mathieson & Burnstock 1985), coronary (Corr & Burnstock 1991; Keefetal. 1992), and hepatic (Brizzolara& Burnstock 1991) arteries, guinea-pig coronary artery (Keef et al. 1992), and cat middle cerebral artery (Conde et al. 1991). It has been suggested that these vessels relax in response to ATP released as a cotransmitter with NA from sympathetic nerves. In the rabbit coronary artery (Corr & Burnstock 1991) NA coreleased with ATP from sympathetic nerves has been shown to cause dilatation via  13-  adrenoceptors which is consistent with the synergistic nature of P x-purinoceptor- and 2 -adrenoceptor-mediated vasoconstrictor responses seen in other vessels with sympa 1 o thetic NA-ATP cotransmission. 1.5.4.2. E y-purinoceptors and NANC transmission 2 The best example of a blood vessel receiving NANC transmission is the rabbit portal vein. This was the vessel originally used by Su (1975) to provide evidence for the release of ATP from perivascular nerves. He showed that although this release was abolished by tetrodotoxin, a significant proportion was resistant to blockade by guanethidine, suggesting Introduction  28 that this component arose from ATP released from a nonsympathetic source. Other evi dence supporting these observations include: (i) the inability of sympathectomy to affect quinacrine fluorescence (Burnstock et a!. 1984); (ii) mimicry by ATP of the rapid vasodilatation produced by stimulation of the perivascular nerves in the presence of guanethidine and atropine (Kennedy & Burnstock 1 985a); (iii) reduction by reactive blue 2 of vasodilatation mediated by perivascular nerves or produced by exogenous ATP (Reilly  eta!. 1987). Similar evidence for “direct” purinergic vasodilatation has been found for rat intrapulmonary arteries (lnoue & Kannan 1988), skeletal muscle blood vessels (Shimada & Stitt 1984), and some coronary and cerebral resistance vessels (Burnstock 1990c). 1.5.4.3. y-purinoceptors and endothelium-dependent vasodilatation Endothelial cell P y-purinoceptors are believed to be activated by blood-borne ATP 2  —  usually that derived from platelets during aggregation, or from the endothelium itself as a result of increased blood flow (Bodin et a!. 1991; Ralevic et a!. 1992; Hassessian et a!. 1993), ischaemia, hypoxia (Hopwood eta!. 1989; Buxton etal. 1990), or physical damage (Pearson & Gordon 1979). It was recently shown, however, that hypoxia may not stimu late ATP release from rat cerebral cortex capillary endothelium (Phillis eta!. 1993), as previously believed, which is in contrast to what occurs in the ischaemic or hypoxic heart (Borst & Schrader 1991; Headrick et a!. 1992). ATP itself has been shown to induce y-purinoceptors (Yang et 2 release of ATP from cardiac endothelial cells via an action at P 2 and EDRF fol a!. 1994). Numerous in vitro studies have demonstrated release of PGI lowing exposure of cultured endothelial, but not vascular smooth muscle, cells to ATP (De Mey & Vanhoutte 1981; Pearson & Gordon 1989; Pearson & Carter 1990). Further more, the EDRF released in response to ATP has been identified as NO (Palmer et al. 1987), derived from L-arginine (Palmer eta!. 1988), and can be inhibited by haemoglobin, methylene blue (Ralevic eta!. 1991), hydroquinone (Hopwood eta!. 1989), and deriva tives of L-arginine (Mathie et a!. 1991; Kaul et a!. 1992; Toda et a!. 1993). Other studies have demonstrated the ability of ATP to produce endothelium-dependent relaxation even Introduction  29 2 is blocked (Gordon & Martin when the capacity of endothelial cells to synthesize PGI 1983). Therefore, it has been proposed that ATP-induced vasodilatation is largely a con sequence of NO under most conditions (Gordon 1990). It appears that the vasodilator 2 are only seen in response to larger amounts of ATP (Fleetwood & Gordon effects of PGI 1987). As discussed previously, although ATP can cause vasoconstriction via P x-purino2 ceptors, the net effect of intraluminal ATP is usually vasodilatation under normal condi tions when the endothelium is intact (Cohen eta!. 1984; Kaul eta!. 1992). Thus, in the absence of endothelium (mechanical or chemical removal) (Kennedy eta!. 1985; Machaly eta!. 1988; Ralevic & Burnstock 1991b; Ralevic etal. 1992) or in the presence of reactive blue 2 (Taylor et a!. 1989) a vasoconstrictor effect is often unmasked. The eventual metabolites of ATP, ADP and adenosine, can reinforce the vasodilatatory effects of ATP by activating Pay- and Pi -receptors located (predominantly) on the endothelium and smooth muscle, respectively.  Actions of ATP in Vascular Beds and in Whole Animals  1.6.  The majority of studies investigating the vascular actions of ATP have been con ducted in isolated tissues in vitro. Although in vitro observations are useful in the charac terization and identification of receptor subtypes, closer approximations of physiological effects of ATP are obtained from studies using vascular beds and whole animals. Very little has been published on the effects of ATP and its analogues using vascular bed prepa rations; however, it appears that effects are variable not only among different vascular beds but also among different species. There are even fewer studies on the effects of ATP  in whole animals. Nevertheless, evidence to date appears to implicate an important role forATP and related purines in vasoregulation and haemostasis. 1.6.1.  Vascular beds Since vascular beds comprise small resistance arteries and arterioles, observations  obtained from these preparations are physiologically more relevant to the control of Introduction  30 peripheral vascular resistance than are those from isolated vessels. Vasodilator and vasoconstrictor responses of ATP have been demonstrated in a number of vascular beds, including the coronary, hepatic, mesenteric, intestinal, pancreatic, renal, hindlimb, foetal, and facial/nasal. 1.6.1 .1. Coronary vascular bed Although the action of adenosine in heart function and tone has been the subject of intense investigation since Berne (1963) identified adenosine as a physiological regulator of cardiac blood flow, the cardiac actions of ATP have not received much attention until recently due to the general belief that ATP exerted its actions indirectly, following break down to adenosine.  Indeed, ATP’s negative inotropic and chronotropic effects, anti  adrenoceptor effect, and inhibition of adrenergic neurotransmission are mediated by stimu lation of Pi-purinoceptors since they are antagonized by the non-selective adenosine receptor antagonist, 8-phenyltheophylline (Burnstock 1980; Pelleg eta!. 1990). Likewise, part of the vasodilatatory response to ATP has been attributed to adenosine (Ribeiro & Lima 1985), although the potency of adenosine as a coronary vasodilator is only about 25% that of ATP (Burnstock 1980). There is accumulating evidence implicating ATP per se not only as a coronary vasodilator (Olsson & Pearson 1990; Pelleg etai’. 1990), but also as a mediator of positive inotropy (Hoyle & Burnstock 1986; Legssyer et al. 1988; Scamps eta!. 1990; Pelleg etal. 1990). A recent study (Mantelli et a!. 1993) demonstrated that the depressant effect of ATP on atrial contractility is converted to a positive inotropic effect in the presence of either 1,3dipropyl-8-cyclopentylxanth me (A 1 receptor antagonist) or 8-phenyltheophylline. In addi tion, these authors showed that suramin and reactive blue 2 concentration-dependently reduced the positive inotropic effect of ATP. Similarly, Legssyer eta!. (1988) have shown that 8-phenyltheophylline enhances ATP-induced positive inotropy in rat papillary and ven tricular muscles.  Introduction  31 The rat coronary vascular bed responds to ATP by producing a biphasic response, consisting of an initial increase followed by a decrease in perfusion pressure, which is mediated by P2X- and P y-purinoceptors, respectively (Hopwood & Burnstock 1987). Fur 2 thermore, it has been demonstrated that ATP can mediate both endothelium-dependent (Hopwood eta!. 1989) and endothelium-independent (Corr& Burnstock 1991) vasodilata y-purinoceptors, respectively, in coro 2 tion via action at endothelial or smooth muscle P nary vessels. It has been suggested that the vasodilatatory effect of ATP may contribute to myocardial reactive hyperaemia (Giles & Wilcken 1977; Qlsson & Pearson 1990). 1.6.1.2. Hepatic vascular bed In the isolated perfused rabbit liver at basal tone, ATP and its analogues produced vasoconstriction with a potency order consistent with an action at P x-purinoceptors (Ralevic 2  et al. 1991).  In the same study, raising vascular tone with NA revealed ATP-induced  vasodilator responses with a rank order of potency of ATP analogues consistent with an action at P y-purinoceptors. Furthermore, the ability of methylene blue (which antago 2 nizes smooth muscle guanylyl cyclase and, possibly, inactivates EDRF) to antagonize responses to ATP, and the inability of 8-phenyltheophylline to attenuate ATP responses remaining after antagonism with methylene blue, demonstrated that the ATP-induced va y-purinoceptors and subsequent 2 sodilatation was due to a direct action on endothelial P -purinoceptors following 1 release of EDRF, and not due to adenosine acting at P ectoenzymatic breakdown of ATP. The attenuation of relaxations to ATP but not those to adenosine by inhibitors of the L-arginine to NO pathway, N-monomethyl-L-arginine and N nitro-L-arginine methyl ester (L-NAME), further confirms that ATP-induced vasodilatation y-purinoceptors (Mathie 2 of the hepatic arterial vascular bed is mediated by endothelial P  et a!.. 1991). Thus, it appears that the role of ATP in the hepatic vasculature is two-fold: constriction via smooth muscle P x-purinoceptors following release as a cotransmitter 2 from sympathetic nerves (Brizzolara & Burnstock 1990), and relaxation via endothelial y-purinoceptors following local release. It has also been proposed that ATP may partici 2 P Introduction  32 pate in the compensatory hepatic arterial vasodilatation in response to reduced portal blood flow  (i.e.  the “buffer response”) (Ralevic  eta!.  1991).  1.6.1 .3. Mesenteric vascular bed Studies similar to those conducted in the hepatic vascular bed have also been un dertaken in the isolated perfused rat mesenteric arterial bed. Ralevic and Burnstock (1991 b) have demonstrated vasoconstrictor responses to ATP in preparations with basal tone and L-NAME-sensitive vasodilator responses in those with raised tone. The vasodilator re sponse was abolished by removal of the endothelium with sodium deoxycholate. In the same study, evidence was presented for the existence of “pyrimidinoceptors” on the vas cular smooth muscle and endothelium which mediate vasoconstriction and vasodilatation, respectively. In addition, it has been shown that both ATP and oçf3-methylene-ATP, at subthreshold and supra-threshold doses, produce potentiation of vasoconstrictor responses to NA, thus indicating a postjunctional synergistic action between NA and ATP via action at c-adrenoceptors and P x-purinoceptors, respectively (Ralevic & Burnstock 1 990b). This 2 finding is supported by  in vitro  experiments using rat mesenteric arteries in which ATP, at  doses not producing contraction, potentiates NA-induced contraction (SjOblom-Widfeldt 1990). Sympathetic cotransmitterATP has also been implicated in the augmented pres sor response to transmural field stimulation during moderate cooling in the rat mesenteric vasculature, since the enhanced constrictor effect seen at 24 °C, but not 37 °C, is abol ished by cj3-methylene-ATP in the presence of prazosin (Yamamoto  et  a!. 1992).  1.6.1.4. Pancreatic vascular bed Dual effects of ATP have been demonstrated in the isolated perfused rat pancreas (Chapal & Loubatieres-Mariani 1983; Hillaire-Buys  eta!.  1991). In this preparation, the  y-mediated vasodilata 2 effect of ATP at basal tone was concentration-dependent, with P tion occurring at low doses and P x-mediated vasoconstriction occurring at high doses. 2 y-purinoceptors revealed a vasoconstrictor response to ATP at a con 2 Also, blockade of P centration without effect  per Se,  while blockade of P x-purinoceptors enhanced ATP-in 2 Introduction  33 duced vasodilatation. Furthermore, theophylline (Pi-purinoceptor antagonist) failed to modify the vasodilator effect of ATP, thus indicating that vasodilator responses to ATP were not mediated by adenosine derived from the breakdown of ATP. 1.6.1 .5. Intestinal vascular bed y-mediated vasodilatation, in preparations with 2 P x 2 mediated vasoconstriction and P low and high perfusion pressures, respectively, have been demonstrated in the autoperfused intestinal circulation of anaesthetized cats (Taylor et a!. 1989). Interestingly, c3-methylene-ATP was found to be a more powerful vasoconstrictor in post-capillary capacitance vessels than in pre-capillary resistance vessels, and produced tachyphylaxis of P2x-purinoceptors in the latter, but not the former (Taylor & Parsons 1991). In the same prepara tion, Taylor and Parsons (1989) demonstrated a prazosin- and yohimbine-resistant neurogenic vasoconstrictor response. The residual vasoconstrictor response was abol ished by c3-methyIene-ATP desensitization; however, a small, slower onset, more sus tained vasoconstriction persisted, the cause of which was not determined although it was suggested that high local concentrations of NA may have overcome cL-ad renoceptor block ade or that there may have been an increase in the concentration of other transmitter substances (e.g. peptides). c3-methylene-ATP desensitization also attenuated neurogenic vasoconstriction in cats not treated with oL-adrenoceptor antagonists, with a 74% decrease in response elicited by 1 Hz but only a 31% reduction in the response to 8 Hz. This observation is consistent with in vitro findings of the frequency- and train length-depend ency of the relative contributions of NA and ATP to the stimulation-induced vasoconstrictor response in rat mesenteric arteries (Sjöblom-Widfeldt 1990). 1 .6.1.6. Renal vascular bed cL-ad renoceptor antagonist-resistant vasoconstrictor responses have been demon strated in the isolated rat kidney such that the cL-j-adrenoceptor antagonists, prazosin and corynanthine, and the nonselective oL-adrenoceptor antagonist, phentolamine, did not sig nificantly reduce vasoconstrictor responses to low frequency but attenuated the responses Introduction  34 to high frequency periarterial nerve stimulation (Schwartz & Malik 1989). The responses to low frequency stimulation and the c-adrenoceptor antagonist-resistant responses at higher frequencies were abolished or significantly reduced after desensitization of 2Xpurinoceptors with oj3-methylene-ATP. In comparison, prazosin alone dramatically re duced the neurogenic vasoconstriction at all frequencies in the rabbit kidney, suggesting species differences in renal vascular responses to periarterial nerve stimulation. Churchill y-medi2 and Ellis (1993) have demonstrated both P x-mediated vasoconstriction and P 2 ated endothelium-dependent vasodilatation in response to 2-methylthio-ATP and cj3-methy- and P 2 ylene-ATP which are specific agonists for the P x-purinoceptors, respectively. 2 1.6.1 .7. Hindlimb vascular bed In the rat hindlimb, a biphasic response to ATP is produced, with vasoconstriction preceding a transient vasodilatation, although the pressor response is likely mediated by 5-hydroxytryptamine (Sakai et al. 1979). In the hindlimb of the cat, ATP and ADP caused vasodilatation, and the rank order of potency of ATP analogues was consistent with an y-purinoceptors (Taylor et al. 1989). This vasodilatation was reduced by 88% 2 action at P by gossypol, a potent and irreversible inhibitor of EDRF (Dudel & Forstermann 1988). In the rabbit hindlimb in vivo, Shimada and Stitt (1984) determined that the increase in blood flow to skeletal muscle produced by hypothalamic stimulation (the “defence reaction”) is -purinoceptors. 2 mediated by ATP acting at P 1.6.2.  Whole animals Bolus injection or infusion of ATP in animals has long been known to induce a pro  found reduction in blood pressure often associated with bradycardia. Gillespie (1934) reported one of the earliest studies demonstrating this effect in anaesthetized cats. Fol lowing bolus injection in the anaesthetized rat the onset of vasodilatation is typically rapid and the duration of the peak depressor response is transient (Delbro & Burnstock 1987). In contrast, the blood pressure response to a bolus injection of ATP in the pithed rat was reported to consist of an initial rise followed by a decrease and a second increase (Schlicker Introduction  35 et al. 1989).  Measurement of cardiac output revealed that peak pressure changes to  bolus injections of ATP and several ATP analogues were at least 80% due to changes in systemic vascular resistance (Sollevi et a!. 1984; Delbro & Burnstock 1984) which con firms the early findings of Rowe et al. (1962). The observation that dipyridamole, an inhibitor of adenosine uptake, significantly prolonged and potentiated the depressor re -purinoceptors are involved as a result 1 sponse t0ATP injection or infusion suggests that P of metabolism of ATP to adenosine (Sollevi eta!. 1984; Delbro & Burnstock 1987). These findings are confirmed by comparison of the arterial and venous ATP and adenosine lev els during inferior vena cava infusion of ATP in dogs  —  very low ATP levels in arterial  samples indicated that virtually all of the nucleotide had been metabolized to adenosine before reaching the arterial section of the vasculature, whereas in venous samples col lected simultaneously, only small amounts of adenosine were present, suggesting that metabolism occurs during passage through the pulmonary and coronary circulation (Sollevi et al. 1984). Consistent with this observation, the same study demonstrated that infusion of adenosine and ATP produced equal decreases in systemic vascular resistance. There are very few studies which have attempted to characterize the receptor sub types responsible for ATP effects in vivo, and even fewer have addressed the functional significance of NA-ATP cotransmission. This, however, appears to be a technical problem since many of the pharmacological tools used successfully in vitro have not performed similarly in intact animals. For example, oj3-methylene-ATP desensitization of P2x-purinoceptors has been shown to lack selectivity in vivo (Schlicker et a!. 1989; Daziel et a!. 1990).  In addition, x,3-methylene-ATP exerts cardiotoxic effects at doses required to  produce desensitization, and even when desensitization is produced by administering the dose of o,f3-methylene-ATP incrementally (to avoid cardiac arrest), receptor desensitiza tion rapidly declines within a few minutes (Schlicker eta!. 1989; Tarasova & Rodionov 1992). As a metabolically-stable agonist, ccj3-methylene-ATP cannot be infused due to rapid tachyphylaxis and cardiotoxicity and, as a bolus injection, has been found to pro Introduction  36 duce unpredictable vascular and cardiac effects which vary according to the route of ad -purinoceptor antagonist, 2 ministration (Delbro et a!. 1 985). The recently identified P suramin, has shown promise as a useful pharmacological tool in vivo. Urbanek et a!. (1990) demonstrated that suramin produces a 6-fold parallel shift to the right of the doseresponse curve for x,j3-methylene-ATP in the pithed rat, while Schlicker et al. (1989), in the same preparation, showed that suramin decreased not only the response to o,f3-methylene-ATP but also the initial rise in blood pressure (but not the subsequent decrease) elicited by ATP or electrical stimulation of the thoracolumbar sympathetic outflow. Never theless, it was suggested that the ability of suramin to reduce the electrically-evoked pres sor response may be the result of inhibition of neurotransmitter release via a presynaptic site rather than blockade of postsynaptic P x-purinoceptors (Sch licker eta!. 1989). There 2 fore, further characterization of the action of suramin is required before the use of this drug can provide substantive evidence of the role of cardiovascular purinoceptors in vivo. An x-ad renoceptor antagonist- resistant response to sympathetic nerve stimulation, analogous to that observed in isolated vessels and vascular beds, has been identified in the pithed rat (Flavahan et a!. 1985). This vasopressor response was abolished by 6hydroxydopamine or guanethidine, indicating a sympathetic nerve origin, and was found to constitute approximately half the control response. However, since the response re maining after reserpine treatment was smaller than the prazosin-plus-rauwolscine-resist ant response, it was suggested that part of the oc-adrenoceptor antagonist resistant pres sor response could be mediated, in part, by ‘y-adrenoceptors. In this study, desensitization with oc,f3-methylene-ATP reduced the nerve-mediated pressor response only after oc-block ade or reserpine pretreatment, but not in drug-free controls; this was interpreted as indi cating a relatively minor role for purinergic cotransmission in rat vasculature. Subse quently, the use of a novel administration regime for oc,3-methylene-ATP resulted in a 60% reduction of the nerve-evoked pressor response in the absence of oc-adrenoceptor block ade, and completely abolished the residual response after cx-adrenoceptor antagonism Introduction  37 (Bulloch & McGrath 1988b). The previous inability of c,f3-methylene-ATP to affect the pressor response before ct-antagonism, or to abolish the residual response after o-an tagonism completely (Flavahan eta!. 1985), was attributed to the transient nature of its action. Thus, the effects of cL,f3-methylene-ATP and cL-antagonists appeared to be addi tive, with 40% of the pressor response attributable to ci-adrenoceptor (mainly xj) stimula -purinoceptor stimulation (Bulloch & McGrath 1986, 1 988a). 2 tion and the remainder to P Similarly, Daziel et al. (1990) demonstrated that the pressor response to sympathetic nerve stimulation in the pithed rat was attenuated by approximately 80% following c3-methylene-ATP desensitization; however, unlike Bulloch and McGrath (1988b) who deemed the desensitization procedure “selective” by virtue of its failure to block responses to NA, Daziel and coworkers (1990) found that cL,f3-methylene-ATP treatment also attenuated the pressor responses to NA, angiotensin II, and vasopressin. In the pithed rat, it has been demonstrated that the purinergic component of the 2 sympathetic pressor response can be attenuated by nifedipine, which blocks L-type Ca 2 channel block channels, whereas the adrenergic component is largely resistant to Ca ade (Bulloch & McGrath 1988a). Nifedipine also attenuated the vasopressor response produced by intravenous bolus administration of cL,3-methylene-ATP. These observations appear to provide an in vivo correlate for in vitro findings which indicate electromechanical 2 channels; see section 1.5.3) coupling (i.e. involving the EJP and voltage-dependent Ca as the mechanism of ATP-induced vasoconstriction. Recently, purinergic transmission was found to contribute significantly to the pressor sinocarotid reflex but only negligibly to the pressor response resulting from stimulation of somatic afferents (Tarasova & Rodionov 1992). This study demonstrated that the pressor responses to sciatic nerve stimulation, and to asphyxia, were strongly depressed by phentolamine and dihydroergotamine, while the average magnitude of the response to carotid artery occlusion remained unchanged in the presence of cL-adrenoceptor block ade. In comparison, ganglion blockade with hexamethonium brought about a pronounced Introduction  38 decrease in all three reflexes (i.e. those to carotid artery occlusion, sciatic nerve stimula tion, and asphyxia). Interestingly, the ability of c-adrenoceptor antagonism to attenuate the sinocarotid reflex was quite variable, with responses ranging from a 70% decrease in pressor response to only a slight decrease or even augmentation of the response. Desen sitization by c,f3-methylene-ATP attenuated the pressor responses resistant to x adrenoceptor blockade, and recovery from desensitization (in 3  -  5 mm) was accompa  nied by restoration of the sinocarotid reflex. Another interesting finding from this study is that the rate of blood pressure elevation, but not the magnitude, in response to sciatic nerve stimulation was lowered in the presence of CL,J3-methylene-ATP. This observation is consistent with studies on isolated vessels which reveal that the initial, phasic component of the response to nerve stimulation is preferentially inhibited by o3-methylene-ATP (Sjöblom-Widfeldt 1990).  1.7.  Aims of the Thesis The vascular effects of ATP and its role in the regulation of blood pressure in whole  animals are not well characterized for reasons discussed above. Although some studies have identified c-adrenoceptor antagonist-resistant vascular responses in whole animals, very few have sought to determine whether these responses have a purinergic compo nent (Flavahan etal. 1985; Bulloch & McGrath 1988a,b; Schlicker eta!. 1989; Daziel eta!. 1990; Tarasova & Rodionov 1992). Therefore, the present thesis is an attempt to study the cardiovascular effects and possible venoregulatory role of ATP in the conscious rat. The specific aims of the experiments described in this thesis are: (i)  To demonstrate resistance to x-adrenoceptor antagonism in the venous system, as reflected by mean circulatory filling pressure;  (ii)  To determine whether purinergic neurotransmission is involved in the control of basal and reflex venous tone;  Introduction  39 (iii)  To determine whether sympathetic NA-ATP cotransmission contributes to the main tenance of venous tone;  (iv)  To characterize the arterial and venous effects of exogenous ATP.  Introduction  40 2.  METHODS & MATERIALS  2.1.  Methods  2.1.2.  Animals Male Sprague-Dawley rats (360 ± 30 g, mean ± s.d., and median  =  360 g) obtained  from Charles River, Canada were used in this study. Rats were kept in the Department of Pharmacology and Therapeutics of the University of British Columbia and given free ac cess to Purina Rat Chow and water. Recommendations from the Canada Council of Animal Care and internationally accepted principles in the care and use of experimental animals were adhered to. 2.1.3.  Surgical preparation of animals for use in MCFP studies  2.1.3.1. Standard preparation Mean circulatory filling pressure (MCFP) was determined by the method of Yamamoto et al. (1980). Animals were prepared while anaesthetized with halothane vaporized in air (5% for induction, 1.5-2.5% for maintenance). PE 50 cannulae (Intramedic, NJ, USA) were inserted into the left iliac artery and right iliac vein for the measurement of blood pressure by a pressure transducer (model P23XL, P23 ID or P23D8, Gould Statham, CA, USA) and the administration (bolus injection or infusion with Harvard Apparatus infusion pump model 975, Harvard Apparatus, MA) of drugs, respectively. These cannulae were advanced approximately 2-3 cm into each vessel. An additional PE 50 cannula was in serted into left iliac vein and advanced 4-5 cm into the inferior vena cava to record central venous pressure (CVP) with a pressure transducer (model P23XL, P231D or P23D8, Gould Statham, CA, USA). Gentle tapping on the ventral mid-abdominal region of the rat pro duced a large, transitory increase in central venous pressure if the iliac central venous cann ula was positioned correctly. A saline-filled, balloon-tipped cannula was placed in the right atrium through the right external jugular vein, and proper positioning of the balloon was initially assessed by care Methods & Materials  41 fully shaking the cannula. Shaking the cannula produced arrhythmias if the balloon was positioned correctly. Proper positioning was further verified by testing the ability of the inflated balloon to stop the circulation completely as described in section 2.1.4. This was shown by a simultaneous, smooth increase in venous pressure and a decrease in mean arterial pressure (MAP) to less than 25 mmHg. Atrial balloons were constructed from PE 50 tubing, 5-0 silk suture and Stretch Tite® plastic wrap as shown below. All cannulae 1 in 9% w/v NaCl) and tunneled with a trochar were filled with heparinized saline (25 iu mIto the back of the neck where they were exteriorized, secured, flame-sealed, and colour coded. Incisions were closed and rats were placed in individual cages, with free access to food and water, to recover from surgery for 18-24 h before being used in experiments.  Construction of Atrial Balloons U  Silk suture is lashed around the Stretch Tite® and the cannula several times then secured; excess Stretch Tite is cut away  // i  Stretch Tite° is folded over a saline filled cannula  J The finished atrial  balloon is test-inflated with 0.1-0.3 ml of saline to test for leaks and proper deflation  After recovery, arterial and central venous lines were reconnected to pressure trans ducers attached to a Grass multichannel polygraph (model 79D, Grass Instrument Co., MA, USA). Mean arterial and central venous pressures were obtained by electronic aver aging. Heart rate (HR) was obtained either manually from the upstroke of the blood pres sure recording or electronically using a Grass tachograph (model 7P44C or 7P4G, Grass Instrument Co., MA, USA). At the conclusion of experiments, rats were sacrificed using i.v. pentobarbitone and post-mortem dissection of rats was used to confirm that the atrial  Methods & Materials  42 balloon and central venous line were positioned properly. Results from rats in which these were improperly positioned were discarded. 2.1 .3.2. Modifications to the standard preparation: experiments involving suramin PE 50 cannula was inserted into the right carotid artery for the injection of suramin or its vehicle (saline). Care was taken not to damage the vagus nerve. 2.1.4.  Measurement and calculation of MCFP MCFP was determined in conscious, unrestrained rats by inflating the implanted  atrial balloon with  0.5 ml of saline in order to arrest circulation. Within 5 sec after circu  latory arrest, MAP decreased to a final arterial pressure (FAP) of 25 mmHg or less and central venous pressure simultaneously increased from baseline to a plateau value. The difference between the baseline and plateau venous pressures is referred to as venous plateau pressure (VPP). MCFP was calculated using equation 1, taken from Samar and Coleman (1978), and using a value of 1/60 for the arterial to venous compliance ratio (Yamamoto et al. 1980).  MCFP=VPP+-. 1 -(FAP—VPP) 60  equation 1  22.  Experimental Design, StatisticalAnalysis, and Presentation of Results  2.2.1.  Experimental design  All experiments followed a randomized, split plot factorial design with repeated meas ures on dose-dependent factors. Groups of experiments were arranged as replicated 2 x 2x  X factorials, with 2 pretreatment levels, 2 treatment levels and X repeated measures  on the treatment (5 or 7 sequential doses). Repeated measurements were conducted on MAP, HR and central venous pressure. Plateau central venous pressures during circula tory arrest were converted to MCFP using the equation defined in section 2.1 .4. In addi  Methods & Materials  43 tion, control values were measured before and after pretreatment and are referred to as “Pretreatment Control” and the “Post-treatment Control”, respectively. 2.2.2.  Statistical analysis  Analyses were performed separately on control values and dose-response relation ships. Control values were analyzed without transformation, whereas dose-response analysis was performed on data expressed as percentage of post-treatment control in order to reduce variance introduced by the control covariate. i.  Analysis of variance with replication was used to test the effect of pretreatment on pre- and post-treatment control means.  ii. To test the between groups pretreatment and treatment main effects and the pretreatment x treatment interaction on the average response, standard repeatedmeasures analysis of variance was used. Between groups a priori, multiple com parisons were made using the Tukey (hsd) procedure. iii. Within groups effects were analyzed with standard repeated measures analysis of variance to test main and interaction effects. The Huynh-Feldt epsilon correc tion for multisample asphericity was used to adjust probabilities. Within groups a priori hypotheses were tested using multivariate profile/trend analysis (referred to as curve analysis in Results). Profile/trend analysis was used to compare dosedependency of responses. The F-value was estimated using Wilks’ lambda. Bonferonni layering was used to adjust probabilities when multiple comparisons were made. The appropriate corrections were made for unequal sample sizes. A p <0.05 was pre selected as the criterion for statistical significance. The statistical package, SYSTAT 5.2.1 for Macintosh (SYSTAT Inc., IL), was used to analyze all data. 2.2.3.  Presentation of results  All values are expressed as mean ± s.e.mean with the number of observations given in brackets, if provided. Values in all tables are referenced to their respective figures. Methods & Materials  44 Points in figures are mean % of post-treatment control with error bars representing ± s.e.mean. Vehicle-treated time controls are presented in the Controls section of the Re sults and in the appropriate figures as shaded lines and symbols. The shaded, control data is repeated in figures along with the solid black, drug-treated data so that main and interactive effects can be distinguished.  Experimental Protocol  2.3.  The general design used in all experiments is presented in section 2.2.1. 2.3.1.  Phentolamine and mecamylamine 1 Rats were pretreated with either saline (1.8 jil minl) or hydralazine (0.3 jimol kg  i.  minl) which was continuously infused through the central venous line for the remain der of the experiment. After a plateau response to the pretreatment was attained (20 mm), a dose-response curve to either saline (1.8- 100 tl minl) or phentolamine (0.035 -  1 minl) was constructed. Saline and phentolamine were infused through 1.9 jimol kg  the right iliac venous line until a plateau response was attained (7 mm), after which MCFP was measured. Rats were allowed 3 mm to recover after each dose, so that MCFP measurements were made at 10 mm  intervals. MCFP measurements were  also taken prior to the administration of the pretreatment (Pretreatment Control) and after a steady state response to the pretreatment was attained (Post-treatment Con trol). MAP and HR were continuously monitored. ii.  Rats were pretreated with a continuous infusion of either 30% ethanol in double1 minl) for 20 mm distilled water (37 p1 minl) or nifedipine (0.3 imol kg  followed by  construction of dose-response curves to either saline (1.8- 100 p1 mimi) or phentolamine (0.035  -  1.9 jimol kg 1 minl) infused through the right iliac venous line. As a precau  tion arising from the light-sensitivity of nifedipine, all bottles, syringes and cannulae containing nifedipine were covered with aluminium foil. Haemodynamic measurements were taken as described in 2.3.1 .(i). Methods & Materials  45 Rats were pretreated with a continuous infusion of either saline (1 .8 jil mini) or  iii.  hydralazine (0.3 p.mol kg-i mini) for 20 mm followed by the construction of a cumula tive dose-response curve to either saline (0.3 ml dose-i) or mecamylamine (0.18 180 -  iimol kg-i) injected through the right iliac venous line. Haemodynamic measurements were taken as described in 2.3.1.(i). In preliminary experiments, the dose range of mecamylamine was selected by meas  iv.  uring the ability of each dose of mecamylammne (0.18  -  180 pmoI kg-i) to inhibit the  reflex bradycardia produced by a methoxamine (80 nmol kg-i)-induced increase in MAP. The left iliac artery and vein were cannulated for measurement of MAP and HR, and administration of drugs, respectively. Methoxamine was i.v. injected at 10 mm intervals, 7 mm following i.v. injection of each dose of mecamylamine which allowed 3 mm for haemodynamic readings to recover from methoxamine. Suramin I  2.3.2.  Rats were pretreated with continuous infusion (through the central venous line) of either saline (1.8 jil mm-i) or hydralazine (0.3 p.mol kg-i mini) followed by construc tion of a cumulative dose-response curve to either saline (0.3 ml dose-i) or suramin (25  -  400 jimol kg-i). Saline and suramin were given as bolus injections through the  carotid arterial line 7 mm before MCFP was measured, not including the time required for injection. Preliminary studies demonstrated that i.a. injection substantially reduced the severe cardiotoxic effects observed when suramin was administered i.v.  High  doses of suramin (100-400 jimol kg-i) were injected slowly over a 2-3 mm period to minimize suramin’s severe cardiotoxic and convulsive effects. Rats were allowed 3 mm  to partially recover from the effects of each dose, so that MCFP measurements  were made at  10 mm intervals. MCFP measurements were also taken prior to and  20 mm after the start of the infusion of hydralazine, which was at the plateau phase of the response to hydralazine. MAP and HR were continuously monitored.  Methods & Materials  46 Pretreatments were administered similarly to that described in 2.3.2.(i) except that the continuous infusion consisted of either 30% ethanol in double-distilled water (37 uI 1 mm-i). Administration of saline or suramin and mini) or nifedipine (0.3 uimol kgmeasurement of haemodynamic variables were the same as described in 2.3.2.(i). As a precaution arising from the light-sensitivity of nifedipine, all bottles, syringes and cannulae containing nifedipine were covered with aluminium foil. Rats were pretreated with either saline (0.3 ml) or mecamylamine (18 iimol kg-i) i.v.  iii.  injected through the central venous line, followed (20 mm  later) by construction of  dose-response curves to either saline or suramin. Administration of saline or suramin and measurement of haemodynamic variables were the same as described in 2.3.2.(i). The degree of mecamylamine-induced ganglionic blockade was assessed for each experiment by comparing the reflex tachycardia induced following i.v. injection of a depressor dose of acetylcholine (2 rig) before and after (at the end of the experiment) administration of mecamylamine. MAP and HR were continuously monitored. 2.3.3.  Phentolamine Rats were pretreated with either saline (0.3 ml) or suramin (200 tmol kg-i) which  was injected slowly through the carotid arterial line. After a plateau response to the pretreatment was attained (10 mm), a dose-response curve to i.v. infusion of either saline (1.8  -  100 tl mini) or phentolamine (0.035  -  1.9 jimol kg-i mimi) at dose-  intervals of 10 mm was constructed. MCFP measurements were also taken prior to and 10 mm after pretreatment with suramin and 7 mm after the infusion of each dose of phentolamine or saline. 2.3.4.  Suramin II Rats were pretreated with either 6% citrate dissolved in double-distilled water (0.4  ml) or reserpine (3 mg kg-i) which was injected i.p. 24 h before the start of the experi ment. On the next day, a cumulative dose-response curve to either saline (0.3 ml dose-i) or suramin (25  -  400 umol kg-i) was constructed. Administration of suramin Methods & Materials  47 and all haemodynamic measurements were the same as described in 2.3.2.(i), except that a pretreatment control measurement could not be taken. 1 for 10 mm Rats were pretreated with either saline (26 il mirn  followed by 1.8 jil  1 for 10 mm 1 for the duration) or guanethidine (loading dose of 7.2 iimol kg-i mirn min 1 minl) which was infused through the followed by a maintenance dose of 0.5 pmol kg central venous line for the remainder of the experiment. At the plateau response to the pretreatment (1 hr), a cumulative dose-response curve to either saline or suramin was constructed. Administration of suramin and all haemodynamic measurements were the same as described in 2.3.2.(i). 2.3.5.  ATP Rats were pretreated with either saline (0.3 ml) or mecamylamine (18 jimol kg-i)  injected through the central venous cannula. Attainment of a plateau response to pretreatment (20 mm) was followed by construction of a dose-response curve to either 1 minl) infused through the right saline (3.5-200 p.1 minl) 0rATP (0.29- 16.8 p.mol kg iliac venous line at dose-intervals of 10 mm. MCFP measurements were also taken prior to and 20 mm after pretreatment with mecamylamine and 7 mm after the infusion of each dose of ATP or saline. Administration of saline or suramin and measurement of haemodynamic variables were the same as described in 2.3.2.(i). The degree of mecamylamine-induced ganglionic blockade was assessed for each experiment by comparing the reflex tachycardia induced following i.v. injection of a depressor dose of acetylcholine (2 p.g) before and after (at the end of the experiment) administration of mecamylamine. MAP and HR were continuously monitored. ii.  Rats were pretreated with either saline (0.3 ml) or suramin (200 p.mol kg-i) which was injected slowly through the carotid arterial line. After a plateau response to the pretreatment was attained (10 mm), a dose-response curve to infusion of either saline or ATP was constructed. Administration of ATP and all measurements of MCFP were identical to those described in 2.3.5.(i). Methods & Materials  48 Rats were pretreated with either saline (0.3 ml) or cibacron blue (13 jimol kg-i) in  iii.  jected through the central venous cannula. After a plateau response to the pretreatment was attained (20 mm), a dose-response curve to either saline orATP was constructed. Administration of ATP and all measurements of MCFP were identical to those de scribed in 2.3.5.(i). Rats were pretreated with either saline (0.5 ml) or 8-phenyltheophylline (27 pmol  iv.  kg-i) which was injected through the central venous cannula. After a plateau response to the pretreatment was attained (20 mm), a dose-response curve to either saline or ATP was constructed. Administration of ATP and all measurements of MCFP were identical to those described in 2.3.5.(i).  2.4.  Drugs All drugs were prepared fresh daily and dissolved or diluted with normal saline (9%  NaCI w/v in double-distilled water), except where noted. Sonication was used to dissolve hydralazine, mecamylamine, and phentolamine in vehicle. Acetylcholine chloride, adeno sine 5-triphosphate disodium, cibacron blue 3GA, guanethidine, hydralazine HCI, mecamylamine HCI, cj3—methyleneATP Li salt, nifedipine, and 8-phenyltheophylline were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Citric acid and sodium chloride were obtained from Fisher Scientific Co. (ON, Can). Sodium suramin was obtained from CB Chemicals (Woodbury, CT, USA). Methoxamine, sodium pentobarbitone, phentolamine, and reserpine were obtained from Burroughs Wellcome Inc (ON, Can), MTC Pharmaceu ticals (ON, Can), CIBA Geigy (ON, Can) and Carl Roth (Germany), respectively. Nifedipine was dissolved in 100% ethanol then diluted with double-distilled water to 30% ethanol. Doses of 8-phenyltheophylline were prepared as individual aliquots and suspended in saline. Reserpine was dissolved in 6% citrate in double-distilled water.  Methods & Materials  49 3.  RESULTS  3.1.  Controls: Time and Volume Effects of Vehicle in the Absence and Presence of Various Drug Treatments Pre- and post-treatment control values of MAP, HR, and MCFP for time/volume con  trol experiments in vehicle- or drug-treated groups are presented in Table 1. The data shown in Table 1 and Figures 1 .1 to 1 .6.3 is presented in this section in order to illustrate the time and volume effects of vehicle in the absence and presence of drug treatment. The dose-response curves illustrated in the figures of this section reappear (as shaded lines and symbols) for the purpose of comparison along with the appropriate groups in sections 3.2 to 3.6 in which the time/volume effects are described.  Results  C,)  CD C,,  DDW (6% Citrate) Reserpine  Saline S u ram in Mecamylamine Cibacron Blue 8-Phenyitheophylline  1.5  1.6  98 103 106 100 102  ±2 ±3 ±3 ±2 ±2  —  104±2  101±2 104±2 103±3 104±3  107 ± 3 106±2  101 ± 1 106 ± 2 105 ± 3 109 ±2 104±2  (mmHg)  5.3 5.3 5.7 5.6 5.5  ± ± ± ± ±  —  0.1 0.1 0.3 0.1 0.1  5.3±0.2  5.3±0.2 5.4±0.2 5.2±0.2 5.6±0.2  5.2 ± 0.1 5.5±0.2  5.8±0.2 5.3±0.2  5.4 ± 0.1 5.5 ± 0.2 5.6 ± 0.1  (mmHg)  —  377 351 362 383 391  ± ± ± ± ±  16 13 6 15 14  403±15 357± 17  418± 15 475± 17 393±15 393± 10  371 ± 9 477±8  396 ± 18 477 ± 8 355±11 420± 12 420±8  (beats/mTh)  102 ± 2 112±5 72 ± 2 103 ± 2 105 ± 3  103±3 83±3  102±2 77±3 76±4 79±2  107±4 78±2  102 ±3 70±2  100 ± 2 78 ± 2 114±2  (mmHg)  5.1 4.6 4.4 5.7 5.5  ±0.2 ± 0.2 ± 0.3 ± 0.2 ± 0.2  5.2±0.1 5.4±0.2  5.1 ±0.3 6.5±0.2 3.6±0.1 5.4± 1.1  5.1±0.1 6.7±0.2  5.2±0.2 6.5±0.3  5.2 ± 0.1 6.7 ± 0.2 5.9 ± 0.2  (mmHg)  Post-treatment Controls HR MCFP MAP  Values are mean ± s.e.mean. DDW = double distilled water, EtOH = ethanol, = not measured. Figures referenced show the volume- or infusion rate-response curves as % of control value for each treatment. Doses of drugs were as ), guanethidine (7.2 iimol kg-i mirn 1 1 for 10 mm followed by 0.5 .imol kg-i mini), follows: cibacron blue (13 tmol kg ) 1 min mecamylamine , (18 tmol kgi), nifedipine (0.3 jimol kg1 hydralazine (0.3 imol kg1 minl), 8phenyltheophylline (27 iimol kgi), reserpine (3 mg kg-i 24 h prior to study), suramin (200 iimol kg-i).  ± ± ± ± ±  18 17 12 8 19  416±12  Saline Hydralazine Mecamylamine G u an eth i dine  1.4  383 432 406 375 396  408± 11 421 ±14 418±18 425± 17  Saline Hydralazine  1.3  —  383 ± 12 414±12  DDW (30% EtOH) Nifedipine  (beats/mm)  1.2  Saline Hydralazine Suramin  Treatment Group  Pretreatment Controls MAP HR MCFP  404 ± 20 414 ± 12 412 ± 10 410± 10 380±8  Figure Reference 1.1  unrestrained Sprague-Dawley rats.  n 7 7 6 6 9 6 7 6 6 6 5 6 6 6 6 5 7 7  both before (Pretreatment Control) and after treatment (Post-treatment Control) with either vehicle or drug in conscious,  Table 1. Summary of heart rate (HR), mean arterial pressure (MAP) and mean circulatory tilling pressure (MCFP)  C,’ 0  51 110 0 -4-,  0 0 0 —  90  :i:  80 110  -  0  100  -  90  80 110  .  0  o 100 0 ‘4-  0  0-  U  90  0 80  -1  1 0 Saline (iji/min), Log  2  Figure 1.1. Dose-response curves of the effects of saline on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unre ) (0) or suramin (200 1 1 min strained rats treated with saline (•), hydralazine (0.3 imol kg ) (A); each point represents mean ± s.e.mean, n 6. 1 jimol kg  Results  52 110 0 4-  C 0 0  100  4-  0  90 I  80 110 0 4-’  C 0 0  100  9-  0  90  80  cl  110 0  I— 4-  C  o 100 0  4-  0  0  U  90  0 80  0  2 1 Cumulative Volume Saline (ml)  Figure  12. Dose-response curves of the effects of saline on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unre strained rats continuously infused with vehicle (30% ethanol in double-distilled water) (•) ) (0); each point represents mean ± s.e.mean, n 1 or nifedipine (0.3 imol kg 1 min  6.  Results  53 110  -  b00  •— 90  -  I  80 110 0  100  90  80 110 0 -I-.  C  o 100 C)  0  0-  LL  90  C-)  80  0  1 0.5 Cumulative Volume Saline (ml)  1.5  Figure 1.3.  Dose-response curves of the effects of saline on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unre 1 min ) (0); 1 strained rats continuously infused with saline (•) or hydralazine (0.3 iimol kg each point represents mean ± s.e.mean, n  6.  Results  54  130  120  -  -  0 -I-’  -  C 0 ()  o  T  ‘I  _100  A  90  80  I  0  1 Cumulative Volume Saline (ml)  I  2  Figure 1.4.1. Dose-response curves of the effects of saline on heart rate (HR) in con  ) (0), 1 kg min scious, unrestrained rats treated with saline (•), hydralazine (0.3 pmol 1 1 min 1 for 10 mm followed ) (A) orguanethidine (3 pmoI kg 1 mecamylamine (18 pmol kg ) (s); each point represents mean ± s.e.mean, n 5. 1 1 min by 0.5 imoI kg  Results  55  130  120  A 0 -I  C 0 C) 0  0  100  90  80  -  0  1 Cumulative Volume Saline (ml)  2  Figure 1.4.2. Dose-response curves of the effects of saline on mean arterial pressure  1 (MAP) in conscious, unrestrained rats treated with saline (•), hydralazine (0.3 tmoI kg 1 for 10 1 min ) (A) or guanethidine (3 jtmol kg 1 ) (0), mecamylamine (18 tmol kg 1 min 1 min ) (Li); each point represents mean ± s.e.mean, n 5. 1 mm followed by 0.5 tmol kg  Results  56  I  l30  120  1  0  110  A  0 LL  C-)  90  80  I  I  0  1 Cumulative Volume Saline (ml)  2  Figure 1.4.3. Dose-response curves of the effects of saline on mean circulatory filling  pressure (MCFP) in conscious, unrestrained rats treated with saline (•), hydralazine (0.3 1 1 min ) (A) or guanethidine (3 imol kg 1 1 min ) (0), mecamylamine (18 jimol kg 1 iimol kg for 10 mm followed by 0.5 iimol kg 1 min ) 1  (s); each point represents mean ± s.e.mean, n 5.  Results  57 110 0 C 0 C.) 0 ‘—  90  80 110 0  100  90  80 110  0-  U  90  C-)  80  I  I  0  2 1 Saline (ml) Cumulative Volume  Figure 1.5.  Dose-response curves of the effects of saline on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unre 1 24 h prior to study) strained rats treated with DDW (6% citrate) (•) or reserpine (3 mg kg (0); each point represents mean ± s.e.mean, n  =  6.  Results  58  i  120  110 0 0 0  100  90  80  I  0  1 Saline (p1/mm), Log  I  2  Figure 1.6=1. Dose-response curves of the effects of saline on heart rate (HR) in con  ) (0), 1 scious, unrestrained rats treated with saline (•), suramin (200 pmol kg ) (L) or 8-phenyltheophylline 1 ) (A), cibacron blue (13 iimol kg 1 mecamylammne (18 imol kg (27 jimol kg ) (•); each point represents mean ± s.e.mean, n 5. 1  Results  59  140  A  130  —  0  120  C 0 ()  110 tl  100  90  80  0  1 Saline (p1/mm), Log  2  Figure 1.6.2. Dose-response curves of the effects of saline on mean arterial pressure  ) 1 (MAP) in conscious, unrestrained rats treated with saline (•), suramin (200 p.mol kg ) () or 81 ) (A), cibacron blue (13 iimol kg 1 (0), mecamylammne (18 pmoI kg ) (I); each point represents mean ± s.e.mean, n 5. 1 phenyltheophylline (27 pmol kg  Results  60  120  -  110 0 4-’  C 0 0  100 0  U  C-)  90  80  0  1 Saline (p1/mm), Log  2  Figure 1.6.3. Dose-response curves of the effects of saline on mean circulatory filling  pressure (MCFP) in conscious, unrestrained rats treated with saline (•), suramin (200 ) (A) or81 ) (A), cibacron blue (13 jimol kg 1 ) (0), mecamylammne (18 jimol kg 1 iimol kg ) (n); each point represents mean ± s.e.mean, n 5. 1 phenyltheophylline (27 jimol kg  Results  61  Resistance of MCFP to a-Adrenoceptor Antagonism in Rats with Normal and  3.2.  Reflexly-Increased Venous Tone Effect of phentolamine in the absence and presence of hydralazine (Figure 2.1)  3.2.1.  The control values for haemodynamic variables are given in Table 2. Saline did not significantly alter MAP, HR, or MCFP whereas hydralazine decreased MAP (23 ± 1 %) and increased both HR (19±2%) and MCFP (31 ±2%) compared to post-saline control values  (n = 14). Overtime, continuous hydralazine infusion produced no further significant changes in MAP, HR, or MCFP. Phentolamine, in the absence of hydralazine, clearly and significantly decreased MAP and increased HR in a dose-dependent manner, but had no effect on MCFP. Curve analysis of the MAP curves revealed a significant difference between phentolamine groups in the absence and presence of hydralazine in addition to a significant hydralazine phentolamine interaction which, together with visual inspection of the curves, suggest that hydralazine potentiated the depressor effect of low doses of phentolamine. In the pres ence of hydralazine, phentolamine produced dose-dependent bradycardia which was not significantly different from the decrease in HR observed in the hydralazine-treated control group.  However, a significant hydralazine-phentolamine interaction suggests that  hydralazine converted the phentolamine-induced tachycardia into bradycardia. In the pres ence of hydralazine, phentolamine produced a very modest, but insignificant, decline in MCFP. 3.2.2.  Effect of phentolamine in the absence and presence of nifedipine (Figure 2.2)  The control values for haemodynamic variables are given in Table 2. Vehicle (30% ethanol in double-distilled water) did not significantly alter MAP, HR, or MCFP. Nifedipine treatment significantly decreased MAP (27 ± 2%) and increased MCFP (19 ± 3%), but did not affect HR compared to the post-treatment control values of vehicle-treated groups (n 15). During continuous nifedipine infusion, MAP increased slightly but significantly over  Results  62 time. In contrast, HR and MCFP were not significantly affected, although HR showed a slight, gradual decrease and MCFP a slight, gradual increase. In the vehicle-treated group, phentolamine dose-dependently and significantly de creased MAP and increased HR, but had no effect on MCFP. The phentolamine-induced depressor effect was not significantly different in the presence of nifedipine compared to that observed in the absence of nifedipine. Phentolamine produced a very modest de crease in HR in the presence of nifedipine; however, this effect was insignificant when compared to the effect of saline in the nifedipine-treated control group. A significant inter action between nifedipine and phentolamine suggests that nifedipine converted the phentolamine-induced tachycardia into bradycardia.  A moderate but significant  phentolamine-induced decrease in MCFP was revealed in the presence of nifedipine. 3.2.3.  Effect of mecamylamine in the absence and presence of hydralazine (Figure 2.3)  The dose-response effect of mecamylamine-mediated blockade of the reflex bradycardia accompanying a methoxamine-induced increase in MAP was examined. In the absence of mecamylamine, methoxamine increased MAP by 61 ± 5 mmHg and de creased HR by 182±11 beats min (n = 6). At the dose range tested (0.18- 180 jimol kg-i), mecamylamine potentiated the pressor effect of methoxamine by 0 to 65 ± 16% (n  =  6)  and inhibited methoxamine-induced bradycardia by 8 to 100 ± 1% (Figure 2.4). The control values for haemodynamic variables are given in Table 2. Saline had no significant effect on MAP, HR, or MCFP. Hydralazine treatment significantly decreased MAP (27 ± 3%) and increased both HR (22 ± 2%) and MCFP (19 ± 3%) compared to the respective post-treatment control values in saline-treated groups (n  14). During continu  ous hydralazine infusion, HR declined slightly but significantly over time, while MAP and MCFP were stable. In the absence of hydralazine, mecamylamine markedly decreased HR, MAP, and MCFP in a dose-dependent and significant manner. The mecamylamine-induced de crease in MCFP was not significantly altered by hydralazine. In comparison, hydralazine Results  63 treatment considerably attenuated the mecamylamine-induced depressor effect such that the response was no longer significantly different from that in the hydralazine-treated con trol rats given saline infusion.  Curve analysis indicated a significant hydralazine  mecamylamine interaction, suggesting that hydralazine potentiated the bradycardic effect of mecamylamine.  Results  C,)  C,)  CD  Mecamylamine 13 Saline 1 .3 Hydralazine Saline Hydralazine Saline Pooled Hydralazine Pooled  2.3  383 414 398 376 391 387  ± ± ± ± ± ±  ± ± ± ± ± ± 12 12 19 12 11 11  10 8 6 17 5 9 107 106 104 104 105 105  109 104 102 107 103 105 ± ± ± ± ± ±  ± ± ± ± ± ± 3 2 5 1 3 1  2 2 2 3 2 1  101 ± 1 106 ± 2 102 ± 2 102 ± 2 101±1 104±1  404 ± 20 414 ± 12 401 ± 6 425 ± 18 403±11 419±10 410 380 401 413 406 394  (mmHg)  (beats/mTh)  ± ± ± ± ± ±  0.2 0.2 0.1 0.2 0.1 0.1 5.2 ± 0.1 5.5 ± 0.2 5.7±0.3 5.3 ± 0.2 5.4 ± 0.2 5.4 ± 0.1  5.8 5.3 5.3 5.2 5.6 5.3  5.4 ± 0.1 5.5 ± 0.2 5.3 ± 0.1 5.6 ± 0.2 5.4±0.1 5.6±0.1  (mmHg)  Pretreatment Controls HR MAP MCFP  371 477 394 451 382 464  420 420 395 434 407 424  ±9 ±8 ± 23 ± 11 ± 12 ±8a  ± 12 ±8 ±7 ± 12 ±8 ±7  396 ± 18 477 ± 8 395 ± 8 467 ± 16 396±10 472±9a  (beats/mm)  107 78 103 76 105 77  ± ± ± ± ± ±  4 2 6 3 3 2a  100 ± 2 78 ± 2 101 ±2 78 ± 1 101±1 78±1 102 ± 3 70 ± 2 101 ±2 79 ± 1 101 ±2 74 ± i a  (mmHg)  5.1 6.7 5.6 6.1 5.4 6.4  5.2 6.5 5.2 6.0 5.2 6.2  0.2 6 0.3 9 0.1 6 0.2 6 0.1 12 a 15 0.2 ±0.1 6 ± 0.2 7 6 ± 0.4 ±0.2 7 ± 0.2 12 ± 0.2 a 14  ± ± ± ± ± ±  n 5.2 ± 0.1 7 6.7 ± 0.2 7 5.2 ± 0.1 6 7.0 ± 0.1 7 5.2±0.1 13 6.8±0.la 14 (mmHg)  Post-treatment Controls HR MCFP MAP  Values are mean ± s.e.mean. DDW = double distilled water, EtCH = ethanol. Figures referenced show the doseresponse curves as a percentage of the Post-treatment Control for each group. Treatments followed by superscript numbers are the respective vehicle-treated time controls and are also referenced to the figures showing the time-control curves. a significant difference from saline control, p < 0.05  DDW (30% EtCH)l. 2 .2 1 Nifedipine DDW (30% EtCH) Nifedipine DDW (30% EtCH) Pooled Nifedipine Pooled  Phentolamine  2.2  Figure Treatment Group Ref 2.1 Phentolamine 11 Saline 1 .1 Hydralazine Saline Hydralazine Saline Pooled Hydralazine Pooled  before (Pretreatment Control) and following treatment (Post-treatment Control) with either vehicle, hydralazine (0.3 iimol ) in conscious, unrestrained Sprague-Dawley rats. 1 1 minl) or nifedipine (0.3 imol kg-i min kg-  Table 2. Summary of heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) both  0)  65 120  -  _110 0 C 0 C) ‘4-  0  90 80 70 60 120  0  110 100 90  0  80 70 60  -  -  120 0 .4-,  C  o 100 C.)  ‘4-  0  90  0  80  IL  0  :4-  70 60  -5 -7 -6 -8 Phentolamine (mol/min/kg) or Vehicle Time-Control, Log  Figure 2.1. Dose-response curves of the effects of phentolamine (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats continuously infused ) (triangles); each point rep 1 1 min with either saline (circles) or hydralazine (0.3 tmol kg resents mean ± s.e.mean, n  6.  Results  66 120 .110 0  100  90 —  80 70 60  -  120 0  100 90 80 70 60 120 h10 o 100 C.)  c9 U  C.)  70 60  -5 -7 -6 -8 Vehicle Time-Control, Log Phentolamine (mol/min/kg) or  Figure 2.2. Dose-response curves of the effects of phentolamine (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats continuously infused with either vehicle (30% ethanol in double-distilled water) (circles) or nifedipine (0.3 imol 1 min kg ) (triangles); each point represents mean ± s.e.mean, n 1  6.  Results  67 120 0 j::  100  0 C)  80 I  70 60 50 120  __..110 0  • 100 0  90 0  80 70. 60 50 120 :=110 0  •E 100 90 80 IL  0  70 60 50  -4 -3 -5 -6 -7 Time-Control, Log Mecamylamine (mol/kg) or Vehicle  Figure 2.3. Dose-response curves of the effects of mecamylamine (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats continuously infused ) (triangles); each point rep 1 1 min with either saline (circles) or hydralazine (0.3 tmol kg resents mean ± s.e.mean, n  6.  Results  68  200  200  0  -I-,  C  o °150  1503  100  100  C  (0 Cl) ci)  -  ci) o  ci) o  -  D  50  50  >< H  -  .  I-  0 1E-7  1E-6  1E-5 Mecamylamine (mollkg), Log  0 1E-3  Figure 2.4.  Dose-response curves of the effects of mecamylamine on changes in heart rate (HR) (A) and mean arterial pressure (MAP) (•) produced by a test dose of methoxamine (80 nmol kg’) in conscious, unrestrained rats; each point represents mean ± s.e.mean, n  =  6.  Results  69 Possible Role of Purinergic Neurotransmission in Basal and Reflexly-lncreased  3.3.  Venous Tone Effect of suramin in the absence and presence of hydralazine (Figure 3.1)  3.3.1.  The control values for haemodynamic variables are given in Table 3. Saline did not alter MAP, HR, or MCFP whereas hydralazine treatment decreased MAP (28 ± 2%) and increased both HR (18 ± 2%) and MCFP (25 ± 2%) compared to post-treatment control values of saline-treated groups (n  =  16). Neither MAP, HR, nor MCFP changed signifi  cantly over time during continuous hydralazine infusion. In both saline- and hydralazine-treated groups, suramin significantly and dose-de pendently decreased HR, but had no significant effect on MCFP. Suramin produced dosedependent pressor effects in the absence and presence of hydralazine which, according to curve analysis, were significantly different from the corresponding saline control curves. The MAP curves for suramin in the absence and presence of hydralazine were also sig nificantly different from each other which suggests that hydralazine attenuated the suramin induced pressor effect at low doses of suramin. Effect of suramin in the absence and presence of nifedipine (Figure 3.2)  3.3.2.  The control values for haemodynamic variables are given in Table 3. Vehicle treat ment (30% ethanol in double-distilled water) did not significantly alter MAP, HR, or MCFP whereas nifedipine treatment produced a significant decrease in MAP (33 ± 2%) and in creased both HR (6 ± 2%), and MCFP (25 ± 3%) (n  =  16). During continuous nifedipine  infusion, there were only a slight, gradual but insignificant increase in both MAP and MCFP, and a slight, gradual but insignificant decrease in HR over time. In the vehicle-treated group, suramin produced a significant dose-dependent increase in MAP which was accompanied by a dose-dependent, but insignificant, decrease in HR and no change in MCFP. Nifedipine did not significantly alter the suramin-induced pressor effect.  Similarly, the suramin-induced bradycardia also persisted in the presence of  nifedipine but was significantly greater than that occurring in the absence of nifedipine  —  Results  70 this did not appear to be the result of potentiation, since no significant nifedipine-suramin interaction occurred, but rather the result of the time-dependent slight bradycardic effect of nifedipine. According to curve analysis, nifedipine treatment revealed a slight but sig nificant increase in MCFP when compared to either the nifedipine-treated control or the vehicle-treated suramin group. 3.3.3.  Effect of suramin in the absence and presence of mecamylamine (Figure 3.3)  The control values for haemodynamic variables are given in Table 3. Saline did not significantly affect MAP, HR, or MCFP whereas mecamylamine treatment significantly decreased both MAP (31 ± 3%) and MCFP (30 ± 2%), and insignificantly decreased HR (6 ± 3%), compared to post-treatment control values from saline-treated groups (n = 12). The dose of mecamylamine used in these studies inhibited acetylcholine-induced tachy cardia by 79 ±9% (n = 12). In the presence of mecamylamine, HR did not change signifi cantly over time; however, MAP and MCFP each exhibited a marked and significant in crease, immediately following the balloon inflation for the post-treatment control response, which was maintained for the duration of the experiment. In the absence of mecamylamine, suramin significantly and dose-dependently de creased HR and increased MAP, but had no effect on MCFP. Mecamylamine treatment did not significantly alter the suramin-induced bradycardia observed in the absence of mecamylamine, whereas it revealed a significant dose-dependent decrease in MCFP. The suramin-induced pressor effect persisted in the presence of mecamylamine but was insig nificant compared to the respective mecamylamine-treated control group, although it was significantly greater than the vehicle control in the absence of mecamylamine.  Results  CD  U)  U)  Saline .4 1 1 .4 Mecamylamine Saline Mecamylamine Saline Pooled Mecamylamine Pooled  Suramin  3.3 408 ± 11 418± 18 396±11 393±23 396±11 405±15  101 ± 2 103±3 111±3 102±5 111±3 103±3  109±2 104±2 111±4 104± 1 110±2 104± 1  5.3 ± 0.2 5.2±0.2 5.3±0.1 5.5±0.2 5.2±0.1 5.4±0.1  5.8 ±0.2 5.3±0.2 5.8±0.2 5.4±0.2 5.8±0.1 5.4±0.2  5.3±0.2 5.4±0.2 5.3±0.1 5.1±0.1 5.2±0.1 5.3±0.1  101±2 104±2 111±3 102±1 108±2 104±1  408±11 421 ±14 396±11 416±7 400±8 415±6 410 ± 10 380±8 409±18 378±8 410±10 381±5  (mmHg)  (mmHg)  (beats/mTh)  418 ± 15 393± 15 390±10 357± 13 399±9 375±11  420 ± 12 420±8 410±14 460± 12 414±9 440±8a  418±15 475± 17 390±10 462±9 399±9 469±6a  (beats/rn/n)  102 ± 2 76±4 111±2 75±6 108±2 75±4a  102±3 70±2 105±5 70±2 104±3 70±1 a  102 ±2 77±3 111±2 77±2 108±2 7-f-a  (mmHg)  5.1 ± 0.3 6 3.6±0.1 6 5.3±0.1 13 3.9±0.2 6 5.3±0.1 19 3.7±0.la 12  5.2±0.2 6 6.5±0.3 6 5.4±0.2 7 6.9±0.3 7 5.3±0.1 13 6.6±0.2a 13  n 5.1 ±0.3 6 6.5±0.2 6 5.3±0.1 13 6.5±0.2 9 5.3±0.1 19 6.6±0.la 16 (mmHg)  Post-treatment Controls MCFP HR MAP  Values are mean ± s.e.mean. DDW = double distilled water, EtOH = ethanol. Figures referenced show the dose-response curves as a percentage of the Post-treatment Control tor each group. Treatments followed by superscript numbers are the respective vehicle-treated time controls and are also referenced to the figures showing the time-control curves. a significant difference from saline control, p < 0.05  DDW (30% EtCH)l. 2 1 .2 Nitedipine DDW (30% EtOH) Nifedipine DDW (30% EtCH) Pooled Nifedipine Pooled  Suramin  3.2  Figure Treatment Group Ref. 3.1 Suramin 1 .4 Saline 1 .4 Hydralazine Saline Hydralazine Saline Pooled Hydralazine Pooled  Pretreatment Controls HR MAP MCFP  before (Pretreatment Control) and following treatment (Post-treatment Control) with either vehicle, hydralazine (0.3 pmol kg-i mimi), nifedipine (0.3 jimol kg-i mini) or mecamylamine (18 jimol kg-i) in conscious, unrestrained Sprague-Dawley rats.  Table 3. Summary of heart rate (HR), mean arterial pressure (MAP) and mean circulatory tilling pressure (MCFP) both  72 140  -  120 100  140  .  120  -  ioo <  80 60  -  -  140 0  120  0 0  100  -  Suramin (mol/kg) or Vehicle Time-Control, Log  Figure 3.1. Dose-response curves of the effects of suramin (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats continuously infused with either ) (triangles); each point represents mean 1 1 min saline (circles) or hydralazine (0.3 tmol kg ± s.e.mean, n  6.  Results  73 140 0 0  120  ci  100 80 60 140 0 0 0  120 100  0  <  80 60 140  0  ::  Suramin (mol/kg) or Vehicle Time-Control, Log  Figure 3.2. Dose-response curves of the effects of suramin (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats continuously infused with either ) 1 1 min vehicle (30% ethanol in double-distilled water) (circles) or nifedipine (0.3 jimol kg (triangles); each point represents mean ± s.e.mean, n  6.  Results  74 140 120 100 80 60 140 0 C 0 C)  •c5 100 0  <  80 60 140  0  120  ..;  Suramin (mol/kg) or Vehicle Time-Control, Log  Figure 3.3. Dose-response curves of the effects of suramin (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats treated with either saline (circles) or ) (triangles); each point represents mean ± s.e.mean, n 6. 1 mecamylamine (18 iimol kg  Results  75  Does Suramin Reveal an cc-Adrenoceptor Antagonist-Sensitive Component of  3.4.  Venous Tone? 3.4.1.  Effect of phentolamine in the absence and presence of suramin (Figure 4.1)  The control values for haemodynamic variables are given in Table 4. While saline did not alter MAP, HR, or MCFP, suramin treatment significantly decreased HR (8 ± 2%) and increased both MAP (15 ± 2%) and MCFP (12 ± 2%) (n  =  12). Overtime, there was  no change in MAP, HR, or MCFP, in either the saline- or suramin-treated group although MCFP slightly decreased in the suramin-treated group. A preliminary experiment demon strated that suramin treatment virtually abolished the pressor response to i.v. injection of oL,3-methylene-ATP (P x-purinoceptor agonist) for at least 3 hours (data not shown). 2 In the absence of suramin, phentolamine significantly and dose-dependently de creased MAP and increased HR, but had no effect on MCFP. Suramin treatment did not significantly affect either the phentolamine-induced tachycardia or MCFP.  The  phentolamine-induced depressor effect, however, was significantly greater in the pres ence than in the absence of suramin.  Results  Cl)  U)  CD  (mmHg)  101±1 105±3 102±2 104±1 101±1 104±1  (beats/mTh)  404±20 412±10 401±6 418±18 403±11 415±10 5.4 5.6 5.3 5.3 5.4 5.5  ± ± ± ± ± ±  0.1 0.1 0.1 0.3 0.1 0.1  (mmHg)  396 355 395 376 396 365  ± ± ± ± ± ±  18 11 18 4 10 6a  (beats/mm)  100 114 101 117 101 116  ±2 ±2 ±2 ±1 ±1 ±la  (mmHg)  5.2 5.9 5.2 5.7 5.2 5.8  ± ± ± ± ± ±  o.i  0.1 0.2 0.1 0.2 0.1  (mmHg)  Post-treatment Controls MCFP HR MAP  n 7 6 6 6 13 a 12  Values are mean ± s.e.mean. Figures referenced show the dose-response curves as a percentage of the Posttreatment Control for each group. Treatments followed by superscript numbers are the respective vehicle-treated time controls and are also referenced to the figures showing the time-control curves. a significant difference from saline control, p <0.05  Figure Treatment Group Ref. 4.1 Phentolamine 1 .1 Saline 1 .1 S u ram in Saline S u ram in Saline Pooled Suramin Pooled  Pretreatment Controls HR MAP MCFP  both before (Pretreatment Control) and following treatment (Post-treatment Control) with either saline or suramin (200 imol kg-i) in conscious, unrestrained Sprague-Dawley rats.  Table 4. Summary of heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP)  0)  77 120 ___.110  -  -  0 0 C)  90—  80 70 60  -  -  -  120 D110 0  10 0 C-)  90 80 70 60 120 110 o 100 C-)  -  -  -  90ci. LL  C.)  80 70 60  -  -  -  -5 -6 -7 -8 Phentolamine (mol/min/kg) or Vehicle Time-Control, Log  Figure 4.1. Dose-response curves of the effects of phentolamine (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats treated with either sa ) (triangles); each point represents mean ±s.e.mean, 1 line (circles) orsuramin (200 imol kg n6.  Results  78  3.5.  Does Sympathetic Cotransmission Contribute to Venous Tone?  3.5.1.  Effect of suramin in the absence and presence of reserpine (Figure 5.1)  The control values for haemodynamic variables are given in Table 5. Reserpine treatment had no effect on MCFP, but significantly decreased both MAP and HR by 21 ± 2% and 11 ± 3%, respectively, of the post-treatment control values for the vehicle (6% citrate in double-distilled water)-treated groups (n  =  12). MAP, HR, and MCFP did not  change significantly over time in the reserpine-treated control group. In the vehicle-treated group, suramin significantly and dose-dependently decreased HR and increased MAP, but had no effect on MCFP. Neither the bradycardia nor the pressor effect of suramin was significantly affected by reserpine. In contrast, reserpine revealed a modest suramin-induced decrease in MCFP which was significantly different from the slight increase that was produced by suramin in the absence of reserpine. 3.5.2.  Effect of suramin in the absence and presence of guanethidine (Figure 5.2)  The control values for haemodynamic variables are given in Table 5. Saline did not alter MAP, HR, or MCFP. Guanethidine treatment significantly decreased the post-treat ment control value for MAP (22 ± 2%) compared to the saline-treated groups, but had no significant effect on either MCFP or HR (n = 13). During continuous guanethidine infusion, MAP, HR, and MCFP were stable over time, none of the measurements differing signifi cantly from the saline-treated control group. In the absence of guanethidine, suramin dose-dependently and significantly decreased HR and increased MAP, while MCFP was unchanged by suramin. In the presence of guanethidine, neither the suramin-induced bradycardia nor MCFP differed significantly from the saline-treated suramin group. In contrast, the suramin-induced pressor effect was significantly greater in the presence than in the absence of guanethidine. Further more, it appeared that this increased pressor response was the result of a guanethidine suramin interaction rather than of any time-dependent effect of guanethidine on MAP. Results  C’)  CD C’)  Suramin  5.2  SaIine 14 14 Guanethidine Saline Guanethidine Saline Pooled Guanethidine Pooled  DDW(6% Citrate)l. 5 15 Reserpine DDW(6%Citrate) Reserpine DDW(6% Citrate) Pooled Reserpine Pooled 408±11 425±17 396± 11 401 ± 7 400±8 410 ± 6  (beats/mm)  101±2 104±3 111±3 111 ±2 108±2 108 ± 2  (mmHg)  5.3±0.2 5.6±0.2 5.3±0.1 5.4 ± 0.1 5.2±0.1 5.4 ± 0.1  —  —  —  —  —  —  (mmHg)  Pretreatment Controls MAP MCFP HR  403±15 357 ± 17 381 ±14 342 ± 20 392±10 349 ± 13 a 418±15 393±10 390± 10 41 1 ± 7 399±9 404 ± 6  (beats/mm)  103±3 83 ± 3 111±3 87 ± 2 108 ±2 85 ± 2 a 102 ±2 79±2 111±2 87 ± 3 108±2 84 ± 2 a  (mmHg)  5.1 ±0.3 5.4±1.1 5.3±0.1 5.4 ± 0.2 5.3±0.1 5.4 ± 0.1  5.2±0.1 5.4 ± 0.2 5.4±0.1 5.2 ± 0.1 5.3±0.1 5.3 ± 0.1  (mmHg)  Post-treatment Controls MCFP HR MAP  6 5 13 8 19 13  6 6 7 6 13 12  n  Values are mean ± s.e.mean. DDW = double distilled water. Figures referenced show the dose-response curves as a percentage of the Post-treatment Control for each group. Treatments followed by superscript numbers are the respective vehicle-treated time controls and are also referenced to the figures showing the time-control curves. a significant difference from saline control, p < 0.05  Suramin  Treatment Group  5.1  Ref.  Figure  both before (Pretreatment Control) and following treatment (Post-treatment Control) with either vehicle, reserpine (3 mg 1 mm1 for 10 mm followed by 0.5 imol 1 1 24 h prior to study), or guanethidine (3 pmoI kgkgkg- minl) in conscious, unrestrained Sprague-Dawley rats.  Table 5. Summary of heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP)  CD  80 130 0 0 0  110  90• 70 I 50 130 0 0 C)  110  90. a<70 50 130 0  -  Suramin (mol/kg) or Vehicle Time-Control, Log  Figure 5.1. Dose-response curves of the effects of suramin (black symbols) or vehicle (shaded symbols) on hearl rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats treated with either vehicle (6% 1 24 h prior to study) (trian citrate in double-distilled water) (circles) or reserpine (3 mg kg gles); each point represents mean ± s.e.mean, n 6.  Results  81 160 —  0  140 120 100  a: 180 60 160  I  0 0 0 0  100 0  80 60 160 1140 0 0  120  0  —  0  a  80 60  -5  -4 Suramin (mol/kg) or Vehicle Time-Control, Log  -3  Figure 5.2. Dose-response curves of the effects of suramin (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats treated with either saline (circles) ) (triangles); 1 1 min 1 min 1 for 10 mm followed by 0.5 imol kg or guanethidine (3 imol kg each point represents mean ± s.e.mean, n  5.  Results  82  3.6.  Characterization of the Cardiovascular Effects of Exogenous ATP  3.6.1.  Effect of ATP in the absence and presence of mecamylamine (Figure 6.1)  The control values for haemodynamic variables are given in Table 6. While saline had no effect on MAP, HR, or MCFP in any of the groups, mecamylamine treatment signifi cantly decreased MAP (25 ± 2%), HR (9 ± 2%), and MCFP (30 ± 3%) compared to posttreatment control values from saline-treated groups (n = 12). The dose of mecamylamine used in these studies inhibited acetylcholine-induced tachycardia by 86 ± 11 % (n  =  12). In  the presence of mecamylamine, neither HR nor MCFP changed significantly with time; however, MAP gradually and significantly increased with time. In the absence of mecamylamine, infusion of ATP significantly and dose-depend ently decreased both HR and MAP. Curve analysis indicated that MCFP was significantly lower in the ATP than the saline control group and that this downward trend was slightly, but significantly, more pronounced in the presence than in the absence of mecamylamine. The ATP-induced bradycardia was not significantly affected by mecamylamine; however, a significant mecamylamine-ATP interaction suggested that the ATP-induced depressor effect was potentiated by mecamylamine. 3.6.2.  Effect of ATP in the absence and presence of suramin (Figure 6.2)  The control values for haemodynamic variables are given in Table 6. Suramin treat ment produced an increase in MAP (13 ± 2%) and decreases in both MCFP (13 ± 3%) and HR (10 ± 3%), all of which were significantly different from post-treatment control values in saline-treated groups (n = 12). Overtime, HR, MAP, or MCFP did not change in either the saline- or suramin-treated control groups. Effect of ATP in the absence of treatment (suramin)—as in section 3.5.1. Neither the ATP-induced depressor effect, bradycardia, nor decline in MCFP was significantly affected by suramin. With respect to the bradycardia, however, curve analysis indicated a signifi cant difference between the ATP dose-response curves in the absence and presence of Results  83 suramin, suggesting that the ATP-induced decrease in HR was slightly potentiated by suramin. 3.6.3.  Effect of ATP in the absence and presence of cibacron blue (Figure 6.3)  The control values for haemodynamic variables are given in Table 6. Cibacron blue treatment had no significant effect on post-treatment control values for haemodynamic variables compared to saline-treated groups (n  =  13), nor did it have a significant effect  over time. Effect of ATP in the absence of treatment (cibacron blue)  —  as in section 3.5.1.  Cibacron blue did not alter the dose-dependent bradycardic effect of ATP. In contrast, the ATP-induced depressor effect was moderately attenuated by cibacron blue as suggested by a significant cibacron blue-ATP interaction and a significant difference in between ATP dose-response curves constructed in the absence and presence of cibacron blue. Cibacron blue also revealed a slight but significant ATP-induced increase in MCFP according to curve analysis. 3.6.4.  Effect of ATP in the absence and presence of 8-phenyltheophylline (Figure 6.4)  The control values for haemodynamic variables are given in Table 6. Treatment with 8-phenyltheophylline failed to exert any significant effect on control values for MAP, HR, or MCFP compared to the saline-treated groups (n = 14), nor did it have any significant effect over time. Effect of ATP in the absence of treatment (8-phenyitheophylline)  —  as in section 3.6.1.  In the presence of 8-phenyltheophylline, the ATP-induced depressor effect was clearly and significantly attenuated, as confirmed by a significant 8-phenyltheophylline-ATP inter action and a significant difference in profile between the ATP dose-response curves in the absence and presence of 8-phenyltheophylline. Similarly, 8-phenyltheophylline abolished the ATP-induced bradycardia and, according to curve analysis, revealed a slight increase in HR. The ATP-induced decline in MCFP persisted in the presence 8-phenyltheophylline and was slightly but significantly more pronounced acccording to curve analysis. Results  C’)  i  5.5 ± 0.1 5.0 ± 0.2 5.2 ± 0.1  8-Phenyltheophylline 396 ± 19 102 ± 2 6 • 1 8-Phenyltheophylline 392 ± 11 106 ± 2 8-Phenyltheophylline Pooled 392 ± 12 104 ± 2  391 ± 14 404 ± 16 397 ± 10  383 ± 15 360 ± 13 373±10  351 ± 13 367± 14 359 ± io a  362±6 362 ± 6 362±0.4a  377±16 420± 10 398±11  ‘beats/min)  105 ± 3 107 ± 3 106 ± 2  103 ± 2 102 ± 3 102 ±2  112±5 119±3 116 ± 3 a  72±2 80 ± 2 77±2a  102±2 104±3 103±2  6 6 12  n  5.5 ± 0.2 5.3 ± 0.2 5.4 ± 0.1  5.7 ± 0.2 5.2 ± 0.2 5.4±0.2  7 7 14  7 6 13  4.6±0.2 6 4.6±0.3 6 4.6 ± 0.2 a 12  4.4±0.3 5 3.7 ± 0.2 7 4.0±0.2a 12  5.1 ±0.2 5.5±0.2 5.3±0.1  ‘mmHg,)  Post-treatment Controls HR MAP (mmHg) MCFP  Values are mean ± s.e.mean. Figures referenced show the dose-response curves as a percentage of the Post treatment Control for each group. Treatments followed by superscript numbers are the respective vehicle-treated time controls and are also referenced to the figures showing the time-control curves. Treatment groups referenced 6.1-6.4 apply to all other ATP-treated groups. a significant difference from saline control, p < 0.05  ATP  6.4  5.6 ± 0.1 5.2 ± 0.1 5.4±0.1  100 ± 2 102 ± 2 100 ±2  375 ± 8 393 ± 23 389±10  Cibacron Blue 16 Cibacron Blue Cibacron Blue Pooled  ATP  6.3  5.3±0.1 5.4±0.3 5.3 ± 0.2  103±3 105±3 104 ± 2  432±17 422± 11 427 ± 10  Suramin 6 1 Suramin Suramin Pooled  ATP  6.2  5.7±0.3 5.4 ± 0.3 5.5±0.2  Mecamylamine 16 Mecamylamine Mecamylamine Pooled  ATP  106±3 107 ± 2 107±2  mmHg,)  406±12 408 ± 10 407±7  (‘mmHg,)  5.3±0.1 5.7±0.2 5.5±0.1  383±18 426± 10 404±12  (beats/mm)  Pretreatment Controls MAP HR MCFP 98±2 107±3 102±2  Saline 16 Saline Saline Pooled  Treatment Group  6.1  6.1-6.4ATP  Ref  Figure  conscious, unrestrained Sprague-Dawley rats.  both before (Pretreatment Control) and following treatment (Post-treatment Control) with either saline, mecamylamine ), suramin (200 jimol kgi), cibacron blue (13 pmol kg-i) or 8-phenyltheophylline (27 imol kg-i) in 1 (18 iimol kg1 min  Table 6. Summary of heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP)  85 130 D110 0  90 70 I  50 30 130  0 4-.  0 C-) 0  70 U  50 30 130 110 90. 0  0  0  50 30  -7  -5 -6 ATP (mol/kg/min) or Vehicle Time-Control, Log  Figure 6.1. Dose-response curves of the effects of ATP (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats continuously infused with either ) (triangles); each point represents 1 1 min saline (circles) or mecamylamine (18 imol kg mean ± s.e.mean, n  5.  Results  86 110 0 -I  90  0  70  ci:  50  C 0 Ci  -  -  30 110 0 -I-,  C 0 0  90  0  70  0  50 30  110 0 C  0 Ci 0  70. 0  U  C-)  30  -  -7  -5 -6 ATP (mol/kg/min) or Vehicle Time-Control, Log  Figure 6.2. Dose-response curves of the effects of ATP (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats treated with either saline (circles) or suramin (200 jimol kg ) (triangles); each point represents mean ± s.e.mean, n 6. 1  Results  87 110 0  90 0 0  70 50 30 110 0  90 0 0  70 <  50 30 110  -  0 C 0 0  7 1E 4-  0  ATP (mol/kg/min) or Vehicle Time-Control, Log  Figure 6.3. Dose-response curves of the effects of ATP (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats treated with either saline (circles) or cibacron blue (13 jimol kg ) (triangles); each point represents mean ± s.e.mean, n 6. 1  Results  88 110 0  p90. 0 C)  70 50 30 110 0  90 0 C)  70 a <  50 30 110  ATP (mol/kg/min) or Vehicle Time-Control, Log  Figure 6.4. Dose-response curves of the effects of ATP (black symbols) or vehicle (shaded symbols) on heart rate (HR), mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP) in conscious, unrestrained rats treated with either saline (circles) ) (triangles); each point represents mean ±s.e.mean, 1 or 8-phenyltheophylline (27 pmol kg n6.  Results  89  DISCUSSION  4.  It has been demonstrated that ATP plays an important role as a cotransmitter of NA in numerous sympathetically innervated blood vessels and vascular beds (see Burnstock 1990d; Westfall  eta!.  1990; Olsson & Pearson 1990). In comparison, very few studies  have demonstrated such a role for ATP in whole animals (Flavahan McGrath 1988a,b; Schlicker  eta!.  1989; Daziel  eta!.  et  a!. 1985; Bulloch &  1990; Tarasova & Rodionov 1992)  and, apparently, in none of these have conscious animals been used. In an attempt to  correlate observations made  in vitro  with those made  in vivo,  this study assessed the role  of ATP in the control of MAP, HR, and MCFP in conscious, unrestrained rats by examining -purinoceptors, and 2 - and P 1 the effects of receptor antagonists (of o-adrenoceptors, P autonomic ganglia), chemical sympathectomy (by reserpine or guanethidine), and ATP per Se.  Furthermore, we compared the contribution of endogenous ATP and NA in main  taining basal vascular tone with that during drug-induced vasodilatation and concomittant elevation of sympathetic nerve activity. The role of ATP in the venous system, estimated by the effects of pharmacological manipulations on MCFP, was of particular interest in this study. MCFP was defined by Guyton as “the pressure that would be measured at all points in the entire circulatory system if the heart were stopped suddenly and the blood were redistributed instantane ously in such a manner that all pressures were equal” (Guyton  et  a!. 1973). It has been  demonstrated that MCFP is a measure of total body vascular capacitance and is depend ent on blood volume, unstressed volumes, and arterial and venous compliances (reviewed in Tabrizchi & Pang 1992; Rothe 1993). However, since venous compliance is much greaterthan arterial compliance (Guyton  eta!.  1973; Yamamoto  eta!.  1980), MCFP largely  reflects overall venous smooth muscle tone. It has been demonstrated that MCFP is a measure of the ratio of blood volume to the overall compliance of the circulatory system; therefore, in order for MCFP to provide an accurate measure of venous tone, blood vol Discussion  90 ume must remain constant. The method of measuring MCFP used in this study was introduced by Yamamoto et al. (1980) and is especially advantageous since it does not require extensive surgery and an accompanying prolonged recovery period. More impor tantly, since experiments can be conducted in conscious, unanaesthetized rats with intact cardiovascular reflex mechanisms, more physiologically relevant conclusions can be drawn. Indeed, it has been demonstrated that MCFP measured in pentobarbitone-anaesthetized rats may not accurately reflect total body venous tone since, under these conditions, central venous pressure failed to equilibrate with portal venous pressure during circulatory arrest (Tabrizchi et al. 1993).  4.1.  Resistance of MCFP to ct-A drenoceptor Antagonism in Rats with Normal and Reflexly-lncreased Venous Tone This set of experiments was undertaken to determine the role of ct-adrenoceptors  and the autonomic nervous system in the maintenance of vascular tone (see Results 3.2). Blockade of ct-adrenoceptors by phentolamine produced vasodilatation in both normal and vasodilator (hydralazine or nifedipine)-treated rats. These results indicate that basal arterial tone, and that remaining following administration of vasodilator drugs, is main tained via ct-adrenoceptor activation, presumably by NA released from perivascular sym pathetic nerves. In addition, it was observed that hydralazine, but not nifedipine, potentiated the vasodilatatory effect at low phentolamine doses, suggesting that hydralazine may in crease the contribution of ct-adrenoceptors to the maintenance of MAP. The significance of this finding is unclear. In rats treated with vehicle, phentolamine produced tachycardia whereas in both hydralazine- and nifedipine-treated rats, phentolamine caused modest but insignificant bradycardia. Reflex activation of the sympathetic nervous system is presumably respon sible not only for the phentolamine-induced tachycardia, but also for the tachycardia ob served following treatment with either hydralazine or nifedipine. Indeed, D’Oyley et a!. Discussion  91 (1989) demonstrated attenuation of hydralazine-induced tachycardia following ganglion blockade with hexamethonium in the conscious rat, while nifedipine has been found to induce reflex tachycardia in anaesthetized and conscious rats (Nordlander 1985; Waite et al. 1988). It is possible, though inconclusive according to the present results, that the moderate phentolamine-induced bradycardia observed in the presence of both hydralazine and nifedipine was the result of blockade of cardiac oc-adrenoceptors. Under basal conditions phentolamine did not alter MCFP, while hydralazine treat ment revealed only a negligible, insignificant decline in MCFP in response to blockade of c-adrenoceptors. In contrast, nifedipine revealed a significant phentolamine-induced de crease in MCFP. Previous studies have demonstrated that both hydralazine (D’Oyley et al. 1989) and nifedipine (Waite eta!. 1988) have minor direct venodilatatory effects but are capable of very effectively increasing venous tone indirectly via activation of autonomic reflexes. The present results suggest that neither basal nor reflexly-increased venous tone is appreciably maintained by the activation of c-adrenoceptors. This is in accord with previous findings that MCFP is largely resistant to prazosin and rauwolscine (c1- and c*2adrenoceptor-selective antagonists, respectively); however, the same study found that in the presence of a reflexly-induced increase in MCFP, both OL-adrenoceptor antagonists significantly decreased MCFP (D’Oyley and Pang 1989) which does not agree with the present findings.  Similarly, Ito and Hirakawa (1984) were able to demonstrate a  phentolamine-induced decrease in MCFP in pentobarbitone-anaesthetized open chest dogs. In this same preparation, however, prasozin did not affect MCFP unless venous tone had been elevated by infusion of NA (Ito & Hirakawa 1984).  In another study,  phenoxybenzamine (non-selective a-adrenoceptor antagonist) failed to affect MCFP in pentobarbitone-anaesthetized dogs either in the absence or presence of infused adrena line (Hirakawa eta!. 1984). Clearly, there are considerable discrepancies between the effects of o-adrenoceptor antagonism on MCFP and several studies have failed to show that x-adrenoceptor blockade lowers MCFP. Discussion  92 Although this study showed that the phentolamine-induced decline in MCFP in the presence of hydralazine was negligible, that observed in the presence of nifedipine was small but significant. The electrophysiological mechanism of ATP-induced vasoconstric tion is well-characterized and can be described as “electromechanical coupling” since ATP acts by opening the intrinsic ion channel of the P2x-purinoceptor which subsequently 2 channels, ultimately leading to activation of the initiates the EJP and voltage-gated Ca +-dependent contractile machinery of vascular smooth muscle (see Introduction 1.2.3 2 Ca 2 completely abolished ATP and 1.5.3.2). The finding that removal of extracellular Ca mediated contractions in dog saphenous vein (Salag et al. 1990) supports the proposal that ATP produces venoconstriction via electromechanical coupling. Moreover, nifedipine has been shown to selectively block purinergic rather than adrenergic nerve-mediated vasopressor responses in pithed rats (Bulloch & McGrath 1988b). In comparison, the mechanism of NA-induced contraction can be described as “pharmacomechanical cou pling” since contraction is evoked either without membrane potential changes or accom panied by a slow depolarization. In the case of NA-induced contraction, the source of 2 contractile Ca 2 may be intracellular stores or nifedipine-resistant receptor-operated Ca channels. Therefore, the results of the present experiments may suggest that prevention 2 channels removes ATP-mediated reflex of Ca 2 influx through nifedipine-sensitive Ca venomotor tone thereby unmasking the venodilator action of phentolamine. In the pres ence of hydralazine, ATP-mediated venoconstriction may obscure phentolamine-induced venodilatation. The failure of phentolamine to appreciably reduce MCFP, either under basal condi tions or in the presence of hydralazine, was not due to an absence of sufficient basal tone since mecamylamine-induced ganglion blockade markedly and dose-dependently reduced MCFP in both the absence and presence of hydralazine. This is in keeping with the previous observation that hexamethonium-induced ganglion blockade produces a similar dose-dependent depression of MCFP (D’Oyley & Pang 1990). In light of these observa Discussion  93 tions, it appears that the weak venodilatatory activity of phentolamine in the presence of raised venomotor tone is indicative of a small role for c-adrenoceptors in the maintenance of neurogenic venous tone. Mecamylamine also produced marked decreases in MAP and HR in the absence of hydralazine. However, while the HR response to mecamylamine was unaltered by hydralazine, the mecamylamine-induced reduction in MAP was virtually abolished.  This latter effect was unexpected since hydralazine failed to prevent a  phentolamine-induced depressor effect. It is possible to speculate that the mecamylamine induced decrease in venomotor tone (MCFP) and therefore venous return stimulated an increase in vasopressin release which served to obscure the mecamylamine-mediated depressor effect since it is well-known that decreased stretch of the atria promotes the release of vasoactive peptides such as angiotensin II and vasopressin (Share 1988; Keeton & Campbell 1981) and, moreover, that vasopressin exerts negligible effects on MCFP while producing potent arterial vasoconstriction (Pang & Tabrizchi 1986).  4.2.  Possible Role of Purinergic Neurotransmission in Basal and Reflexly-Increased Venous Tone In order to determine whether purinergic tone was responsible for phentolamine-,  but not mecamylamine-, resistant normal and reflexly-increased venous tone, experiments .purinoceptor antagonist (see Results 2 were performed using suramin, a non-selective P 3.3). The finding that suramin increased MAP and decreased HR similarly in hydralazine and nifedipine-treated rats indicates that the mechanisms responsible for the suramin 2 induced effects on MAP or HR involve neither nifedipine-sensitive voltage-gated Ca . Ca ) channels nor hydralazine-sensitive mechanisms (perhaps involving intracellular 2 Although the effects of suramin on MAP and HR were not systematically investigated, results from several different experiments in this study implicate direct excitatory vasomotor and cardiodepressant activities, as will become apparent later in this discussion.  Discussion  94 MCFP was unchanged by suramin in the both the absence and presence of hydralazine treatment. In the presence of nifedipine, however, suramin produced a very modest but insignificant increase in MCFP. When designing this experiment, it was ex x-purinergic tone (in the 2 pected that if suramin decreased MCFP via antagonism of P absence or presence of hydralazine), then nifedipine treatment should eliminate any x-purinoceptor-mediated vaso 2 suramin-induced decrease in MCFP by interfering with P constriction at a site beyond the receptor. This, however, was obviously not the case. It therefore appears that endogenous ATP is unimportant under basal conditions and after elevation of venomotor tone. The selectivity of suramin in vivo has previously been estab lished in the pithed rat (Schlicker et al. 1989; Urbanek et al. 1990). However, in light of reports of unselective action in vitro (Nally & Muir 1992) it is possible that in the conscious rat preparation used in this study, the effect of suramin may have involved action(s) apart -purinoceptors. 2 from antagonism of P  If suramin was, indeed, selective for  2 -purinoceptors, it is possible to speculate that any antagonism of postjunctional P 2 P purinoceptors was obscured by a prejunctional action of suramin which would have en hanced release of sympathetic transmitters (primarily of NA). A presynaptic action of suramin would explain the discrepancy between these results and those from experi ments involving phentolamine in the presence of nifedipine from which it was concluded that inhibition of a nifedipine-sensitive purinergic component unmasked phentolamine sensitive venomotor tone. Indeed, a prejunctional action of suramin was postulated for its effect in the pithed rat (Schlicker et a!. 1989).  In addition, it is possible that suramin  directly increased venomotor activity which is consistent with suramin’s direct excitatory vasomotor activity for the maintenance of MAP as proposed earlier. Ganglion blockade by mecamylamine did not alter either the suramin-induced bradycardia or pressor effect which suggests that neither requires intact autonomic gan glia. Therefore, it appears that the bradycardia is predominantly the result of a direct cardiodepressant action of suramin rather than an indirect reflex-induced withdrawal of Discussion  95 sympathetic tone to the heart. Furthermore, it can be concluded that suramin did not increase MAP by interfering with neurogenic vascular tone. This observation is in accord ance with the ability of suramin to elicit a sustained increase in MAP in the pithed rat (Schlicker eta!. 1989; Urbanek eta!. 1990). Interestingly, mecamylamine treatment revealed an appreciable depressant effect of suramin on MCFP. According to the results of the acetylcholine test for blockade, near complete ganglion blockade was obtained in these experiments (79 ± 9%). It follows, therefore, that the suramin-induced decrease in MCFP was a direct effect of suramin rather than an indirect effect involving antagonism of sympathetic or purinergic tone, or a reflex response to the suramin-induced pressor effect. Although the possibility of such indirect actions is disputable considering the high degree of ganglion blockade, it is not an untenable hypothesis. Despite a significant reduction in the acetyicholine-induced tachy cardia in the presence of ganglion blockade, found by other investigators (Waite et al. 1988; Glick et a!. 1992), it cannot be definitively stated that this was an accurate measure of the degree of ganglion blockade since there are no thorough studies examining the adequacy of this criterion. Thus, the blunting of cardiac responses to acetylcholine-in duced reflex may not accurately represent the overall degree of bockade of autonomic ganglia and, in particular, blockade of ganglia responsible for maintaining vascular tone. In light of the proposed mechanism of action of ATP-induced vasoconstriction (i.e. electro mechanical coupling), it is possible that only minimal innervation is sufficient for cotrans mitter ATP to exert a constrictor effect since the ATP signal (i.e. receptor-ligand binding) will be greatly amplified by voltage-dependent mechanisms. If this is the case, then even the very small fraction of neurally-released ATP that presumably remains in the presence of mecamylamine should be sufficient to produce venoconstriction. In addition, the pat tern of nerve activity during ganglion blockade may have been altered such that purinergic transmission was favoured over adrenergic transmission. The stimulation parameter-de pendence of the ratio of cotransmitter NA and ATP is well-characterized (see Introduction Discussion  96 1.5.3.3). Furthermore, the results from these experiments do not rule out the possibility that perivascular purinergic nerves may exist separately from sympathetic nerves and may be differentially affected by mecamylamine.  4.3.  Does Suramin Reveal an cx-Adrenoceptor Antagonist-Sensitive Component of Venous Tone? Since neither phentolamine nor suramin above was successful in depressing MCFP,  experiments were conducted to determine if concurrent administration of both antagonists revealed such an effect (see Results 3.4). Thus, phentolamine dose-response curves were constructed in the absence and presence of a high dose of suramin.  The  phentolamine-induced tachycardia was not affected by suramin and was probably a reflex response to the accompanying depressor effect. Interestingly, this depressor effect was significantly greater in the presence of suramin which suggests that antagonism of arterial postsynaptic P x-purinoceptors may have been involved. In other words, there may exist 2 an o-adrenoceptor antagonist-resistant MAP response which is due to excitatory purinergic tone and is inhibited by suramin. In contrast, MCFP remained unaffected by phentolamine in the pressence of suramin.  Assuming suramin antagonized postsynaptic P2X-  purinoceptors in the venous system, these results suggest that purinergic venomotor tone was not responsible for opposing any venodilatatory activity of phentolamine. It is unlikely that the dose of suramin was not sufficient to produce blockade of venous P2x-purinoceptors since a preliminary experiment demonstrated that the pressor response to i.v. injection of o,f3-methylene-ATP was almost completely attenuated for at least 3 h. In addition, it has 1 (half that been shown previously in the pithed rat that suramin at a dose of 100 tmoI kg used in the present experiments) produced a parallel shift to the right (by a factor of 6) of the dose-response curve for cj3-methylene-ATP, but not NA or neuropeptide Y (Urbanek et a!. 1990). In addition, Schlicker et a!. (1989) reported that suramin blockade persisted for at least 30 mm  in the pithed rat. Discussion  97 One might speculate that in the venous system suramin acts primarily by antagoniz -purinoceptors on sympathetic nerve terminals, thereby enhancing release of NA 2 ing P and, possibly, ATP. Such an action for suramin has recently been demonstrated, for the first time, by Allgaier et al. (1994). These authors found that suramin, but not 8-(psulphophenyl)-theophylline (Pi-purinoceptor antagonist), completely prevented ATP-in duced inhibition of NA release from chick sympathetic neurons. Interestingly, the same study also demonstrated that x,f3-methylene-ATP had no effect on NA release, while 2y-purinoceptor agonist) produced a facilitation of NA release which 2 methylthio-ATP (P y-purinoceptor antagonist). Similarly, phentolamine is 2 was prevented by cibacron blue (P -autoreceptors 2 known to block not only postsynaptic c-adrenoceptors but also presynaptic c (Starke et al. 1989), following which release of both NA and ATP are enhanced (Bulloch & Starke 1990; MacDonald eta!. 1992; Msghina etal. 1992; Bao 1993). Thus, concurrent administration of suramin and phentolamine may have resulted in a substantial enhance ment of NA and ATP release such that blockade of postsynaptic receptors was overcome and venoconstriction was elicited (see Bevan et al. 1987).  However, the ability of  endogenously-released agonists to overcome receptor antagonism is highly unlikely on the basis of relatively very slow on-off rates for exogenous antagonists in addition to very effective physiological mechanisms for clearance of endogenous neu rotransm itters such as ATP and NA. Another possible explanation for the inability of concurrently adminis tered suramin and phentolamine to depress MCFP is an inaccessibility to antagonists of excitatory adrenergic and/or purinergic receptors in the venous system. These results might also be interpreted as evidence for the putative existence of phentolamine- and suramin-insensitive -y-adrenoceptors; however, studies have previously demonstrated an absence of such effects in veins (Laher eta!. 1986; Hirst & Jobling 1989). Earlier experiments were described in which phentolamine produced a significant depression of MCFP in the presence of nifedipine which, presumably, blocked purinergic tone at a site beyond the P x-purinoceptor. If suramin exerted primarily postsynaptic 2 Discussion  98 inhbition of P2x-purinoceptors, one could argue that phentolamine, in the presence of suramin, should have produced a decrease in MCFP similar to that produced in the pres ence of nifedipine. However, this was not so. The cause of this apparent discrepancy may simply be that the NA to ATP ratio is higher in ref lexly-increased versus basal sympa thetic venomotor tone. There is a good deal of evidence which suggests that the pattern of nerve stimulation influences the ratio of released NA and ATP from sympathetic nerves (Kennedy eta!. 1986b; Sjöblom-Widfeldt eta!. 1990, Sjöblom-Widfeldt & Nilsson 1990; Evans & Cunnane 1992). It is therefore possible that the pattern of sympathetic nerve activity is altered following reflex activation of venomotor tone, as following infusion of nifedipine, such as that the proportion of ATP to NA release is increased.  4.4.  Sympathetic Cotransmission and Venous Tone The use of chemical sympathectomy (e.g. by reserpine, guanethidine, or 6-  hydroxydopamine) in the study of sympathetic NA-ATP cotransmission has provided key evidence in support of cotransmission in the cardiovascular system (see Introduction 1.5.3.1). This set of experiments was therefore designed to approach the question of sympathetic cotransmission in vivo in a manner analogous to that used to demonstrate cotransmission in vitro. It is important to recognize that reserpine interferes with the NA but not the ATP uptake mechanism of chromaffin granules (Winkler et a!. 1981) while guanethidine selectively renders the membrane of catecholaminergic terminal axons inexcitable (Hausler & Haefely 1979). In the present experiments, reserpine failed to alter control values for MCFP even though both MAP and HR were significantly decreased. Furthermore, the dose of reserpine used in these experiments was previously shown to abolish catecholamine stores by at least 98% (Gillespie & McGrath 1974; Brizzolara & Burnstock 1990) without affecting the release of ATP or the x-adrenoceptor antagonist resistant component in several vessels and tissues (Kugelgen & Starke 1985; Warland & Burnstock 1987; Kirkpatrick& Burnstock 1987). Thus, the unaltered MCFP in reserpinized Discussion  99 rats might be interpreted as supporting the existence of a non-catecholamine transmitter in the maintenance of venous tone. Indeed, this hypothesis is supported by the observa tion that suramin produced a slight but significant decrease in MCPF following reserpine, but not guanethidine, treatment. Guanethidine has previously been shown to block not only the adrenergic but also the purinergic component of perivascular sympathetic nerve stimulation in several arteries (Kennedy et a!. 1986; Evans & Cunnane 1992). Taken together, the present results suggest that sympathetically-released ATP, acting at 2Xpurinoceptors, may participate in maintaining venous tone, though perhaps only to a small extent, following depletion of NA from sympathetic nerve terminals. The finding that there was no significant difference in post-treatment control MCFP values between reserpine- and guanethidine-treated groups, however, casts doubt on proposals concerning NA-ATP cotransmission in the venous system since these results point to the possible involvement of neurotransmitters other than ATP and NA in the main tenance of venous tone. That suramin failed to lower MCFP in guanethidine-treated rats further suggests that ATP is not involved, leaving the possibility that a “non-adrenergic, non-purinergic” element may be responsible for maintaining venous tone. Indeed, non adrenergic, non-cholinergic (NANC) neurotransmission is well-characterized in the portal vein of several species (Burnstock et aL 1984; Kennedy & Burnstock 1 985a; Brizzolara et a!. 1993) and has been shown to be resistant to guanethidine (Burnstock et a!. 1979)  —  it  is possible, though highly speculative, that analogous nerves serve the venous system and release transmitters whose actions are resistant to blockade of the conventional cc adrenoceptor or P x-purinoceptor. It is important to recognize, however, that guanethidine 2 posseses local anaesthetic activity and consequently may have paralyzed all neurotrans mission, in which case non-neurogenic mechanisms would be responsible for sustaining venous tone.  It is also possible to speculate that had reflexes been stimulated, the  contribution of ATP in reserpinized rats might have been increased and a larger suramin sensitive component might have become apparent since, as discussed previously, the Discussion  100 pattern of nerve stimulation may be altered during increased sympathetic nerve activity such that the ATP to NA ratio of released transmitter is increased. x-purinoceptor-mediated vasoconstriction to exogenous ATP has been demon 2 P strated in vitro in the human and dog saphenous vein (Salag et al. 1990; Rump & von Kugelgen 1994), in the dog maxillary internal vein (Salag et a!. 1992), and in the dog cutaneous vein (Flavahan & Vanhoutte 1986). In addition, it has been demonstrated that x,3-methylene-ATP is particularly active on capacitance vessels of the cat intestinal circu lation in vivo, producing greater venoconstriction than either injected noradrenaline or high frequency stimulation of sympathetic nerves (Taylor & Parsons 1989). Neverthe less, as in this study, it has proved difficult to demonstrate a purinergic component of neurogenic venoconstriction (Rump & von Kugelgen 1994). The explanation for this could be that exogenous ATP and its analogues stimulate primarily extrajunctional P2Xpurinoceptors. Consistent with a relatively minor role for ATP in the maintenance of ve H]c3-methylene-ATP (P2X3 nous tone is the demonstration that the densities of [ purinoceptor radioligand) binding sites in veins, such as the rabbit mesenteric vein and inferior vena cava (but not the portal vein) are relatively very low compared to those in muscular arteries such as the rat mesenteric, tail, and central ear arteries (Bo & Burnstock 1993). Interestingly, suramin-induced bradycardia was not affected by either reserpine or guanethidine which indicates that the response is dependent on neither sympatheticallyreleased NA nor functional sympathetic nerves. These results are in accordance with the inability of mecamylamine-induced ganglion blockade to affect suramin-induced decrease in HR. Therefore, it appears that suramin possesses direct cardiodepressant activity. Similarly, the suramin-induced increase in MAP was not altered by reserpine and was even potentiated by guanethidine. These results suggest that the pressor effect of suramin does not require a functioning sympathetic nervous system.  Discussion  101 4.5.  Cardiovascular Effects of Exogenous ATP Since there have been no previous investigations into the effects of exogenous ATP  on venous tone as reflected by MCFP, experiments were conducted in our conscious rat model to determine the effects of infused ATP on MCFP in the absence and presence of various receptor antagonists. In vehicle-treated rats, ATP produced a profound decrease in MAP which persisted, apparently unaffected, in the presence of mecamylamine and suramin.  y..-purinoceptor antagonist) and 82 In contrast, both cibacron blue (P  phenyltheophylline (Pi -purinoceptor antagonist) attenuated the ATP-induced decrease in MAP; however, only the effect of the latter reached statistical significance. Although nei ther antagonist has been characterized extensively in vivo, the same dose of 8phenyltheophylline and one-half the dose of cibacron blue used in this study have previ ously been shown to significantly attenuate vasodilatation due to adenosine and ATP, respectively, in fetal lambs (Konduri et al. 1992, 1993). The present results suggest that i.v. infusion of ATP produces vasodilatation primarily via adenosine acting at Pi-purinoc eptors which would require ATP to be extensively metabolized (presumably by ectonucleotidases in the cardiac and pulmonary vascular beds) before reaching the arte rial circulation. This proposal is consistent with the finding that, in anaesthetized dogs, arterial levels of ATP during i.v. infusion are negligible while venous ATP levels are ap proximately 90% of the expected plasma concentration for any given infusion rate (Sollevi etaL 1984). ATP infusion also produced a marked bradycardia which was unaltered in the pres ence of mecamylamine or cibacron blue but completely abolished by 8-phenyltheophylline. 8-phenyitheophylline even revealed a modest but statistically insignificant ATP-induced tachycardia while suramin very slightly enhanced the ATP-induced decline in HR. It there fore appears that the ATP-induced bradycardia is primarily (if not totally) the result of adenosine acting at cardiac Pi-purinoceptors, probably of the Ai -subtype (see Pelleg et a!. 1990).  The very slight ATP-induced tachycardia observed in the presence of 8Discussion  102  phenyltheophylline may have been a reflex response to ATP-induced vasodilatation (i.e. -adrenoceptors) or, possibly, ATP may have increased HR via stimu 1 mediated cardiac f3 y-purinoceptors. Indeed, Mantelli etal. (1993) demonstrated that block 2 lation of cardiac P -purinoceptors converted an ATP-induced negative inotropic effect to a 1 ade of inhibitory A positive inotropic effect that could be antagonized by suramin or cibacron blue. This ex planation is consistent not only with the slight enhancement of the ATP-induced bradycardia y-purinoceptors), but also with the failure of 2 by suramin (presumably via blockade of P mecamylamine to alter the ATP-induced bradycardia. It is possible that the inability of ganglion blockade to modify the cardiac effects of ATP is indicative of a presynaptic action of exogenous ATP, and/or adenosine, as an inhibitor of neurotransmitter release from sympathetic nerve terminals (see Westfall et al. 1990b). Based on the data presented, y-purinoceptors in the response to ATP 2 however, the involvement of stimulatory cardiac P is highly speculative since the decrease in HR produced byATP was unaffected by cibacron blue. The response of MCFP to ATP infusion, although insignificant, consisted of a very small increase followed by a decline. The decrease in MCFP was slightly more pro nounced in the presence of either mecamylamine or 8-phenyltheophylline, while suramin had no effect on the response of MCFP to ATP. The presence of cibacron blue, in con trast, revealed a slight but insignificant ATP-induced increase in MCFP. The slight ATP induced decline in MCFP was most likely the result of ATP, and not adenosine since (i) during i.v. ATP infusion only 10% of venous ATP is degraded to adenosine (Sollevi eta!. 1984) and (ii) Glick et a!. (1992) demonstrated that i.v. infusion of adenosine per se produces relatively much larger decreases in MCFP than did ATP in the present study. Presumably the venous response to ATP is complex and may involve some of the following factors in addition to others: (i) reflex increase in sympathetic (or possibly other) tone, (ii) stimulation of presynaptic purinoceptors by ATP and adenosine, thereby opposing the hypotension induced reflex by decreasing release of excitatory transmitters (i.e. NA and ATP), Discussion  103 (iii) endothelium-dependent and/or -independent venodilatation by ATP (and, possibly, adenosine), and (iv) ATP-induced venoconstriction. Consequently, the response of MCFP to ATP in the absence of any pretreatment was presumably the net effect of a number of different ATP-dependent mechanisms which potentially may act synergistically or antago nistically. It is probably safe to assume that if ATP has no venous dilator effect its profound depressor effect should have evoked a substantial increase in reflex venous tone similar to hydralazine, which has virtually no venous effects and increased MCFP markedly (D’Oyley  et a!. 1989). If this was not the case, then ATP must have exerted venodilatatory actions which obscured this reflex tone such that the net effect was not very different from the control. The finding that mecamylamine revealed only a slightly more pronounced ATP-induced decline in MCFP supports the idea that ATP (and/or its metabolite adenosine), when in fused alone, stimulates sympathetic presynaptic Pu rinoceptors, thereby decreasing exci tatory transmitter release (see Westfall eta!. 1990b). Although, this proposal is question able in light of the possibility that ATP was unable to produce further venodilatation under conditions in which venomotor tone had already been significantly depressed by mecamylamine, the observation that 8-phenyltheophylline produced an effect on the ATP induced decline in MCFP similar to that of mecamylamine not only supports the notion of a presynaptic site of action of ATP, but also implicates ATP, and not adenosine, as the mediator of the slight decrease in MCFP. The observation that cibacron blue revealed a slight but insignificant increase in MCFP in response to ATP might be the result of (i) inhibition of presynaptic autoinhibitory purinoceptors, thus enhancing reflex venoconstrictor tone, and/or (ii) inhibition of y-purinoceptors responsible for venodilatation. If, in fact, cibacron blue 2 postsynpatic P acted presynaptically then this action would be in keeping with the notion of a measureable neuromodulatory role of i.v.-infused ATP in the venous system. A postynaptic site of ac tion for cibacron blue, on the other hand, is also a reasonable hypothesis since this action, Discussion  104  too, would facilitate reflex venoconstriction. However, the failure of suramin to produce effects similar to cibacron blue, either presynaptically or postsynaptically, casts serious doubt on such an action of cibacron blue or even on the in vivo selectivity of these antago nists. Still, it could be argued that this apparent discrepancy originates from the additional x-antagonistic property of suramin but not cibacron blue. It should be recognized, how 2 P -purinocep2 ever, that suramin is a competitive antagonist with low affinity for vascular P tors (Leff et a!. 1990)  —  this might have enabled ATP to easily displace suramin, thus  accounting for suramin’s inability to alter responses to ATP.  4.6.  1.  Summary and Conclusions In the conscious rat, phentolamine (a non-selective x-adrenoceptor antagonist) was  found to be a more effective arterial than venous vasodilator in both basal conditions and during drug (hydralazine or nifedipine)-induced vasodilatation and reflex venoconstriction. While MCFP was not significantly decreased by phentolamine either under basal condi tions or during hydralazine treatment, phentolamine did significantly decrease MCFP in the presence of nifedipine. Following suramin treatment, the phentolamine-induced de pressor effect was significantly greater whereas MCFP remained unchanged. Under basal conditions, mecamylamine very effectively reduced both MAP and MCFP whereas in the presence of hydralazine-induced vasodilatation and elevated venomotor tone, ganglion blockade reduced MCFP but not MAP.  CONCLUSIONS:  Arterial tone is primarily maintained by ot-adrenoceptor activation in both  basal conditions and during increased sympathetic nerve activity, whereas venous tone, although dependent on autonomic nerve activity, is completely resistant to blockade of c adrenoceptors under basal conditions and partially resistant in the presence of elevated venomotor tone. The finding that phentolamine produced a greater decrease in MCFP in Discussion  105  the presence of nifedipine than in the presence of hydralazine suggests that the oL-adrenoceptor antagonist-resistance of MCFP may be purinergic in origin. Blockade of purinergic mechanisms at receptor level by suramin may not have revealed an c-antago nist-sensitive component as did nifedipine (via blockade at site beyond the purinoceptor) because of differences in the ratio of NA to ATP released from sympathetic nerve termi nals as a result of different patterns of nervous activity under basal conditions and during elevated sympathetic nerve activity. It is unclear why phentolamine but not blockade of autonomic reflexes by mecamylamine reduced MAP in the presence of hydralazine.  2.  -purinoceptors by suramin produced a dose-dependent increase in 2 Blockade of P  MAP and decrease in HR neither of which was affected by hydralazine, nifedipine, mecamylamine, reserpine, or guanethidine. Suramin failed to reduce MCFP in the ab sence or presence of hydralazine, nifedipine, or guanethidine. In contrast, mecamylamine treatment revealed a significant dose-dependent decrease in MCFP, while reserpine treat ment revealed a slight but significant decline in MCFP.  CONCLUSIONS:  The mechanisms responsible for the suramin-induced pressor effect and  2 2 through nifedipine-sensitive voltage-gated Ca bradycardia involve neither influx of Ca . 2 channels, nor hydralazine-sensitive mechanisms possibly involving intracellular Ca Furthermore, neither the suramin-induced MAP nor HR effect is dependent on the integ rity of the sympathetic or autonomic nervous system. Suramin may antagonize presynaptic purinoceptors in vivo, thus accounting for its inability to reduce basal or reflexly-increased MCFP. The ability of suramin to decrease MCFP in reserpinized rats may indicate a role for sympathetic NA-ATP cotransmission in the venous system, while the appreciable suramin-induced decrease in MCFP during near-complete ganglion blockade may reflect the role of “electromechanical coupling” and the existence of purinergic mechanisms in the venous system. Discussion  106  3.  l.v. infusion of ATP produced profound depressor and bradycardic effects. The ATP  induced depressor effect was unaffected by mecamylamine and suramin whereas block -purinoceptors by 8-phenyltheophylline clearly and significantly attenuated this 1 ade of P y-purinoceptors by cibacron blue only slightly and insignificantly 2 response. Blockade of P attenuated the depressor effect of ATP. ATP-induced bradycardia was not affected by mecamylamine or cibacron blue whereas 8-phenyltheophylline completely abolished this response and even revealed a slight, but insignificant, increase in HR in response to ATP. Suramin slightly but insignificantly enhanced the ATP-induced bradycardia. ATP produced a slight but insignificant depression of MCFP which was unaltered in the presence of suramin, and slightly but insignificantly enhanced both during mecamylamine-induced ganglion blockade and following 8-phenyltheophylline treatment.  Cibacron blue, in  constrast, revealed a slight but insignificant ATP-induced increase in MCFP.  CONCLUSIONS:  I.v. infusion of ATP produces a profound depressor effect predominantly via  activation of Pi-purinoceptors following breakdown to adenosine. ATP per se may also contribute to the depressor effect, though only minimally, presumably by activation of y-purinoceptors. The ATP-induced bradycardia is apparently mediated ex 2 endothelial P clusively by adenosine while ATP per se may exert a direct stimulatory effect on the heart via P y-purinoceptor activation. 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