PEPTIDERGIC, PURINERGIC AND ADRENERGIC NEUROTRANSMITTERS
IN THE CONTROL OF THE CORONARY ARTERY
by
Laura Anne Corr
Department of Anatomy and Developmental Biology
and Centre for Neuroscience,
University College London
Gower Street,
London WC1
and
Department of Cardiac Medicine,
National Heart and Lung Institute,
Dovehouse Street,
London SW3
Thesis submitted for the degree of Doctor of Philosophy
University of London
1 ABSTRACT OF THESIS
In addition to the "classical" transmitters, noradrenaline and acetylcholine, autonomic perivascular nerves are now known to contain several peptide and purine neurotransmitters. These may act directly on the vascular smooth muscle, as cotransmitters or neuromodulators, and may also stimulate endothelial responses.
This thesis presents a study of the smooth muscle and endothelial responses of the coronary artery to such neurotransmitters. It also describes changes in the peptidergic innervation and in the responses to these neurotransmitters which occur under physiological and pathophysiological conditions.
Acetylcholine produced endothelium-dependent relaxation of the rabbit epicardial coronary artery at low concentrations, but smooth muscle constriction dominated at higher concentrations. Noradrenaline relaxed this vessel by action on smooth muscle 0-adrenoceptors which were identified as 0j; the endothelium played no role in mediating responses to the 0-adrenergic agonists. There was very little alpha-receptor-mediated vasoconstrictor response to noradrenaline; ATP too was found to act directly as a vasodilator of the smooth muscle in this vessel, supporting the hypothesis that noradrenaline and ATP may be inhibitory co-transmitters in the coronary artery. Examination of the purinoceptor subtypes demonstrated not only Pj- but also P2y-purinoceptors on the smooth muscle and on the endothelium, while P2^-purinoceptors were found only on the smooth muscle.
2 The responses of the coronary arteries of female rabbits to these adrenergic and purinergic transmitters, as well as some of the more recently recognised peptidergic neurotransmitters, were found to undergo profound changes during development of sexual maturity. Coronary arteries from the New Zealand white
(NZW) rabbits were compared with those from Watanabe Hereditable
Hyperlipidaemic (WHHL) rabbits to assess the long-term effects of atherosclerosis on the responses to these agents. This study showed that endothelium-dependent responses were depressed in the young WHHL animals. Maturational changes in the atherosclerotic rabbits in the responses to non-endothelium-dependant agonists mirrored closely those seen in normal rabbits but the responses to the endothelium-dependant vasodilators differed markedly, showing a compensatory recovery during maturation while the response in the normal animals declined.
Using guanethidine to produce long-term sympathectomy in neonatal rats, compensatory changes in peptide-containing nerves were examined using immunohistochemical and assay techniques. Non-sympathetic NPY-containing nerves were identified in the ileum, bladder, heart and vas deferens but blood vessels were supplied entirely by sympathetic NPY-containing nerves. Despite the reduction of sympathetic NPY, the non-sympathetic NPY-containing nerves did not proliferate to compensate for the loss of the sympathetic nerves. However, marked and selective increases were found in nerves immunoreactive for the sensory neurotransmitter calcitonin gene-related peptide, but not for substance P or vasoactive intestinal polypeptide.
The pattern of receptors exhibited by the coronary artery and the changes in innervation and responses which were demonstrated in health and disease are discussed.
3 TO MY PARENTS, NUALA AND GERRY,
WHOM I LOVE VERY MUCH ACKNOWLEDGEMENTS
I am most grateful to my supervisors, Professor Geoffrey Burnstock and
Professor Philip Poole-Wilson, for their interest, encouragement and advice which
often extended to matters beyond the scope of this thesis.
My thanks also to the many people who helped me during this period. In
particular I thank my friend and collaborator Judy Aberdeen, with whom all the
work in Chapters 10 and 11 was carried out; Jill Lincoln, who performed the
catecholamine assays with HPLC in Chapters 10 and 11; Pam Milner, who assayed
the peptides using an ELISA in Chapters 10 and 11; Sian Harding and Charles
Hoyle who provided generous and helpful advice; Mollie White, Annie Evans and
Rosie Cole for their friendly support and secretarial skills; Gill Knight for her
cheerful companionship and practical help; Marie Phillips and Edith Quinn for
their technical help and advice; Frances Cribben and Phillippa Chara tan for
editorial assistance; the staff of the photographic department; the staff of the joint
animal house; and the many other colleagues who provided practical support and
helped make the time so personally rewarding. I am especially grateful to
Bradford Marks for the love and laughter.
I am indebted to the Medical Research Council for financial support.
Other than the work in Chapters 10 and 11 acknowledged above, this thesis is
entirely my own work.
5 CONTENTS
Abstract ...... 2
Acknowledgements...... 5
Contents ...... 6
List of Tables ...... 15
List of Figures ...... 16
CHAPTER 1 INTRODUCTION ...... 17
CHAPTER 2 GENERAL BACKGROUND ...... 21
2.1 A CLINICAL PERSPECTIVE ...... 21
2.1.1 Coronary Heart Disease ......
21
2.1.2 Changing Concepts ...... 22
2.1.3 The Role of Coronary Vasospasm ...... 23
2.2 STRUCTURE AND FUNCTION OF THE CORONARY ARTERIES . 26
2.2.1 The Coronary Circulation ...... 26
2.2.2 Factors Controlling Coronary Blood Flow ...... 27
2.2.3 Structure of the Epicardial Coronary Artery ...... 28
2.3 THE ROLE OF ENDOTHELIUM ...... 30
2.3.1 Traditional Concepts ...... 30
2.3.2 New Concepts ...... 32
i Endothelial Control of Vasomotor Tone ...... 32
ii The Endothelium as a Source of Vasoactive Agents ...... 34
6 2.4 THE ROLE OF THE AUTONOMIC NERVOUS SYSTEM 35
2.4.1 Sympathetic Nervous System ...... 35
i Anatomy ...... 35
ii Autonomic Neuroeffector Junction ...... 36
iii Sympathetic Neurotransmitters and Receptors
- Classical View ...... 38
iv New Concepts
- Cotransmission and Neuromodulation ...... 40
2.4.2 Parasympathetic Nervous System ...... 42
i Anatomy ...... 42
ii Parasympathetic Neurotransmitters and Receptors
- Classical View ...... 43
iii New Concepts ...... 45
2.4.3 Sensory-Motor Nervous System ...... 47
i Anatomy ...... 47
ii Sensory-Motor Neurotransmitters and Receptors ...... 47
2.4.4 Intrinsic Nevous System ...... 49
2.4.5 Neuronal Plasticity ...... 50
CHAPTER 3 GENERAL METHODOLOGY ...... 54
3.1 PHARMACOLOGY ...... 54
3.1.1 Choice of Preparation ...... 54
3.1.2 Method ...... 56
3.1.3 Administration of Drugs ...... 59
7 3.2 HISTOCHEMISTRY ...... 60
3.2.1 Fluorescence Histochemistry for Noradrenaline ...... 60
i Introduction ...... 60
ii Method for Whole Mounts ...... 61
iii Method for Sections ...... 62
iv Assessment of Innervation ...... 62
3.2.2 Immunohistochemistry ...... 63
i Introduction ...... 63
ii Method for Whole Mounts ...... 64
iii Method for Sections ...... 65
iv Assessment of Innervation ...... 65
3.3 ASSAY ...... 66
3.3.1 Inhibition Enzyme-Linked Immunosorbant Assay of Peptides .. 67
3.3.2 HPLC with Electrochemical Detection of Noradrenaline ...... 69
3.4 CHEMICAL SYMPATHECTOMY ...... 71
3.4.1 Guanethidine Sympathectomy ...... 72
3.4.2 6-Hydroxydopamine Sympathectomy ...... 72
3.5 PREPARATION OF DATA AND STATISTICAL ANALYSIS ...... 73
3.5.1 Pharmacology ...... 73
3.5.2 Histochemistry and Assay ...... 73
3.6 SOURCES OF MATERIALS ...... 74
3.7 PREPARATION OF SOLUTIONS ...... 76
3.7.1 General ...... 76
3.7.2 Histochemistry ...... 77
3.7.3 Assay ...... 80
8 CHAPTER 4 ENDOTHELIAL RESPONSES OF THE RABBIT CORONARY ARTERY TO BETA-ADRENERGIC AGONISTS
AND ACETYLCHOLINE ...... 83
4.1 SUMMARY ...... 83
4.2 INTRODUCTION ...... 84
4.3 METHODS ...... 85
4.3.1 Materials ...... 86
4.3.2 Statistics ...... 86
4.4 RESULTS ...... 86
4.4.1 Responses to Acetylcholine ...... 86
4.4.2 Responses to Adrenergic Agonists ...... 87
4.5 DISCUSSION ...... 88
CHAPTER 5 BETA-ADRENERGIC RELAXATION OF THE RABBIT
CORONARY ARTERY IS MEDIATED BY AN HOMOGENEOUS
POPULATION OF BETAj-ADRENOCEPTORS ...... 100
5.1 SUMMARY ...... 100
5.2 INTRODUCTION ...... 100
5.3 METHODS ...... 102
5.3.1 Materials ...... 102
5.3.2 Statistics ...... 103
5.4 RESULTS ...... 103
5.4.1 Effect of jS|-Antagonists and Propranolol ...... 104
5.4.2 Effect of ICI 118,551 ...... 104
5.5 DISCUSSION ...... 105
9 CHAPTER 6 VASODILATOR RESPONSE OF THE SMOOTH MUSCLE OF
THE RABBIT CORONARY ARTERY TO THE SYMPATHETIC
COTRANSMITTERS NORADRENALINE AND ADENOSINE
TRIPHOSPHATE ...... 117
6.1 SUMMARY ...... 117
6.2 INTRODUCTION ...... 118
6.3 METHODS ...... 119
6.3.1 Pharmacology ...... 119
6.3.2 Materials ...... 119
6.3.3 Statistics ...... 119
6.4 RESULTS ...... 120
6.4.1 At Raised Tone ...... 120
6.4.2 At Basal Tone ...... 121
6.5 DISCUSSION ...... 122
CHAPTER 7 PURINOCEPTOR SUBTYPES ON THE SMOOTH MUSCLE AND
ENDOTHELIUM OF THE RABBIT CORONARY ARTERY .... 133
7.1 SUMMARY ...... 133
7.2 INTRODUCTION ...... 134
7.3 METHODS ...... 135
7.3.1 Pharmacology ...... 135
7.3.2 Materials ...... 136
7.3.3 Statistics ...... 136
7.4 RESULTS ...... 137
7.4.1 At Basal Tone ...... 137
7.4.2 At Raised Tone ...... 138
7.5 DISCUSSION ...... 139
10 CHAPTER 8 RESPONSES OF CORONARY ARTERIES TO
NEUROTRANSMITTERS - CHANGES WITH SEXUAL
MATURITY IN THE FEMALE RABBIT ...... 154
8.1 SUMMARY ...... 154
8.2 INTRODUCTION ...... 155
8.3 METHODS ...... 156
8.3.1 Materials ...... 157
8.3.2 Statistics ...... 157
8.4 RESULTS ...... 157
8.4.1 Contractile Responses ...... 157
8.4.2 Relaxant Responses ...... 158
8.5 DISCUSSION ...... 159
8.5.1 Vasoconstrictor Responses ...... 160
8.5.2 Vasodilator Responses ...... 161
i Response to Noradrenaline ...... 161
ii Responses to Vasodilator Peptides ...... 162
8.5.3 The Role of Sexual Maturation ...... 164
CHAPTER 9 RECOVERY OF IMPAIRED ENDOTHELIUM-MEDIATED
RELAXATION IN CORONARY ARTERIES DURING EARLY
DEVELOPMENT OF ATHEROSCLEROSIS ...... 173
9.1 SUMMARY ...... 173
9.2 INTRODUCTION ...... 174
9.3 METHODS ...... 176
9.3.1 Materials ...... 176
9.3.2 Statistics ...... 176
11 9.4 RESULTS ...... 176
9.4.1 Effects of Hypercholesterolemia ...... 177
9.4.2 Changes with the Development of Atherosclerosis
i Contractile Responses ...... 178
ii Relaxant Responses ...... 179
9.5 DISCUSSION ...... 180
9.5.1 Effects of Hypercholesterolemia ...... 181
9.5.2 Effects of Development of Early Atherosclerosis ...... 183
i Contractile Responses ...... 183
ii Relaxant Responses ...... 185
CHAPTER 10 MARKED INCREASES IN CALCITONIN GENE-RELATED
PEPTIDE-CONTAINING NERVES IN THE DEVELOPING
RAT FOLLOWING LONG-TERM SYMPATHECTOMY
WITH GUANETHIDINE ...... 202
10.1 SUMMARY ...... 202
10.2 INTRODUCTION ...... 203
10.3 METHODS ...... 204
10.3.1 Long-Term Guanethidine Sympathectomy ...... 204
10.3.2 Acute 6-Hydroxydopamine Sympathectomy ...... 204
10.3.3 Selection of Tissues for Study ...... 204
10.3.4 Fluorescence Histochemistry ...... 205
10.3.5 Immunohistochemistry ...... 205
10.3.6 Assay ...... 206
10.3.7 Materials ...... 206
10.3.8 Statistics ...... 206
12 10.4 RESULTS 206
10.4.1 Long-Term Sympathectomy with Guanethidine ...... 206
10.4.2 Short-Term Sympathectomy with 6-Hydroxydopamine ...... 209
10.5 DISCUSSION ...... 209
CHAPTER 11 NEUROPEPTIDE Y IN NON-SYMPATHETIC NERVES IN
THE RAT: CHANGES DURING NORMAL MATURATION
BUT NOT AFTER LONG-TERM SYMPATHECTOMY
WITH GUANETHIDINE ...... 222
11.1 SUMMARY ...... 222
11.2 INTRODUCTION ...... 223
11.3 METHODS ...... 224
11.3.1 Long-Term Guanethidine Sympathectomy ...... 224
11.3.2 Acute 6-Hydroxydopamine Sympathectomy ...... 224
11.3.3 Tissues Studied ...... 225
11.3.4 Fluorescence Histochemistry ...... 225
11.3.5 Immunohistochemistry ...... 225
11.3.6 Assay ...... 226
11.3.7 Materials ...... 226
11.3.8 Statistics ...... 226
11.4 RESULTS ...... 226
11.4.1 Control Levels of Neuropeptide Y:
Effect of Normal Maturation ...... 226
11.4.2 Effect of Guanethidine Sympathectomy on
6-Week-Old Animals ...... 227
13 11.4.3 Effect of Guanethidine Sympathectomy on
20-Week-Old Animals ...... 228
11.4.4 Effect of Acute Sympathectomy by 6-Hydroxydopamine ...... 229
11.5 DISCUSSION ...... 230
CHAPTER 12 FINAL DISCUSSION ...... 242
12.1 USE OF THE RABBIT CORONARY MODEL ...... 242
12.1.1 Smooth Muscle and Endothelial Responses ...... 242
12.1.2 Effects of Nerve Stimulation ...... 243
12.2 CORONARY RESPONSES TO NEUROTRANSMITTERS ...... 244
12.2.1 Responses to the Classical Neurotransmitters ...... 244
12.2.2 Purinergic Control of the Rabbit Coronary Artery ...... 245
12.2.3 Coronary Responses to the Neuropeptides ...... 247
12.3 PLASTICITY OF VASOMOTOR RESPONSES ...... 247
12.3.1 Physiological Changes ...... 247
12.3.2 Pathophysiological Changes ...... 249
12.4 PLASTICITY OF INNERVATION ...... 250
12.5 CONCLUDING REMARKS ...... 251
REFERENCES ...... 252
A list of publications to date arising from work associated with this thesis is given inside the back cover.
14 LIST OF TABLES
Table 4.1 ...... 92
Table 5.1 ...... 110
Table 5.2 ...... I ll
Table 5.3 ...... 112
Table 6.1 ...... 126
Table 7.1 ...... 145
Table 8.1 ...... 166
Table 9.1 ...... 188
Table 10.1 ...... 214
Table 10.2 ...... 215
Table 10.3 ...... 216
Table 11.1 ...... 234
Table 11.2...... 235
Table 11.3 ...... 236
LIST OF FIGURES
Figure 2.1 ...... 53 Figure 4.3 ...... 96
Figure 3.1 ...... 82 Figure 4.4 A,B ...... 97
Figure 4.1 ...... 93 Figure 4.4 C ...... 98
Figure 4.2 A,B ...... 94 Figure 4.5 ...... 99
Figure 4.2 C ...... 95 Figure 5.1 ...... 113
15 Figure 5.2 A,B ...... 114 Figure 9.1 ...... 189
Figure 5.2 C,D ...... 115 Figure 9.2 ...... 190
Figure 5.3 ...... 116 Figure 9.3 ...... 191
Figure 6.1 ...... 127 Figure 9.4 A,B ...... 192
Figure 6.2 ...... 128 Figure 9.4 C ...... 193
Figure 6.3 ...... 129 Figure 9.5 A ...... 194
Figure 6.4 ...... 130 Figure 9.5 B ...... 195
Figure 6.5 ...... 131 Figure 9.6 ...... 196
Figure 6.6 ...... 132 Figure 9.7 ...... 197
Figure 7.1 ...... 146 Figure 9.8 A,B ...... 198
Figure 7.2 ...... 147 Figure 9.9 A,B ...... 199
Figure 7.3 ...... 148 Figure 9.10 A ...... 200
Figure 7.4 A,B ...... 149 Figure 9.10 B ...... 201
Figure 7.5 A,B ...... 150 Figure 10.1 ...... 217
Figure 7.5 C,D ...... 151 Figure 10.2 ...... 218
Figure 7.6 ...... 152 Figure 10.3 ...... 219
Figure 7.7 ...... 153 Figure 10.4 ...... 220
Figure 8.1 ...... 167 Figure 10.5 ...... 221
Figure 8.2 ...... 168 Figure 11.1 ...... 237
Figure 8.3 ...... 169 Figure 11.2 ...... 238
Figure 8.4 ...... 170 Figure 11.3 ...... 239
Figure 8.5 ...... 171 Figure 11.4 ...... 240
Figure 8.6 ...... 172 Figure 11.5 ...... 241
16 CHAPTER 1
INTRODUCTION
Coronary arteries are capable of vasomotor responses which may profoundly affect myocardial blood flow. For many years, the control of coronary arterial vasomotor tone has been the subject of intense research both in vivo and in vitro. The autonomic neural control of the coronary arteries has aroused particular interest but it has proved very difficult to separate the effects of sympathetic and parasympathetic nerve stimulation on the coronary arteries from the secondary effects due to metabolic changes in the myocardium and from the changes consequent upon stimulation of baroreceptor reflexes. The use of small animal isolated heart preparations - such as the Langendorff model - removed the effect of baroreceptor reflexes, but not that of the metabolising myocardium. Isolated coronary arteries from larger animals, such as the pig and, in particular, the dog, have allowed many of the effects of the "classical" neurotransmitters, noradrenaline and acetylcholine, to be studied but often gave rise to conflicting results.
The discovery in 1981 by Furchgott and Zawadski of the essential role played by the endothelium in mediating vasomotor relaxant responses to acetylcholine has resolved many of the discrepancies arising from these early studies, but raised more questions about the role of cholinergic nerves in the circulation. Furthermore, important variations in the responses of certain species have become apparent; the dog coronary artery relaxes to acetylcholine via the endothelial release of "endothelium derived relaxant factor" - possibly nitric oxide
17 - while in the pig coronary artery, acetylcholine gives a purely vasoconstrictor response by direct action on the smooth muscle. It thus appears that apparantly qualitative differences in the responses of these vessels may arise from essentially quantitative differences in the distribution of receptors expressed on the smooth muscle and endothelium; while the coronary arteries of both the pig and the dog may have receptors to acetylcholine on both the smooth muscle and the endothelium, the preponderance of the receptors on either smooth muscle or endothelial cells allows for very different vasomotor responses in the vessels.
In recent years, the discovery of the existence of many small peptides acting as neurotransmitters in autonomic nerves, and the realisation that purines such as ATP may also act as sympathetic and sensory neurotransmitters, has transformed our understanding of the mechanisms of autonomic nervous control.
Furthermore, it is now known that many of these neurotransmitters may also cause release of vasoactive substances from the endothelium. It is therefore likely that the presence and distribution of receptors to these agents on smooth muscle and endothelium, like those to acetylcholine, determines the nature of the responses in any given vessel or region of a vessel.
Large animals are expensive, difficult to manipulate experimentally and, as described above, do not necessarily provide good models of coronary arterial responses. In this thesis I set out to establish the use of isolated rabbit coronary arteries as a model for the study of coronary arterial vasomotor control. Several factors made this species attractive: much of the work in isolated hearts has been carried out in the rabbit; it is relatively inexpensive; it can be manipulated experimentally; and certain strains provide good genetic models for human atherosclerosis. I aimed, firstly, to establish the nature and distribution of the
18 receptors to the classical neurotransmitters noradrenaline and acetylcholine, to see how this species compares to other better known models and to establish how in this animal, as in the pig and the dog, the nature of the response is determined by the character and distribution of these receptors. This work is described in
Chapters 4 and 5. In the light of recent discoveries about the interaction of adenosine triphosphate and noradrenaline in the sympathetic nervous system, I next studied the responses of the rabbit coronary artery to the purinergic neurotransmitters; subsequently, the purinoceptor subtypes mediating these responses and their distribution on the smooth muscle and endothelium were identified. These results and some of their implications are discussed in Chapters
6 and 7. Little was known of the nature of the receptors mediating responses to the peptidergic neurotransmitters when this thesis was started but the effects of several of these agents on the smooth muscle and endothelium of the rabbit coronary artery were also identified as part of a wider study and are reported in
Chapter 8. By this work, I hoped to gain insight into the pattern of receptors which give rise to the characteristic responses of the coronary artery to these neurotransmitters.
It has been known for some years that the type and density of nerves innervating blood vessels is not fixed but may change under different physiological and pathophysiological circumstances; so, too, may the receptors mediating responses to these agents - for example, down regulation of the /^-adrenoceptors in the myocardium is known to occur in heart failure. In the same way, the nature and distribution of receptors mediating responses to the neurotransmitters in blood vessels is not rigid; changes in responses to noradrenaline have been described with aging and diseases such as hypertension. Hence study of the "normal" coronary
19 artery may not reveal changes in the pattern of innervation or receptors which could give rise to pathological responses.
Using the rabbit model, I examined in this thesis some of the long-term effects of physiological changes (normal maturation) and disease (atherosclerosis) on the responses of the isolated coronary artery. These studies are reported in
Chapters 8 and 9 in this thesis. I also examined the potential for plasticity in peptidergic nerves innervating the cardiovascular system and other organs; for technical reasons this work was carried out in rats (see Chapter 3). By destroying in neonates a selected population of nerves (noradrenergic sympathetic nerves) and subsequently allowing the animals to mature to adulthood, the potential for reinnervation by other extrinsic nerves and by intrinsic nerves - which contain certain neurotransmitters in common with sympathetic nerves - was established.
This is described in the final two experimental chapters of the thesis, Chapters 10 and 11.
The results of this thesis, and some conclusions which may be drawn from them, are considered in the General Discussion in Chapter 12.
2 0 CHAPTER 2
GENERAL BACKGROUND
In this chapter, there is a brief outline of the role played by the epicardial coronary arteries in controlling myocardial blood flow and a description of some of the elements which are involved in the control of coronary vasomotor tone.
The chapter does not attempt to be a comprehensive review, but merely to give a perspective to the work presented in this thesis.
2.1 A CLINICAL PERSPECTIVE
2.1.1 Coronary Heart Disease
Ischaemic heart disease is one of the most important diseases of developed countries, both in terms of its prevalence and of its human and economic cost; it accounts for 40-50% of all deaths in the Western World. In 1988, the mortality rate for ischaemic heart disease in the U.K. was approximately 500/100,000
(Shaper, 1988), with morbidity many times greater. The term ischaemic heart disease covers a group of clinical syndromes that include angina pectoris, acute myocardial infarction, ischaemic cardiac insufficiency and sudden ischaemic cardiac death; in almost all cases the common denominator is restriction of myocardial blood supply through the large, epicardial coronary arteries.
21 Coronary atherosclerosis, which is known to affect selectively the epicardial coronary arteries, is generally considered to be the cause of ischaemic heart disease. Traditionally, rigid stenoses of these arteries were thought to become inexorably more severe causing angina when myocardial oxygen demand exceeded supply, for example during exertion; if this imbalance persisted, it resulted in irreversible acute myocardial ischaemia and infarction. As a consequence of this conceptual approach, the focus of research into ischaemic heart disease prior to the last decade was primarily on the myocardium and it was from this vantage point that the pharmacology, physiology, pathology and therapy were discussed.
2.1.2 Changing Concepts
There is certainly no doubt that the presence of atherosclerotic lesions in the coronary arteries correlates strongly with all the ischaemic syndromes but it is not sufficient, or even necessary, as a cause of myocardial ischaemia. Lesions may be present in individuals who have no detectable symptoms or signs of myocardial ischaemia, while a minority of patients develop angina or acute myocardial infarction without the presence of coronary atherosclerosis (Cheng et al. 1973).
Furthermore, coronary atherosclerosis is very prevalent in developed countries - postmortem studies suggest that, on average, 20-30% of the intimal surface of coronary arteries is covered with raised fibrous plaques and fatty streaks by the age of 40-44 years and there is extensive overlap in the degree of coronary atherosclerosis found in individuals who died accidentally and in those who died of ischaemic heart disease (Strong & Guzman, 1980). Recent work has emphasised that myocardial infarction may occur in areas of myocardium supplied by coronary arteries which do not have critical fixed stenoses (Hackett et al. 1987), while
2 2 severely stenotic coronary arteries may produce stable exertional angina for many years without precipitating myocardial infarction (Maseri et al. 1986).
Over the past decade, other acute ischaemic stimuli have been shown to play a major role in modulating myocardial oxygen supply; the most important of these are coronary thrombosis and coronary vasospasm. Thrombosis of the coronary arteries at the site of an atherosclerotic lesion (which is not necessarily severe) appears to be a very significant factor precipitating acute myocardial infarction, as evidenced by the almost invariable finding of thrombus at coronary arteriography during myocardial infarction and the decrease in mortality following prompt treatment of acute myocardial infartion with thrombolytic agents (Gruppo
Italiano per lo Studio della Streptochinasi nell’Infarto Miocardico (GISSI), 1986;
ISIS-2 Collaborative Group, 1988; GISSI, 1990). However, alteration in epicardial coronary vasomotor tone has been clearly demonstrated to produce angina in the absence of any increase in myocardial oxygen demand and may represent a major pathogenetic component in exertional and unstable angina and acute myocardial infarction (Deanfield et al. 1983; Maseri et al. 1986; Hackett et al. 1987). Far from being the passive conduits of blood as they were once considered, the epicardial coronary arteries are now aknowledged to play an important active role in modulating myocardial oxygen supply which may alter under pathophysiological conditions.
2.1.3 The Role of Coronary Vasospasm
The concept of coronary vasospasm is not new. Indeed, the notion that angina pectoris may be caused by transient vasoconstriction of the coronary arteries was put first forward with the hypothesis of generalised arterial
23 constriction proposed by Brunton (1867) and Fothergill (1879), but it took almost one hundred years before it was accepted as a primary cause of myocardial ischaemia.
The struggle for the acceptance of the role of coronary vasospasm has recently been reviewed by Maseri and Chiercha and is briefly summarised here
(for these references, see Maseri & Chierchia, 1982). In 1889, Hutchard suggested
that angina pectoris was always the result of myocardial ischaemia caused by an
organic or functional narrowing of the coronary arteries and Osier, in the
Lumleian lectures of 1910, considered coronary spasm or narrowing the likeliest
cause for angina occuring at rest. However, it was Danielopolus in the 1920s who
first introduced the theory of an imbalance between supply and demand, resulting
either from a sudden reduction in myocardial blood flow or from a sudden
increase in myocardial oxygen demand in the presence of fixed critical coronary
arterial narrowing. In 1925 Gallavardin identified examples of both mechanisms:
on the one hand patients who had angina at rest but at post-mortem had normal
coronary arteries, and on the other patients who had severe angina on exertion
who generally had marked atherosclerosis of the coronary arteries. He also
recognised that these two mechanisms frequently existed in the same patients.
But in 1928 the concept of coronary spasm as a cause of myocardial
ischaemia began to fall into disrepute with the dogmatic statement by Keefer and
Resnik that coronary arteries in patients with angina were so severely fibrosed and
calcified that it was not possible for them to go into spasm. When, in the 1940s,
Blumgart and coworkers found severe atherosclerosis at post mortem in nearly all
anginal patients the notion of fixed coronary obstruction became ingrained to the
extent that coronary spasm was called "the resort of the diagnostically destitute".
24 Coronary arteriography became the ’gold standard’ for the diagnosis of ischaemic
heart disease and coronary vasospasm was not even considered in classic 1960’s
textbooks such as those by Freidberg and Wood.
Around 1960, Printzmetal first described a ’variant’ form of angina
characterised by transmural myocardial ischemia not related to increased
myocardial oxygen demand which he proposed was due to "increased tonus at the
site of an atherosclerotic plaque" (Printzmetal et al. 1959). At this time, Gensini
and his colleagues documented vasospasm angiographically (Gensini et al. 1962)
and Vlodaver and Edwards later confirmed that coronary arteries with eccentric
stenoses retained elasticity and the potential for constriction (Vlodaver & Edwards,
1971). MacAlpin and his colleagues revived the concept of spasm in 1973 to
explain the lack of myocardial ischaemia on exercise despite increased myocardial
oxygen demand in patients who had Printzmetal’s angina at rest (MacAlpin et al.
1973) and subsequently many other workers demonstrated coronary vasospasm in
isolated reports (Dhurandhar et al. 1972; Froment et al. 1973; Oliva et al. 1973)
and in systematic studies of patients with variant angina, including patients who
had no detectable organic epicardial coronary stenoses (Yasue et al. 1974; Maseri et
al. 1975; 1976; 1977). The syndrome was originally thought to be rare but it was
reported with increasing frequency and by 1980 was even said to account for 20 -
30% of patients admitted to coronary care units because of unstable angina
(National Cooperative Study Group, 1980).
In the late 1970s, careful angiographic documentation of epicardial
coronary vasospasm led Maseri and coworkers to propose that many cases of
angina at rest other than Printzmetal’s variant angina could also be caused by a
transient impairment of coronary blood supply and, as Gallavardin had recognised
25 in 1925, many patients could exhibit more than one form of angina (Maseri et al.
1975; Maseri et al. 1977). Subsequently, it was found that changes in dynamic coronary tone could precipitate or exacerbate exertional angina (Yasue et al. 1976;
Schang & Pepine, 1977; Maseri et al. 1978; Epstein & Talbot, 1981; Willerson et al.
1986) and even acute myocardial infarction (Maseri et al. 1986; Hackett et al.
1987) .
These findings focussed increased attention on the mechanisms responsible for the control of large coronary artery tone, in particular on neurohumoral
influences through the autonomic innervation and the responses to circulatory
vasoactive agents. In this thesis, I have examined the responses of the epicardial
coronary arteries to some of these factors, looking in particular at the more
recently discovered neurotransmitter substances and the important role now known
to be played by the endothelium. I have also explored some of the changes which
may occur in both the innervation and the responses of the epicardial coronary
arteries under differing physiological and pathophysiological conditions.
2.2 STRUCTURE AND FUNCTION OF THE CORONARY ARTERIES
2.2.1 The Coronary Circulation
The basic anatomic patterns of the major coronary arteries are comparable
from the smallest to the largest mammals, i.e., from rodents to whales (Gregg &
Fisher, 1963). There are three major vessels located on the epicardial surface of
the heart, the left anterior descending, left circumflex and right coronary arteries,
which take origin from the aorta at the sinuses of Valsalva. The two left coronary
26 arteries arise from a single coronary ostium behind the posterior cusp of the aortic valve and in general supply the anterior wall of the left ventricle (anterior descending) and the left atrium and lateral and posterior portions of the left ventricle (circumflex). The right coronary artery supplies the right atrium, right ventricle and a variable proportion of the posterior portion of the left ventricle.
These large epicardial vessels give rise to smaller intramural arteries which arise at sharp angles to penetrate the myocardium, eventually forming in some species a plexus of interconnecting arteriolar anastomoses and supplying a dense capillary network.
The heart has only a short-lived capacity for anaerobic metabolism, and the metabolic needs of the myocardium can be considered almost solely in terms of oxidative metabolism. Thus, there is a very close relationship between myocardial oxygen supply and demand (Shipley & Gregg, 1945; Eckenhoff et al. 1947a;
Lichtlen et al. 1971; Berne & Rubio, 1979) and, since 65-85% of the coronary arterial oxygen is extracted by the myocardium in a single passage through the coronary vascular bed, there is little capacity for the myocardium to adjust to increased demand by increased oxygen extraction. The primary mechanism by which the heart adjusts is therefore by changes in coronary blood flow.
2.2.2 Factors Controlling Coronary Blood Flow
Unlike other arterial systems, coronary blood flow is affected by the simple fact that the heart is a mechanically active pump. The driving force is determined by the aortic diastolic rather than systolic pressure, and compressive forces generated by the contracting myocardium have a profound effect on blood flow.
However, the heart has a remarkable ability to autoregulate coronary blood flow
27 over a wide range of pressures (Bayliss, 1902) and to match blood supply closely to myocardial demands (Cross, 1964; Berne & Rubio, 1979). The contribution of myogenic and metabolic factors which contribute to this autoregulation has been reviwed; substances produced by metabolising myocardial tissue such as adenosine, adenosine 5’-triphosphate (ATP), prostaglandins, lactic acid, histamine and potassium can directly influence the responses of the coronary arteriolar smooth muscle and appear to be of primary importance in altering total myocardial blood flow to match myocardial metabolic demands (Berne, 1964).
However, the epicardial coronary arteries are relatively protected from these metabolic influences, since they are surrounded by a layer of connective tissue and have a less intimate relationship with the myocardium than the arterioles
(Winbury et al. 1969; Cohen & Kirk, 1973). In these vessels, other elements such as autonomic neural influences and circulating factors which may interact directly with the smooth muscle and endothelium assume greater significance.
2.2.3 Structure of the Epicardial Coronary Artery
There is a striking similarity between the structure of all mammalian blood vessels despite the wide range in size, structure and function. All are essentially composed of three main layers: the tunica intima, the tunica media and the tunica adventitia (Cox, 1982).
The tunica intima or inner layer consists of a single, confluent layer of endothelial cells which are in contact with the blood and which rest on a subendothelial basement membrane (Majno, 1965). It was traditionally considered principally as a non-thrombogenic surface which provided a permeability barrier
28 between the bloodstream and the vascular wall but it is now known that the endothelium plays an important role in the local control of vascular smooth muscle tone (see below). A variable subendothelial layer of collagen, elastin fibrils and some smooth muscle cells and fibroblasts separates the intima from the tunica media and is called the internal elastic lamina. The intimal region of larger coronary arteries has been reported in the dog to be thicker than the intima of comparably sized arteries elsewhere (Rhodin, 1963).
The tunica media is bounded by the internal elastic lamina on the luminal side and by the external elastic lamina on the adventitial side. It contains the vascular smooth muscle cells which are arranged predominantly in a shallow helical or circular pattern along with some connective tissue, largely elastin and collagen.
These smooth muscle cells are responsible for the development of active tone.
Additionally, they have important synthetic properties as well as the ability to migrate and proliferate (Wissler, 1968); tissue culture studies have shown that they can produce extracellular connective tissue components, such as elastin and collagen. The thickness of the smooth muscle component varies with site and nature of the vessel; the smooth muscle cells may be several layers deep in the muscular arteries, while arterioles have only a single layer of cells and capillaries have none. Furthermore, the proportion of the tunica media which is composed of connective tissue varies greatly; elastic connective tissue predominates in large
’shock-absorbing’ arteries such as the aorta, while the epicardial coronary arteries are ’muscular’ arteries and have only poorly defined elastic laminae (in fact the coronary artery has the lowest elastin to collagen ratio of any vessel of similar size
(Cox, 1982)).
29 The outermost layer of the vessel is the tunica adventitia, composed of a network of connective tissue consisting mainly of collagen and elastin fibres. It contains the vasa vasorum as well as a plexus of autonomic nerves, which do not penetrate into the media but run along the adventitial-medial border outside the external elastic lamina, relatively remote from effector smooth muscle cells
(Dahlstrom et al. 1965; Lever et al. 1965a; Malor et al. 1973; Denn & Stone, 1976)
2.3 THE ROLE OF ENDOTHELIUM
2.3.1 Traditional Concepts
The strategic position of the intima between the lumen of the blood vessels and the vascular smooth muscle suggests that it plays an important role. Indeed, the endothelium displays the features of a large albeit widely distributed organ
(1-1.5 kg in man). For many years, it was considered to be a diffusion barrier moderating vascular permeability. The complex, highly interdigitated structures formed by adjoining endothelial cell membranes formed a relatively tight interconnection (with gaps no wider than 1-2 nM) which prevented the entry of large macromolecules such as plasma proteins; movement of these proteins into the vessel wall thus occurred only by pinocytosis and was an actively regulated process
(Goldberg, 1982).
In addition to the selective transport of proteins, endothelial cells were known to possess important metabolic functions. For example, they contain lipoprotein lipase which regulates the binding of lipoprotein to the vessel wall and
30 maintains homeostasis of vascular lipids (Scow et al. 1976), and they synthesise prostacyclin which inhibits platelet adhesion to the vessel lumen and thus activation of the thrombogenic cascade (Moncada et al. 1977). This role of endothelium as a non-thrombogenic surface was further supported by the finding of proteoglycans within endothelial cells (Gimbrone, 1976) as well as heparin sulphate, antithrombin III (Chan & Chan, 1979) and plasminogen activator
(Loskutoff & Edgington, 1977). However, analyses in the late 1970s and early
1980s of substances produced by cultured endothelial cells resulted in the identification of many factors which promote thrombosis or activation of the coagulation system, such as von Willebrand factor (Jaffe et al. 1974), platelet activating factor (Zimmerman et al. 1985), thromboxane A 2 (Mehta & Roberts,
1983), thromboplastin (tissue factor) and fibronectin (Jaffe & Mosher, 1978), suggesting that the endothelium plays a more subtle role in maintaining a well-regulated balance between coagulation and fibrinolysis.
The endothelium was also known to metabolise several vasoactive factors, including noradrenaline (Hughes et al. 1969; Gillis & Pitt, 1982; Rorie & Tyce,
1985), serotonin (5HT) (Strum & Junod, 1972; Small et al. 1977), adenine nucleotides (Pearson & Gordon, 1985), bradykinin and angiotensin I (Ryan &
Ryan, 1977). Indeed, the high clearance of noradrenaline, 5HT (Thomas & Vane,
1967; Junod & Ody, 1977; Pitt et al. 1982; Gillis, 1985) and adenosine (Nees &
Gerlach, 1983; Pearson & Gordon, 1985) during a single passage through various vascular beds is due mainly to uptake and metabolism by the endothelium.
Despite this, it was not until the work of Furchgott and Zawadski in 1980 the the essential role of the endothelium in the active control of vasomotor tone was appreciated (Furchgott & Zawadski, 1980).
31 2.3.2 New Concepts
2.3.2.i Endothelial Control of Vasomotor Tone
Infusion of acetylcholine into rabbit aorta has long been known to produce vasodilatation which was thought to be a parasympathetic action mediated by smooth muscle muscarinic receptors (see below). However, Furchgott and
Zawadski showed that the endothelium was essential for this relaxation, which was in fact produced by the action of an "endothelium-derived relaxant factor" (EDRF) on the smooth muscle (Furchgott & Zawadski, 1980). This work led to an explosion of interest in EDRF which has been identified as nitric oxide (Ignarro et al. 1986; Palmer et al. 1987).
The endothelium has now been shown to release EDRF in response to a multiplicity of vasoactive substances. Substance P (Furchgott, 1983; Dudel &
Forstermann, 1988; Regoli et al. 1989), histamine (Toda, 1984), 5HT, noradrenaline (Cocks & Angus, 1983; Rubanyi & Vanhoutte, 1985), adenosine
(Rubanyi & Vanhoutte, 1985), ATP (Furchgott, 1981; Kennedy et al. 1985; Martin et al. 1985; Forstermann et al. 1987; Houston et al. 1987; Dudel & Forstermann,
1988), adenosine 5’-diphosphate (ADP) (De Mey & Vanhoutte, 1981; Houston et al. 1987) and many others (Furchgott, 1983; Vanhoutte & Rimele, 1983; Furchgott,
1984; Bassenge & Busse, 1988) have been shown to act at least partially through this mechanism, although there is considerable variation between species and even between different vessels in the same species (Vanhoutte & Miller, 1985; Nyborg
& Mikkelsen, 1990). These substances act on specific receptors on the endothelial cell surface to promote release of EDRF, which diffuses rapidly out to the smooth muscle, causing relaxation by stimulation of soluble guanylate cyclase and hence
32 elevation of cGMP (Bassenge & Busse, 1988). Removal of the endothelium by mechanical abrasion or chemical means results in loss of this relaxation. In some cases this reveals a direct vasoconstrictor response to the agent mediated through receptors on the smooth muscle, which may be of the same or different subclasses to the endothelial receptors (Burnstock & Kennedy, 1985; Burnstock, 1987;
D’Orleans-Juste et al. 1986). As a consequence, it follows that damage to the endothelium may quantitatively and qualitatively alter the response of blood vessels to locally-released or circulating agents (Busse et al. 1985). Diseases which affect the intima in vivo, such as atherosclerosis, may also change the responses to these vasoactive agents (Ginsberg et al. 1984; Kalsner & Richards, 1984).
In addition to EDRF, endothelial cells may also release an endothelium-derived hyperpolarising factor (EDHF) and an endothelium-derived constricting factor (EDCF) (Rubanyi & Vanhoutte, 1984). These have not yet been fully characterised and their function in vivo is not understood; indeed EDCF may not be one single substance and several different types of compound may produce this response. One such factor is endothelin, a 21-residue peptide which has been isolated from cultured porcine aortic endothelial cells (Yanagisawa et al.
1988), and which has been shown to cause a powerful and long-lasting vasoconstriction at extremely low concentrations (Yanagisawa et al. 1988; Larkin et al. 1989; Costello et al. 1990).
These vasoactive endothelium-derived factors can also be released in response to physicochemical stimuli. Flow-mediated shear stress, acting by viscous drag on the luminal surface, has been known to produce dilatation in the femoral artery since 1933 (see Bassenge & Busse, 1988). In 1975, Rodbard suggested that endothelial cells played a key role (Rodbard, 1975) and subsequent workers have
33 confirmed this hypothesis in a number of vessels (Smiesko et al. 1985; Bassenge &
Pohl, 1986); flow-dependent dilation has been demonstrated in the coronary arteries of conscious dogs (Hintz & Vatner, 1983; Hintz & Vatner, 1984) and Holtz and coworkers have confirmed that the mechanism is endothelium-dependent
(Holtz et al. 1983). Other factors such as pulsatile stretch and hypoxia also produce similar endothelium-dependent responses with release of EDRF and prostacyclin - for a fuller review see Bassenge & Busse, 1988.
2.3.2.H The Endothelium as a Source of Vasoactive Agents
Clearly, the blood is the most readily available source of vasoactive substances which may act on the endothelium, either because the substances are present in the circulation (e.g. catecholamines, angiotensin, vasopressin) or because they are released from aggregating platelets (e.g. 5HT, ATP, ADP), while transmitters released from perivascular nerves are unlikely to diffuse across the media and basal lamina without degredation to act on endothelial receptors (see below). However, certain vasoactive substances which are known to produce an endothelial-dependent response, such as acetylcholine and substance P, do not persist in the circulation in appreciable amounts but are rapidly broken down.
In 1985, Parnavelas showed that the enzyme choline acetyltransferase, which is responsible for the synthesis of acetylcholine, could be localised inside the endothelial cells lining the capillaries and small vessels in the rat cortex by using immunohistochemical staining at the electron microscopic level (Parnavelas et al.
1985). This technique has also been used in the past few years to show choline acetyltransferase, substance P, 5HT, vasopressin and angiotensin II inside endothelial cells from several blood vessels including the rat femoral, mesenteric
34 and coronary vessels (Loesch & Burnstock, 1988; Burnstock et al. 1988a; Milner et al. 1989). It has been demonstrated that endothelial cells can synthesise angiotensin II and histamine (Hollis & Rosen, 1972; Kifor & Dzau, 1987).
Recently, Burnstock’s group have shown that 5HT, substance P, ATP and acetylcholine can be released from the rat Langendorff heart in response to hypoxia (Paddle & Burnstock, 1974; Burnstock et al. 1988a; Milner et al. 1989) and substance P has been shown to be released from the endothelial cells of the perfused rat hindlimb (Ralevic et al. 1989) and from cultured endothelial cells
(Lincoln & Burnstock, 1990) in response to increased flow. These studies suggest that physicochemical stimuli may in fact cause local release of vasoactive agents from endothelial cells which then act on the adjacent cells to give rise to the phenomenon of flow-induced dilation.
2.4 THE ROLE OF THE AUTONOMIC NERVOUS SYSTEM
2.4.1 Sympathetic Nervous System
2.4.1.1 Anatomy
Classically, the efferent autonomic nervous system was divided into the sympathetic and the parasympathetic on anatomical, functional, and to a considerable extent, pharmacological grounds. The efferent fibres of the sympathetic system arise in the lateral grey column of the spinal cord from Tj to
L^; from each of these segments small, medullated axons emerge into the corresponding anterior primary ramus and pass via a white ramus communicans into autonomic ganglia outside the central nervous system. Here they synapse with
35 the cell bodies of the long, unmyelinated postganglionic neurones. These chains of ganglia and their nerves form the sympathetic trunks, which commence in the superior cervical ganglion beneath the base of the skull and extend to the coccyx along each side of the vertebral column. The first thoracic ganglion is often fused with the inferior cervical ganglion to form a large stellate ganglion above the neck of the first rib. Some preganglionic neurones from segments T^ to pass straight through the sympathetic chain and form synapses instead with cell bodies of postganglionic neurones in the coeliac, superior mesenteric and inferior mesenteric (hypogastric) prevertebral ganglia and plexuses in the abdomen. Fibres from these then innervate all the abdominal viscera.
The sympathetic nerve supply to the heart is diffuse and derives branches from each of the cervical ganglia and (usually) the upper four thoracic ganglia, forming a superficial and deep cardiac plexus. Branches from these pass with the coronary arteries to supply the myocardium, coronary arteries and conducting system of the heart (Gerova, 1982).
2.4.1 .ii Autonomic Neuroeffector Junction
Terminal portions of autonomic nerve fibres are extensively branched and varicose (Burnstock, 1970; Gabella, 1981). In the model of the autonomic neuroeffector junction proposed by Burnstock and Iwayama (Burnstock &
Iwayama, 1971) these fibres do not form well-defined synapses with particular smooth muscle cells like the skeletal neuromuscular junction but instead release transmitter ’en passage’ from the varicosities during conduction of an impulse.
The effector unit consists of a muscle bundle rather than a single smooth muscle cell and there are no post-junctional specialisations; individual vascular smooth
36 muscle cells in the tunica media are connected by low resistance pathways called
’gap junctions’ which allow electrotonic spread of activity (Burnstock, 1986b).
The distance between the varicosities and the effector bundles of the autonomic neuroeffector junction varies greatly between tissues, from less than
20 nm in muscular, densely innervated tissues such as the vas deferens to more than 2000 nm in the large elastic arteries (Burnstock, 1975b; Burnstock et al.
1980a). This compares to the skeletal neuromuscular junction which generally has a synaptic cleft of 20-50 nm. In the epicardial coronary artery, Gerova has estimated that the distance between the nerve terminals and the smooth muscle may be greater than 1 /un (Gerova, 1982); this wide gap would allow for both prejunctional and postjunctional modulation of neurotransmission by locally released and circulation factors. In small arteries the gap falls to 500 nm and in arterioles it is said to be around 55 - 140 nm (Malor et al. 1973), although Lever et al. have reported denuded nerve terminals approaching the smooth muscle of coronary arterioles in cats, rats, guinea pigs and rabbits in the order of 300 -
700 nm, with the smallest distance as much as 200 nm (Lever et al. 1965a). This is far greater than in the arterioles of other organs (Appenzeller, 1964; Lever et al.
1965b) and is greater than described by other workers in the coronary arteries
(Malor et al. 1973). In precapillaries and capillaries, the denuded areas of the nerve terminals run only 70 - 80 nm from the endothelial cell and face the endothelium rather than the smooth muscle cells of the precapillaries. There are even reports of fusion of the basement membranes of the nerve terminals and endothelium (Thaemert, 1966; Malor et al. 1973). True terminals have been found in several studies to be sandwiched between the vascular cells and the cardiac muscle cells, implying that transmitter release may influence both elements
(Gerova, 1982).
37 2.4.1 .iii Sympathetic Neurotransmitters and Receptors - Classical View
In his classic work reported in 1929, Cannon described the "fight or flight" response to sympathetic stimulation, noting that many vascular beds constricted while others, particularly the coronary and skeletal musclular bed, dilated (Cannon,
1929a). There was also an increase in the rate and force of contraction of the heart. In 1946, von Euler identified noradrenaline as the neurotransmitter released from sympathetic nerves (see von Euler, 1966), and later fluorescence histochemical and electron microscopic studies confirmed the widespread innervation of blood vessels with noradrenaline-containing nerves, although there was a marked variation in the pattern and density of innervation of different vessels (Burnstock, 1970; Burnstock & Iwayama, 1971; Burnstock & Bell, 1974;
Burnstock, 1975b; Burnstock et al. 1980a).
For many years studies on neurohumoral control of the vasculature were thus dominated by consideration of the role of catecholamines released from sympathetic perivascular nerves and the adrenal medulla. Differences in the responses of various vascular beds and organs to catecholamines led to the identification of subtypes of adrenergic receptors on the effector smooth muscle cells. These were classified as a and (3 receptors (Ahlquist, 1948); on the basis of pharmacological studies they were later subdivided into aj and (Linger, 1977;
Starke, 1977; Drew, 1981), and jSj and ^ (Moran, 1966; Furchgott, 1967; Lands et al. 1967a; 1967b).
Postjunctionally, a j- and a d re n o c e p to rs may be found on the smooth muscle (Drew, 1981; Langer & Shepperson, 1982; McGrath, 1982) where they mediate vasoconstriction (Vanhoutte et al. 1981). In addition, a2-adrenoceptors
38 are found prejunctionally where they inhibit transmitter release (Langer, 1979;
Drew, 1981; McGrath, 1981) and they have recently been described on the endothelium of some blood vessels where they may mediate relaxation via production of endothelium-derived relaxant factor (Miller & Vanhoutte, 1985).
Direct relaxation of the vascular smooth muscle is mediated via 0-adrenoceptors
(Vanhoutte, 1978). These are generally characterised as 02» although
0j-adrenoceptors have been described in some vessels (Vanhoutte, 1978); their existence may only be revealed after pharmacological a-adrenoceptor blockade unless the 0-adrenoceptor is the dominant receptor present. 0-adrenoceptors have also been found prejunctionally where they may mediate a positive feedback on adrenergic nerve terminals to enhance transmitter release (Osswald & Guimaraes,
1983); most evidence suggests that these are 02~adrenoceptors (Stjarne & Brundin,
1976; Westfall et al. 1979), although prejunctional 0j-adrenoceptors may exist
(Dahlof et al. 1975). Recent work has also suggested that 0-adrenoceptors may be present on the endothelium of the coronary artery in the dog (Rubanyi &
Vanhoutte, 1985).
The inotropic and chronotropic effects of catecholamines on the heart are said to be mediated via 0j-adrenoceptors. Many attempts have been made to distinguish the primary effects of sympathetic nerve stimulation on the coronary arteries from the secondary effects mediated through increased cardiac metabolism, often with conflicting results. It should be noted, however, that there is considerable evidence for the coexistence of both types of 0-adrenoceptor in one tissue (Carlsson et al. 1972; Ablad et al. 1975; Carlsson et al. 1977; Taira et al.
1977; Belfrage, 1978; Daly et al. 1978).
39 2.4.1.iv New Concepts - Cotransmission and Neuromodulation
For many years it was believed that each nerve could synthesise, store and release only one neurotransmitter: this erroneously became known as Dale’s principle. In fact, Dale’s hypothesis was rather that a single neuron released similar substances at all of its terminals (Dale, 1935). However, following several reports of inconsistencies (Burn & Rand, 1959; Gerschenfeld et al. 1960; Burn &
Rand, 1965; Su et al. 1971; Brownstein et al. 1974; Jaim-Etcheverry & Zieher,
1975), Burnstock directly challenged this theory in 1976 (Burnstock, 1976).
Subsequent experimental evidence has shown that the majority of nerve fibres can store and release more than one neurotransmitter; when these produce postjunctional actions (which are usually synergistic) by acting via their own receptors, they are termed cotransmitters (Potter et al. 1981; Cuello, 1982; Osborne,
1983; Chan-Palay & Palay, 1984; Campbell, 1987; Furness et al. 1989) (see Figure
2. 1).
As early as 1960, Burnstock and his colleagues demonstrated the existence of a population of neurones which were non-adrenergic and non-cholinergic
(Burnstock, 1972). He subsequently identified the transmitter utilised by these nerves as ATP (Burnstock, 1972; 1979a). In addition, ATP had long been known to coexist with noradrenaline in sympathetic nerves (Stjarne & Lishajko, 1966;
Geffen & Livett, 1971) and subsequent studies had demonstrated costorage and corelease of the two substances (Su et al. 1971; Langer & Pinto, 1976). Once it was known that ATP itself could act as a neurotransmitter, extensive evidence accumulated showing that ATP and noradrenaline act as cotransmitters in many tissues including the vas deferens (Sneddon & Burnstock, 1984a; Burnstock &
Sneddon, 1985; Kasakov et al. 1988; Stjarne, 1989), kidney (Schwartz & Malik,
40 1989), skeletal muscle (Shimada & Stitt, 1984), skin (Flavahan & Vanhoutte, 1986) and a number of blood vessels (see Burnstock, 1988b). Purinergic receptors have been classified as Pj and P2, activated preferentially be adenosine and ATP, respectively (Burnstock, 1978b). Subtypes of ATP receptors on vascular smooth muscle and endothelium have been termed P2X an^ ^2Y’ *n Seneral>
^2X"pur^noceptors mediate vasoconstriction while P2Y mediate relaxation
(Burnstock & Kennedy, 1985).
In recent years, it has become clear that ATP is not the only cotransmitter with noradrenaline which is found in abundance in the sympathetic nervous system. Advances particularly in immunohistochemical techniques have identified approximately 16 new putative transmitters, including peptides and monoamines as well as the purines. One of these is a 36 amino acid polypeptide called neuropeptide Y (NPY) which was first isolated from porcine brain by Tatemoto in
1982 (Tatemoto et al. 1982a; 1982b) and which is now known to be widely distributed throughout the sympathetic nervous system (Lundberg et al. 1982a;
Hokfelt et al. 1983; Lundberg & Hokfelt, 1983; Lundberg et al. 1983; Ekblad et al.
1984; Gray & Morley, 1986). The actions of NPY on blood vessels varies markedly; in the cerebral arteries (Edvinsson, 1985; Hanko et al. 1986), skeletal muscle arteries (Pernow et al. 1987) and the coronary vascular bed (Allen et al.
1983; Clarke et al. 1987), NPY appears to have a pronounced direct vasoconstrictor effect but in most other vessels its primary role appears to be as a neuromodulator; that is, it modifies the process of neurotransmission (Burnstock, 1985; 1986a).
Neuromodulation is said to occur, for example, when a substance acts prejunctionally to increase or decrease the amount of transmitter released by the nerve varicosity, or postjunctionally to alter the time course or extent of action of
41 the neurotransmitter (Burnstock, 1987) (see Figure 2.1). At a prejunctional level,
NPY exerts an inhibitory effect on the release of noradrenaline (Dahlof et al.
1985; Pernow et al. 1986) while postjunctionally, it acts in many vascular beds to enhance noradrenaline-induced vasoconstriction by a mechanism which has been shown to be calcium-dependent (Edvinsson et al. 1984; Lundberg et al. 1985).
However, in canine cerebral vessels NPY has recently been reported to inhibit contractions to noradrenaline (Suzuki et al. 1988).
2.4.2 Parasympathetic Nervous System
2.4.2.i Anatomy
Efferent fibres of the parasympathetic nervous system arise from both a cranial outflow, passing along cranial nerves III, VII, IX and X, and a sacral outflow from S2 - S4. Unlike the sympathetic system, the parasympathetic preganglionic fibres are long, unmyelinated fibres which synapse with ganglion cells lying close to, or within, the viscera supplied. The postganglionic fibres are correspondingly short and myelinated. The most important component of the cranial parasympathetic system is conveyed with the Xth cranial nerve- the vagus- and fibres from this supply the heart via the nodose ganglion as well as the lungs and the gastrointestinal system. The vagal fibres to the heart converge on the pretracheal plexus and then pass with the sympathetic nerves to the cardiac plexuses (Mizeres, 1957). The sacral outflow passes in the pelvic splanchnic nerves to supply the pelvic organs.
Woollard’s early observations of vagal innervation of the coronary arteries of the dog, based on nerve profiles remaining after sympathetic denervation
42 (Woolard, 1926) has been extended to other species, including man (Nonidiez,
1939; Hirch & Borghard-Erdle, 1961). Coronary arteries of rats, cattle and rabbits were said to contain substantial amounts of acetylcholinesterase (Navaratnam &
Palkama, 1965); the innervation was reported to be so dense in humans that Hirsch and Borghard-Erdle wrongly concluded that the coronary arterioles were supplied only by vagal fibres (Hirch & Borghard-Erdle, 1961). In fact, the vagus nerve does not even contain all the parasympathetic postganglionic fibres innervating the coronaries. In 1976, Denn and Stone observed nondegenerated fibres containing acetylcholinesterase within the coronary wall after sectioning of the vagus (Denn &
Stone, 1976) and Gerova showed that, although cholinergic fibres were seen in the coronary arterial wall, stimulation of the peripheral portion of the cut vagus nerve did not induce any change in the diameter of the conduit coronary artery (Gerova,
1982). One possible extra vagal source of these fibres is intracardiac cholinergic ganglia which have been detected within the atria (Tcheng, 1951; Jacobowitz,
1967; Yamauchi et al. 1975; Ellison & Hibbs, 1976); it has been shown that the postganglionic fibres from these ganglia pass across the atrioventricular groove
(Blomquist et al. 1987) to supply not only the myocardium but also the intramural coronary arteries (Jacobowitz, 1967; Osborne & Silva, 1970).
2.4.2.H Parasympathetic Neurotransmitters and Receptors- Classical View
The principle transmitter released from parasympathetic nerves is acetylcholine. Cholinergic nerves were originally identified by by histochemical techniques (Koelle, 1963; Bell & McLean, 1967; Burnstock & Robinson, 1967;
Eranko, 1967; Bell, 1968; 1969); these were based on the premise that functional cholinergic nerves contain high levels of acetylcholinesterase but subsequent work showed in some species a high level of acetylcholinesterase staining in adrenergic
43 nerves (Eranko et al. 1970; Eranko & Eranko, 1971; Barajas et al. 1974; Barajas &
Wang, 1975; Barajas et al. 1976). A more recent method uses immunohistochemical localisation of the enzyme choline acetyltransferase
(Eckenstein & Thoenen, 1982) which is more reliable, but even at the ultrastructural level the distinction between sympathetic and parasympathetic nerves is not readily apparant (Pace, 1977). These technical difficulties have meant that less is known about the distribution of parasympathetic nerves than sympathetic nerves; it appears that perivascular parasympathetic nerves do innervate the coronary arteries (Navaratnam & Palkama, 1965; Schenk & Badawi,
1968; Denn & Stone, 1976; Pillay & Reid, 1982) but the supply is sparser than that of sympathetic nerves (Burnstock, 1975a).
Stimulation of the parasympathetic nervous system has traditionally been considered to give rise to effects on the viscera which oppose those produced by sympathetic stimulation. In particular, sympathetic stimulation caused constriction of most vascular smooth muscle, while parasympathetic stimulation was thought to produce vasodilatation via the action of acetylcholine on muscarinic receptors on the smooth muscle (Uvnas, 1966; Burnstock, 1980b). Certainly acetylcholine had long been known to dilate the coronary arteries of many species via an atropine-sensitive mechanism (Wiggers, 1909; Smith et al. 1926; Katz et al. 1938;
Eckenhoff et al. 1947b; Herxheimer, 1960), although in vitro contraction of rabbit coronary arteries to acetylcholine was later reported (Gellai & Detar, 1974).
However, with the recent advances in our understanding of the functions of vascular endothelium, and the interaction of neural and humoral factors on vascular smooth muscle, the role of parasympathetic cholinergic neurotransmission
(and indeed almost all vasoactive agents) on vasomotor tone has been reappraised.
44 2.4.2.iii New Concepts
In their classic paper in 1980, Furchgott and Zawadski confirmed that exogenous acetylcholine did cause relaxation of the rabbit aorta but demonstrated that this was through the release of EDRF and not by the action of acetylcholine on smooth muscle muscarinic receptors (Furchgott & Zawadski, 1980).
Subsequently, vasodilation to acetylcholine in many vessels has been shown to be entirely endothelium-dependent; the smooth muscle response to acetylcholine, if any, is generally vasoconstrictor, although considerable species variation exists
(Kalsner, 1985; 1989a).
It has not been clearly shown that stimulation of parasympathetic nerves produces vasodilation via the endothelium, and indeed conceptually it is difficult to see how acetylcholine released from perivascular nerves can cross the elastic laminae and the tunica media to reach the endothelium, except perhaps in the precapillaries. It now appears that the striking hypotension produced by injection or infusion of acetylcholine into the circulation, which historically provided the most impressive support for the classification of acetylcholine as a vasodilator, is not related to cholinergic neurotransmission but to the action of endothelium-derived relaxant factor (MacAlpin, 1980). However, parasympathetic stimulation may produce vasodilation by other mechanisms, such as neuromodulation and cotransmission. For example acetylcholine may cause neuromodulation via prejunctional muscarinic receptors producing inhibition of noradrenaline release (Loffelholz & Muscholl, 1969; Langer et al. 1975; Story et al.
1990). Furthermore, like NPY and noradrenaline in the sympathetic nervous system, cotransmission occurs in parasympathetic nerves between acetylcholine and another neurotransmitter peptide, vasoactive intestinal polypeptide (VIP).
45 VIP is a 28 amino acid polypeptide, widely distributed in peripheral and central neurones (Said, 1982), which acts as a neurotransmitter causing direct relaxation of vascular smooth muscle (Larsson et al. 1976; Duckies & Said, 1982;
Lee et al. 1984; Unverferth et al. 1985; Brum et al. 1986) through specific VIP binding sites (Barnes et al. 1986). Recent work has demonstrated that it is colocalised with acetylcholinesterase (Lundberg & Hokfelt, 1983) and choline acetyltransferase (Leblanc et al. 1987) and Lundberg and his colleagues have suggested that vasodilation in many exocrine glands (e.g. nasal, salivary, pancreatic and sweat glands) is mediated by corelease of acetylcholine and VIP from postganglionic neurones in both parasympathetic and sympathetic pathways
(Lundberg et al. 1980; Lundberg, 1981; Lundberg et al. 1982b). The best documented evidence for cotransmission of acetylcholine and VIP is in the cat salivary gland, where the transmitters are costored in parasympathetic nerves
(probably in separate vesicles) and coreleased on nerve stimulation. At low frequencies of stimulation acetylcholine is preferentially released, causing increased salivary excretion from acinar cells but only minor vasodilation, while at higher frequencies VIP is released and produces marked vasodilation (Bloom & Edwards,
1980; Lundberg, 1981). VIP may also neuromodulate the actions of acetylcholine, increasing both the postjunctional effect on acinar cell secretion and the release of acetylcholine from nerve varicosities via prejunctional receptors (see Burnstock,
1987).
46 2.4.3 Sensory-Motor Nervous System
2.4.3.1 Anatomy
As well as the efferent sympathetic and parasympathetic fibres, there is an afferent component to the autonomic nervous system. Afferent fibres conveying sensory information (e.g. pain) may pass from the viscera along both the sympathetic and parasympathetic pathways and reach the central nervous system without synapsing. However, it has been known for many years that stimulation of sensory fibres in the dorsal roots of the spinal column, or stimulation of the distal end of cut sensory nerves, could give rise to a motor response producing vasodilation in the skin; this phenomenon was called antidromic vasodilation. In
1927, Lewis described the "axon reflex" in which antidromic impulses in primary afferent sensory neurones pass down collateral branches and cause vasodilation by distal release of the sensory neurotransmitter (see Dale, 1935; Burnstock, 1977).
More recently, work with the sensory neurotoxin, capsaicin, has demonstrated that these sensory neurones not only initiate an axon reflex via collateral branches, but can also release the stored transmitter from the primary sensory nerve terminal itself, allowing a direct motor action at the site of the sensory stimulus (Maggi &
Meli, 1988). The extent of peripheral nerve endings of primary sensory neurones found in association with blood vessels is striking (Dhital & Burnstock, 1987).
ii Sensorv-motor Neurotransmitters and Receptors
Substance P, the oldest-known neuropeptide, was discovered by von Euler and Gaddem in 1931 (von Euler & Gaddum, 1931). It is widely distributed in the central and peripheral nervous system; its localisation in cell bodies of sensory
47 ganglia (e.g. jugular, nodose and trigeminal) as well as nerve fibres in the dorsal horn of the spinal cord and the periphery led Lembeck to first suggest that substance P was the transmitter in primary sensory neurones (Lembeck, 1953).
Subsequent studies substantiated this hypothesis and demonstrated loss of substance
P-immunoreactivity following treatment with capsaicin (Duckies & Buck, 1982;
Furness et al. 1982; 1984a; Saria et al. 1985; Duckies, 1986). Specific receptor sites for substance P have been identified on vascular smooth muscle and endothelium
(Regoli et al. 1984a; 1984b; 1989).
In addition to substance P, other neurotransmitters have been shown to be present in capsaicin-sensitive sensory nerves. Calcitonin gene-related peptide
(CGRP), a 37 amino acid neuropeptide, is also widely distributed in sensory neurones in the central nervous system and the periphery (Rosenfeld et al. 1983;
Hanko et al. 1985; Mulderry et al. 1985; Wharton et al. 1986) and in some species appears to be extensively costored with substance P (Gibson et al. 1984; Gibbins et al. 1985; Lee et al. 1985a; Lundberg et al. 1985; Gulbenkian et al. 1986). Like substance P, CGRP is a potent vasodilator and specific CGRP-binding sites have been reported in the media and intima of rat coronary arteries and aorta (Sigrist et al. 1986). CGRP may also modulate the effects of substance P, potentiating release of substance P and inhibiting its breakdown (Le Greves et al. 1985; Oku et al. 1987).
In 1959, Holton demonstrated that ATP was released on antidromic stimulation of sensory nerves (Holton, 1959). ATP is now known to coexist with substance P in sensory nerves (Burnstock, 1977) and to be an important sensory neurotransmitter (Jahr & Jessel, 1983; Salt & Hill, 1983; Fyffe & Perl, 1984). It is possible in the future that ATP will be found to play a role in mediating cardiac
48 pain (angina pectoris) as has been suggested for adenosine (Crea et al. 1990), and to produce the antidromic coronary vasodilation described by Lewis (Lewis, 1947).
2.4.4 Intrinsic Nervous System
Many studies of different organs have shown that after destruction or removal of the extrinsic nerve supply certain populations of ganglia and nerve fibres persist. These nerves form an ’intrinsic’ nervous system, which has been most extensively characterised in the gut by Furness and Costa (Furness & Costa,
1987). They have identified cell bodies in intramural myenteric and submucous ganglia which are immunoreactive for NPY and which project both to the underlying circular smooth muscle and to the mucosa (Furness et al. 1983).
Furthermore, these intrinsic NPY-immunoreactive neurones in the gut also appear to store VIP (Ekblad et al. 1985) and, in the guinea-pig, may also contain choline acetyltransferase (Furness et al. 1984b). The neuropeptides substance P (Furness et al. 1980) and CGRP (Clague et al. 1985; Gibbins et al. 1985) have also been identified in capsaicin-insensitive intrinsic neurones of the gut, although they do not appear to be colocalised in the same fibres (Mulderry et al. 1988). VIP- and substance P-immunoreactive intrinsic fibres, but not CGRP-immunoreactive fibres, appear to supply gastrointestinal blood vessels (Holzer et al. 1980; Cuello et al. 1981; Galligan et al. 1988). While the exact function of these intrinsic nerves is unknown, they seem to be responsible for a wide range of functions including the control of motility, secretion, absorption and local blood flow (Gershon, 1981;
Costa et al. 1985; Hellstrom et al. 1985).
Intrinsic neurones have also been identified in the heart (Hassall &
Burnstock, 1986). Hassall and Burnstock have identified a population of
49 intracardiac neurones which do not contain dopamine beta hydroxylase (a marker for sympathetic adrenergic neurones) but which are amine (5HT)-handling; tissue culture studies of these ganglia from neonatal animals have shown that they also contain NPY (Hassall & Burnstock, 1984; Hassall & Burnstock, 1987). In the guinea pig, Baluk and Gabella recently further identified a few intrinsic neurones which were immunoreactive for substance P (Baluk & Gabella, 1990). Again, the projection and function of these intrinsic neurones is unknown.
As can be seen from these references, much of the work on intrinsic neurones has been undertaken during the period when the work in this thesis was carried out. Intrinsic neurones have now been identified in many more organs, including the bladder (James & Burnstock, 1988) and trachea (Springall et al. 1988) and recent studies using denervation by transplantation suggest an involvement of intrinsic neurones in perivascular innervation in the respiratory tract (Springall et al. 1988).
2.4.5. Neuronal Plasticity
It is evident that the autonomic nervous system is far more complex than was previously understood. Many of the neurotransmitter substances described above occur in combinations which make it difficult to ascribe to them a definite classification under the traditional divisions of the nervous system. Rather, it appears that different sets of autonomic neurones have a unique ’chemical coding’ specific for each type of nerve fibre and for its neuroeffector target (i.e. other neurons, blood vessels, organs etc.). But the factors which control this chemical coding are as yet poorly understood.
50 It is known that nerve fibres in the autonomic nervous system may change the neurotransmitters expressed within them under certain circumstances (Landis &
Keefe, 1983), such as during development or after heterotopic transplantation of undifferentiated cells (Landis, 1980; Le Douarin et al. 1975). Furthermore,
Burnstock has shown that mature autonomic and sensory ganglia may also change the expression of their neurotransmitters and can spread to reinnervate denervated organs and restore the original nerve density (Burnstock et al. 1978a), although the reinnervating nerve types may be significantly different from the original population (Evans et al. 1979a).
Changes also occur in perivascular nerve density in aging and disease.
Most studies on perivascular innervation during development or aging have focussed on noradrenergic nerves (see Cowen & Burnstock, 1986 for review), although some recent studies have shown developmental changes in the peptidergic perivascular nerves in several different blood vessels and species (Dhall et al. 1986;
Dhital et al. 1988; Mione et al. 1988; Scott & Woolgar, 1988). As yet, there are few pharmacological studies on the functional significance of these changes in innervation. Work by Duckies suggests that vascular adrenergic neuroeffector function may not parallel the age-related decrease in adrenergic innervation
(Duckies & Banner, 1984; Duckies et al. 1985) but no data are available on changes in peptidergic and purinergic responses.
Growth of non-sympathetic nerves after sympathetic denervation has been demonstrated in many studies (Malmfors et al. 1971; Evans et al. 1979a; Gibbins &
Morris, 1988; Rosier & Waterson, 1988). This plasticity of autonomic nerves is under the influence of several factors, such as nerve growth factor (NGF)
(Raynaud et al. 1988) which attracts the nerves to the target cells (Levi-Montalcini
51 & Angeletti, 1963); in tissues which are normally densely innervated, NGF is present in larger amounts (Chamlet et al. 1973; Burnstock, 1981). Sympathetic and sensory neurones have both been shown to utilise, and compete for, this same trophic factor (Levi-Montalcini & Angeletti, 1968; Thoenen & Barde, 1980;
Kessler et al. 1983; Korsching & Thoenen, 1985) but as yet almost nothing is known about the factors which may control trophic responses in peptidergic nerves.
52 Neuromodulation Cotransmission
FIGURE 2.1
Schematic diagram illustrating the principles of cotransmission and neuromodulation between autonomic nerve varicosities and postjunctional smooth muscle. CHAPTER 3
GENERAL METHODOLOGY
3.1 PHARMACOLOGY
3.1.1 Choice of Preparation
Most of the experimental work on coronary arterial responses has been carried out in the dog. Further use of the same species would give the advantage that many of the parameters required to examine this vessel in vitro and much of the basic pharmacology is already well characterised, so newer ideas may be explored more quickly. However, there are several disadvantages: the dog coronary artery does not necessarily give the same responses as other species (e.g. endothelial responses to acetylcholine - see Chapter 4); it is difficult to manipulate the dog experimentally, for example to study aging or atherosclerosis; and it is expensive.
For many years, the Langendorff preparation has been used to examine pharmacological responses of the intact heart; for this smaller animals such as the rat and rabbit are generally used since they are cheaper and easier to manipulate experimentally. Indeed, one of the earliest studies examining responses of the heart to a newly-discovered peptide neurotransmitter (NPY) was performed in the rabbit Langendorff preparation (Allen et al. 1983). Furthermore, there is a strain of rabbit - the Watanabe hereditary hyperlipidaemic - which is an excellent animal
54 model of human familial hypercholesterolaemia, lacking LDL receptors and developing atherosclerosis despite a normal diet (Watanabe, 1980). Thus I chose in this thesis to examine the pharmacological responses of the rabbit.
The responses of the Langendorff preparation reflect the responses of the entire coronary vascular bed, myocardial metabolism and physical compressive forces, as well as stimulation of intrinsic nerves in the heart. While all these mechanisms operate in vivo and are therefore important in the responses of the intact animal, a better understanding of the factors controlling a specific blood vessel may be obtained by examination of the isolated vascular preparation. I wished to focus on the responses of the epicardial coronary arteries to examine the effects on the smooth muscle and endothelium of both the "classical" neurotransmitters, noradrenaline and acetylcholine, and the more recently discovered purinergic and peptidergic neurotransmitters.
Over many years, several different in vitro systems for recording vascular smooth muscle responses have been developed: helically cut preparations (Furchgott
& Bhadrakom, 1953; Bohr et al. 1961), perfusion of intact vessels (de la Lande &
Rand, 1965), longitudinal segments (Hughes & Vane, 1967) and ring segments
(Bevan, 1962; Nielsen & Owman, 1971; Bevan & Osher, 1972). Each of these preparations has some disadvantages. Vascular smooth muscle is generally arranged circularly or in a shallow helix (with the exception of a few blood vessels such as the hepatic portal vein which contains many longitudinal fibres) and control of blood flow in vivo depends more on the diameter than on the length of the vessel. Thus it is not appropriate to study a longitudinal vascular preparation as it is for the vas deferens. In the past, helically cut preparations were often used despite the technical difficulties of cutting spirals at the optimal angle. However
55 since the work of Furchgott and Zawadski highlighted the importance of the endothelium (Furchgott & Zawadski, 1980), damage to the endothelial cells which ineviably occurs during preparation of these strips is now known to be a serious criticism of this model. Perfusion of intact vessels preserves the integrity of the endothelium although it requires quite a long preparation and is unsuitable for vessels which have many side branches, since some loss of pressure always occurs through these. Its disadvantage for the examination of coronary artery responses is the difficulty of removing the surrounding adherent myocardium from a long segment of epicardial vessel without disruption of part of the preparation. Thus I have chosen to examine ring preparations of the rabbit epicardial coronary artery; in these the integrity of the vessel wall is better preserved with less disruption of the muscle, nerves and endothelium. The circumflex branch (diameter approximately 350 /im) was used since this was found to provide the longest homogenous segment of the coronary arterial system in the rabbit. When the work for this thesis started, I could find no reports in the literature where this preparation was used, although perfused isolated rabbit coronary arteries surrounded by a slab of myocardium had been used in one earlier study by de la
Lande (de la Lande et al. 1974).
3.1.2 Method
Male New Zealand white rabbits (2.6-3.8 kg) were used unless otherwise indicated. They were killed by barbiturate anaesthesia and exsanguination. The heart was rapidly removed and the left circumflex coronary artery carefully cut out in a slab of myocardium and placed into modified Krebs solution of the following composition (mM): NaCl 133, KC1 4.7, NaHCO^ 16.3, NaHPO^ 1.35, glucose 7.8, MgSC> 4 0.61, CaC^ 1.89 (modified from Bulbring, 1953) (see Chapter
56 3.7.1). The concentration of CaC^ used is lower than in the standard
Bulbring-modified Krebs solution and was found in preliminary experiments to allow better relaxation of the raised-tone preparations to most agonists. A similar finding has been noted for coronary smooth muscle responses to catecholamines
(Zuberbuhler & Bohr, 1965). The solution was gassed with 95% oxygen and 5% carbon dioxide, which maintained the pH at 7.2-7.4. The vessel was pinned at each end onto Sylgard rubber and dissected free of the surrounding adherent myocardium using fine iridectomy scizzors (John Weiss and Sons, London) under a dissection microscope. Great care was taken not to pull on or stretch the preparations.
Ring segments (approximately 4 mm in length) were cut and carefully threaded onto two fine tungsten wires (diameter 125 nm) which had been prepared as shown in Figure 3.1. The ring segments were mounted horizontally by a modification of the method of Bevan and Osher for the examination of small blood vessels (Bevan & Osher, 1972). A segment of soft rubber tubing hanging on the lower wire was slipped over a rigid metal support and the whole system was then lowered into 6 ml water-jacketed organ baths containing oxygenated modified
Krebs solution maintained at 37°C. The thread attached to the top tungsten wire was hooked onto a force transducer and the rigid support was fixed to a clamp; the transducer could be moved smoothly up or down by a micrometer to apply tension to the preparations. For experiments using peptides, the glass organ baths were coated with silicone to reduce peptide adhesion and, where indicated, indomethacin (10 /xM) was present to prevent fading of the responses throughout the course of the experiment.
57 The segments were allowed to equilibrate for at least 1.5 h, during which time they were washed repeatedly and the resting tension was adjusted to 1 g.
This optimal resting tension was determined from preliminary experiments to provide maximal contractions to 30 mM KC1 without disruption of the endothelium (Duckies & Banner, 1984). Subsequently, the preparations were repeatedly exposed to 30 mM KC1 until successive contractions were reproducible
(usually three to four contractions). For relaxation responses, the tone of the preparation was raised by the addition of 60 /d of 3 M KC1 to the 6ml organ baths to give a final concentration of 30 mM KC1. This was found to give contractions of all the ring preparations which were stable for longer than llOmin and which were reproducible more than eight times in a single preparation. Following washout, preparations contracted with KC1 relaxed promptly back to the basal tension. Other constrictor agonists commonly used to raise the tone of vascular preparations, such as histamine and PGF 2 a , did not consistently give stable or reproducible contractions unless the endothelium was damaged, and noradrenaline does not appreciably contract this vessel (see Chapter 6).
Isometric tension generated by the vascular smooth muscle was measured using a force displacement transducer (Model UF1, Devices, Welwyn Garden City,
U.K.) and recorded on a Grass ink-writing oscilloscope (Model 79). Changes in tension generated in isolated vascular preparations have been shown to correlate with changes in diameter of the vessel (Nagata et al. 1985). Tension is generally expressed by pharmacologists in terms of grams weight (g), although it should be expressed in terms of force (e.g. Newtons, N). In this thesis, tension in expressed in g but conversion to N may be made: 1 g is equivalent to 9.8 mN.
58 Scanning electron microscopy, performed by Marie Phillips, was used in preliminary experiments to examine the endothelial surface of the ring segments of the coronary artery after dissection, after insertion of the tungsten wires, after stretching under the resting tension of 1 g and after exposure to 60 mM KC1 and demonstrated that the endothelium was not removed by these manoeuvres. Where specifically required in the experiments, the endothelium was removed by threading a fine silk surgical thread (6/0) on a blunted, non-cutting, round bodied needle through the lumen of the ring preparations. Haematoxylin and eosin staining and scanning electron microscopy of the preparations confirmed that removal of the endothelium by this method was successful.
3.1.3 Administration of Drugs
Drugs were added to the Krebs solution in the organ baths directly into a gentle stream of the bubbling gas to assist rapid and even dilution. The total volume of drug solution added did not exceed a maximum of 2.5% of the tissue bath volume. Drugs were added cumulatively to the bath if the responses to this were no different than responses to a single addition equivalent to the final concentration of the drug. Responses were allowed to plateau before the next addition of any drug; those drugs which gave rapid responses (e.g. acetylcholine) were added in half-log increments, while those which took longer to reach a plateau (e.g. catecholamines) were added in full-log increments. If the responses demonstrated tachyphylaxis, the drug was added as single doses followed by washout and the preparations were allowed to fully recover between doses. All drugs were dissolved in distilled water, unless otherwise indicated, to provide a stock solution which was then serially diluted in Krebs solution. Catecholamine solutions were dissolved in 10“^ M ascorbic acid to inhibit oxidation.
59 3.2 HISTOCHEMISTRY
3.2.1 Fluorescence Histochemistry for Noradrenaline
3.2.1.1 Introduction
The Falck-Hillarp formaldehyde method for localising biogenic amines allowed transmitter substances to be demonstrated within nerves for the first time
(Falck, 1962b; Falck et al. 1962a). Lindvall and Bjorkland improved the sensitivity of the technique with the introduction of glyoxylic acid (Axelsson et al.
1973; Lindvall & Bjorklund, 1974a) which was better than formaldehyde at promoting fluorescence in the reactions with catecholamines and indoleamines, particularly for the primary amines noradrenaline and dopamine. Falck has estimated that the detection limit for noradrenaline in the terminal varicosities using this technique is approximately 5x10“^ fM (Falck et al. 1982).
There are two steps in the formation of fluorophores from catecholamines with glyoxylic acid: an initial condensation yields a weakly fluorescent
1,2,3,4-tetrahydroisoquinoline-l-carboxylic acid derivative, which is transformed into a strongly fluorescent 2-carboxymethyl-3,4-dihydroisoquinolium derivative by an intramolecularly catalysed reaction with glyoxylic acid (Lindvall et al. 1974b).
Noradrenaline and dopamine yield high levels of florophores, while the secondary amine adrenaline and the indoleamine 5-hydroxytryptamine give little aldehyde-induced fluorescence (Lindvall & Bjorklund, 1974a). The fluorophores from noradrenaline in this reaction are excited maximally at a wavelength of 415 nm and have an emission maximum of 475 nm (Lindvall & Bjorklund, 1974a).
60 Furness and Costa used aqueous solutions of glyoxylic acid on whole mount stretch preparations to demonstrate catecholamines along the entire course of peripheral adrenergic nerves (Furness & Costa, 1975). These preparations require a slight excess of water during the dehydrogenation reaction which is then driven off during development of the fluorophore. Further contact with water must then be prevented (Furness & Costa, 1975). De la Torre and Surgeon later described a similar method for cut sections - the "SPG method"- using a sucrose-phosphate-glyoxylic acid solution (De la Torre & Surgeon, 1976).
Modifications of these techniques were used in this thesis.
3.2.1 .ii Method for Whole Mounts
Catecholamine-containing nerves in the periadventitia of blood vessels were examined as whole mounts. Vessels were cleaned carefully of excess fat, connective tissue and adherent muscle, slit open lengthwise and pinned onto small pieces of Sylgard before being incubated at room temperature in 2% wt/vol glyoxylic acid in 0.1 M phosphate buffered saline (PBS) at pH 7.5 (see Chapter
3.7.1) for 1.5 h. They were counterstained with a solution of 0.005-0.01% pontamine sky blue/0.1% DMSO in 2% glyoxylic acid for 5 to 10 min to reduce background fluorescence (Cowen et al. 1985). After excess pontamine sky blue was rinsed off with glyoxylic acid solution, the preparations were immediately stretched onto clean glass microscope slides with the adventitial side uppermost and allowed to dry until they were just translucent. The slides were then incubated in a dry oven at 100°C for 4 min before being mounted under coverslips in liquid paraffin.
61 3.2.1 .iii Method for Sections
For solid organs where whole mount stretch preparations could not be used, the tissues were rinsed in phosphate buffered saline, blotted dry and embedded in a mountant (OCT compound: Tissue-Tek) on small squares of cork before being rapidly frozen in isopentane cooled in liquid nitrogen. The cork-mounted preparations could then be stored for a period up to a few weeks in liquid nitrogen. Subsequently, 10 /im sections were cut using a -30°C microtome cryostat, and mounted on microscope slides. The sections were allowed to dry and were then dipped into ice cold sucrose-phosphate-glyoxylic acid (SPG) solution
(see section 3.7) approximately five times for one second each time (De la Torre &
Surgeon, 1976). The number of times a specimen was dipped varied with the tissue and was critical for good visualisation of the catecholamine fluorescence without excessive background staining. Excess fluid was quickly removed using absorbant paper and the slides were dried under a flow of cool air for at least 5 min. They were then incubated in an oven at 80°C for exactly 5 min before the sections were mounted under coverslips in liquid paraffin for examination under the microscope.
3.2.1.iv Assessment of Innervation
The sections treated with glyoxylic acid were viewed and photographed with a Zeiss photomicroscope equipped with an epi-fluorescence condenser III RS.
A high-pressure mercury light source (Osram HBO 50) with exciter filter (BP
436/8), barrier filter (LP 470) and dichromatic beam splitter (FT 460) were used.
Sections could be stored at 4°C for at least two months without loss of fluorescence but in general they were viewed within one or two days of
62 preparation. After prolonged storage, the fluorescence would become diffuse and dim.
3.2.2 Immunohistochemistry
3.2.2.I Introduction
The technique of immunohistochemistry provides an extremely sensitive and specific technique for localizing a wide variety of molecules and is extensively used to localise peptide neurotransmitters as well as enzymes involved in the synthesis of the classical neurotransmitters, such as dopamine /?-hydroxylase for noradrenaline and choline acetyltransferase for acetylcholine. The principle of immunohistochemistry is based on the fact that an antibody will bind exclusively with the antigen that stimulated production of that antibody. Tagging of the antibody then allows the bound complex, and hence the antigen, to be localised.
Antibodies are raised by immunising an animal with antigen and are usually of the IgG class. Small, non-immunogenic molecules (haptens) may be rendered immunogenic by coupling them to ’carrier’ proteins (e.g. bovine serum albumin). Furthermore, antibodies can be prepared against other antibodies; this is the basis of the indirect immunohistochemical technique which was used in this thesis (Coons et al. 1955). In this technique, antibodies are raised against the whole IgG class of the particular species which was used to produce the first antibody; addition of this ’second layer’ antibody will bind selectively to the first layer antibody-antigen complex. Conjugation of the second layer antibody, for example to a fluorophore, then allows the complex to be visualised. The most commonly used fluorophore, fluorescein isothiocyanate (FITC), is excited
63 maximally at a wavelength of 490nm and possesses an emission maximum of
510-520 nm (Larsson, 1981).
3.2.2.H Method for Whole Mounts
Blood vessels were examined as whole mounts. They were slit open longitudinally and pinned out onto small pieces of Sylgard using stainless steel micropins before being fixed in 4% wt/vol paraformaldehyde in PBS at pH 7.4 for
1.5 h. The vessels were then dehydrated in a graded series of alcohols (80%, 90%,
100%, 90%, 80%) by immersion for 10 min in each in turn. After three 10 min washes in PBS containing the non-ionic detergent 1% Triton X-100, they then were incubated overnight (12-18 h) in a moist chamber at room temperature with primary antiserum raised in rabbits against the substance being examined. This
’first layer’ antibody was diluted in antibody diluting medium (see 3.7 Preparation of Solutions) to an optimal concentration determined in preliminary experiments.
The tissues were then again washed three times for lOmin with PBS containing 1%
Triton X-100 before incubation for 1 h with the second layer antibody, goat-anti-rabbit immunoglobulin (IgG) conjugated to fluorescein isothiocyanate
(FITC), diluted 1:50 in antibody diluting medium. After this second antibody was rinsed off with PBS, the vessels were counterstained with 0.005-0.01% pontamine sky blue/0.1% DMSO in PBS for 5-10 min to reduce background fluorescence
(Cowen et al. 1985), unpinned, and stretched onto clean glass slides. They were allowed to dry and mounted under coverslips with PBS/glycerol (Citifluor) for subsequent examination.
Dehydration-clearing-rehydration methods and Triton X-100 are used to render the fixed tissues more permeable to antibodies (Larsson, 1981). Bovine serum
64 albumin, lysine and Triton X-100 were included in the antibody diluting medium
(see section 3.7) to minimise background fluorescence (Larsson, 1981).
3.2.2.iii Method for Sections
Tissues that could not be prepared as whole mounts were fixed by immersion in 4% wt/vol paraformaldehyde in PBS, pH 7.4, for 1.5 h. After this, they were rinsed six times in 7% sucrose/azide PBS (see Chapter 3.7) for 30 min each before being placed in 7% sucrose/azide PBS overnight. They were then rinsed, blotted dry, embedded in a mountant (OCT compound) and frozen in cooled isopentane prior to storage in liquid nitrogen. Subsequently, 10 fiin sections were cut on a cryostat as for fluorescence histochemistry. These sections were mounted on poly 1-lysine or gelatin subbed slides (see Chapter 3.7) and allowed to dry thoroughly. They were incubated with the first layer antibody overnight in a moist chamber as described above for whole mounts. After three 10 min washes in PBS (without Triton X-100), they were then incubated with the second layer antibody as above. Finally, the sections were rinsed, counterstained as for whole mounts, and mounted with PBS/glycerol (Citifluor) under coverslips for examination .
3.2.2.iv Assessment of Innervation
Specificity of the immunoreactivity was tested in preliminary experiments by preabsorbing the primary antibody with excess high concentrations (up to 10”^
M) of antigen; in preparations treated with preabsorbed antibody no immunoreactivity was seen. Interference by non-specific binding was excluded by the use of non-immune serum in place of the first layer antibody.
65 All immunohistochemical specimens were examined and photographed using a Zeiss fluorescence photomicroscope equipped with an epi-fluorescence condenser III RS, using the Osram HBO 50 high pressure light source. An exciter filter (BP 450-490), barrier filter (KP 560) and dichromatic beam splitter (FT 510) were used to optimise the system for fluorescein. Histochemical preparations from control and sympathectomised rats (see Chapters 10 and 11 of this thesis) were coded to enable them to be assessed independently without prior knowledge of treatment by me and by my coworker in these experiments, Judy Aberdeen. The subjective assessment of the preparations was in some instances checked by Judy
Aberdeen using a computerised image-analysis system (Seescan Imaging) linked to a Panasonic camera mounted onto the Zeiss photomicroscope.
The slides could be stored for at least six weeks at 4°C without loss of immunofluorescence. However, the fluorescence faded when exposed to light and photographing the sections tended to cause a local loss of fluorescence if prolonged exposures were required. This occured more frequently with Ilford XPI 400 film, which was used until the introduction of Kodak Tmax P3200 film in 1989. This
Kodak film allowed a much shorter exposure time while giving high definition photographs of the slides.
3.3 ASSAY
Tissues in which noradrenaline or peptide concentrations were to be measured were dissected out, rinsed briefly in oxygenated Krebs solution, blotted dry and rapidly frozen in liquid nitrogen until assay. For comparison with immunohistochemical findings, the same specimens of the tissues were taken from
66 a separate group of animals. Control and treated tissues were assayed at the same time.
3.3.1 Inhibition Enzyme-linked Immunosorbant Assay (ELISA) of Peptides
The peptides neuropeptide Y (NPY), calcitonin gene-related peptide
(CGRP), vasoactive intestinal polypeptide (VIP) and substance P were extracted and assayed by Dr. Pam Milner using an inhibition enzyme-linked immunosorbant assay (Milner et al. 1987). The tissue samples were removed from the liquid nitrogen, unwrapped on ice, and carefully weighed or measured. The samples were then plunged into 0.5 M acetic acid in polypropylene tubes in a boiling water bath for 15 min (approximately 20 mg tissue/ml acetic acid) to extract the peptides. They were homogenised and centrifuged for 30 min at 3,500 g; the supernatant was pipetted off, lyophilised and, when necessary, stored at -20°C.
For the assay, Dynatech polystyrene microelisa plates were coated with the appropriate peptide diluted in a 0.1 M bicarbonate-carbonate buffer, pH 9.6, containing 0.2% sodium azide (see Chapter 3.7). These assay peptides were stored at a 1:10 concentration and 10 yu.1 of each were diluted as follows: up to 25 ml for substance P; up to 7.5 ml for VIP; and up to 12.5 ml for CGRP and NPY. 100 jul of the peptide solution was added to each well and incubated for 18 h at 4°C to allow the peptide to adhere to the wells. When the plates were ready, the lyophilised samples were reconstituted in polypropylene (not polystyrene) tubes in
PBS containing 0.05% Tween 20, 0.1% gelatin, 0.02% sodium azide and 100 kallikrein inhibitory units/ml aprotinin (’sample buffer’) at O0C, then centrifuged for 10 min at 3,500 g to remove sediment. Peptide standards were also prepared
67 using the sample buffer at a range of dilutions (0, 5, 10, 25, 50, 100, 250, 500,
1000, and 10,000 pg/well).
The coated ELISA plates were washed three times in PBS containing 0.05%
Tween 20 (PBS-Tween, see Chapter 3.7) to remove excess peptide and incubated for one hour at room temperature in PBS-Tween containing 0.1% gelatin to coat all remaining areas and prevent non-specific binding. The plates were emptied by vigorous inversion onto absorbant paper. Fifty fi\ of extracted sample or standard was added to each well (each sample was assayed in triplicate at two dilutions) followed by 50 fi\ of antiserum raised in rabbits against the peptide, diluted
1:12,500 in sample buffer. The plates were then covered and incubated at 4°C for three days. The principle of this assay is that the antiserum will bind to the peptide in the tissue sample; any surplus unbound antiserum will also bind to the peptide coating the wells. Thus, the higher the concentration of peptide in the tissue sample, the more the antiserum is bound to the sample and the less it is available for binding to the coated plates. After the incubation, the plates were washed three times with PBS-Tween containing 0.02% sodium azide to remove the tissue sample and any antiserum bound to it. Goat anti-rabbit IgG conjugated to alkaline phosphatase, diluted 1:500 in sample buffer, was added to each well and the plates were incubated in a humid chamber at 37°C for two hours. This second layer antibody will bind to any primary antiserum which was conjugated to the peptide coating the wells and hence was not removed when the wells were washed.
The principle is similar to that described under immunohistochemistry. After three washes in PBS-Tween to remove excess goat anti-rabbit IgG, the plates were then washed once in a glycine buffer containing 0.001M MgC^ and 0.001M
ZnC^, pH 10.4 (see Chapter 3.7). 100 /d p-nitrophenol phosphate (1 mg/ml glycine buffer) was finally added to each well and left at room temperature; this
68 compound interacts with the conjugated alkaline phosphatase to produce an intense colour.
The absorbance at 405 nm was read on a Titertek Multiscan automatic spectrophotometer. The readings obtained with the standards were plotted against peptide concentration on logarithmic graph paper to give a straight line from which the sample peptide values were obtained. Wells with all solutions except the coating peptide, antiserum to the peptide or anti IgG-alkaline phosphatase were run through the assay as controls and gave blank values. The minimal detectable concentration was less than 0.5 fmol and the intra-assay variability was around 4%.
3.3.2 HPLC with Electrochemical Detection of Noradrenaline
Noradrenaline levels in the various tissues were determined by Dr. Jill
Lincoln using high performance liquid chromatography with electrochemical detection (Lincoln et al. 1984). The tissue samples were removed from the liquid nitrogen onto ice, weighed or measured and then homogenised in 500 j*l 0.1 M perchloric acid containing 0.4 mM sodium bisulphite and 25 ng/ml dihydroxybenzylamine (DHBA). The DBHA was added as an internal standard; it does not exist biologically but behaves in the same way as noradrenaline or dopamine in terms of its extraction by alumina and its electrochemical properties.
The addition of a known amount of DBHA at this stage thus allows correction for any losses of noradrenaline during the extraction procedure.
After low-speed centrifugation, the supernatant was pipetted off and subjected to alumina extraction using the method of Keller et al. (Keller et al.
1976) with a modification of the addition of 0.1 mM EDTA in the solution used
69 for washing the alumina. This extraction separates the catecholamines from the indoleamines, 5HT and 5HIAA, which are very slow to come off the chromatography system used. Noradrenaline, DHBA and dopamine bind to alumina at neutral pH; when the alumina is washed the 5HT and 5IAA are removed and the catecholamines can then be eluted with 0.1 M perchloric acid containing 0.4 mM sodium bisulphite, which protects the amines from oxidation during the process.
Separation of the compounds was by reverse phase paired-ion high performance liquid chromatography. In reverse phase chromatography, the most polar compounds bind to the column least and thus come off quickest. The mobile phase consisted of 0.1 M sodium dihydrogen phosphate, 0.1 mM EDTA, 5 mM hepatone sulphate (pH 5.0) containing 13% methanol. This liquid phase produced overall charges (i.e. polarity) of the compounds which gave good separation; the methanol reduced the polarity of all the compounds and thus speeded up the run.
EDTA prevents electrochemically active metal ions released from the steel tubing from binding to the column and producing large contaminating peaks on the chromatogram. Hepatone sulphate was used as the ion-pair agent; it alters the polarity of noradrenaline, DBHA and dopamine but to differing extents - dopamine is affected most and thus binds more strongly to the column. The concentration of hepatone sulphate used in the mobile phase was sufficient to allow good separation of the compounds without causing the dopamine to bind excessively and hence reduce the speed and sensitivity of the technique.
Chromatography was carried out at a flow rate of 2.0 ml/min on a radial pak
/x-bondpak C-18 reverse phase column.
70 Noradrenaline and DBHA were detected electrochemically using a glassy carbon electrode set at a potential of +0.72 V. The eluent from the column flowed through a cell containing this electrode; on passing through the potential difference, electrochemically active compounds such as the catecholamines are oxidised resulting in the formation of electrons and hence producing a current.
The magnitude of the current, which is proportional to the concentration of the compound, and the elution time was compared with standards of known concentration to give the sample noradrenaline concentration. Theoretically, the area under the peak should be measured, but for the sharp peaks produced by the catecholamines, peak height was found to be equivalent and was used in this thesis.
3.4 CHEMICAL SYMPATHECTOMY
To determine the potential for plasticity in peptidergic nerves, I wanted in this thesis to examine changes in peptidergic innervation after long-term destruction of the normal sympathetic nerves. Organs innervated with nerve fibres containing different combinations of neurotransmitters were studied. Surgical sympathectomy may be performed where there is a discrete sympathetic nerve supply (as in the cerebral blood vessels) but it is difficult to remove all the sympathetic supply to organs such as the heart. Furthermore, regrowth of the surgically cut nerves might occur before changes in other peptide-containing nerves were seen. For the purposes of this thesis, it was therefore necessary to perform chemical sympathectomy.
71 3.4.1 Guanethidine Sympathetomy
Guanethidine is often used to produce depletion of catecholamine stores from various tissues. However, chronic guanethidine treatment of rats, with doses well in excess of those to produce depletion, results in the selective and long-lasting destruction of postganglionic sympathetic neurones (Jensen-Holm &
Juul, 1971; Heath et al. 1972; Heath & Burnstock, 1977). This has been shown in both neonatal and adult rats (Angeletti, 1971; Burnstock et al. 1971); although qualitatively similar, the destruction in neonates is more rapid and more complete
(Eranko & Eranko, 1971). The mechanism of action of guanethidine as a neurotoxin is not clear, but it appears to be immune-mediated and specific for rats, and has been shown to be ineffective in guinea pigs (O’Donnell & Saar,
1974),hamsters and rabbits (Johnson et al. 1977),mice (Evans et al. 1979b),cats
(Downing & Juul, 1973; Johnson et al. 1977) and pigs (Terris, 1983). In this thesis, the method of Johnson et al. (Johnson et al. 1976) was used: neonatal
Sprague-Dawley rats were injected with guanethidine sulfate (Ismelin; Ciba-Geigy
Laboratories) 50 mg/kg s.c. for 5 days in every 7 from day 8 to day 28 of life.
This has been shown to produce a profound and long-lasting sympathectomy with low morbidity and mortality.
3.4.2 6-hydroxydopamine Sympathectomy
Administration of 6-hydroxydopamine (6-OHDA) produces a rapid and widespread destruction of sympathetic nerve terminals (Thoenen & Tranzer, 1968;
O’Donnell & Saar, 1974). In this thesis, 6-OHDA was used to acutely destroy the sympathetic nerves of immature and mature rats in order to show the degree of innervation of different organs by intrinsic or non-sympathetic nerves which is
72 normally present at these ages. The 6-OHDA was given by intraperitoneal injection of 100 mg/kg of 6-OHDA in 10”^M ascorbic acid, followed the next day by a further injection of 250 mg/kg. The animals were then sacrificed the following day and the tissues examined by histochemistry and assay.
3.5 PRESENTATION OF DATA AND STATISTICAL ANALYSIS
3.5.1 Pharmacology
Vasoconstrictor responses were expressed as the increase in tension above baseline, in g, in response to the agonist; vasodilator responses were calculated as percent relaxation of the KCl-induced tone. The mean responses from a number of ring preparations from each animal were obtained, and the results were expressed as mean + s.e.m. for the number of animals used. The pD 2 for an agonist was calculated by interpolation of the results on the linear part of the curve as the negative log of the concentration of agonist which produced 50% of the maximum response obtained to that agonist. Student’s t-test (paired or unpaired as appropriate) was used for the statistical analysis and a probability of less than 0.05 was considered significant. Where expressed, pA 2 values were determined from Schild plots (Arunlakshana & Schild, 1959), or were calculated according to the equation pA2= log(CR-l)-log[antagonist M] when the Kg was unchanged at different concentrations (Besse & Furchgott, 1976; MacKay, 1978).
3.5.2 Histochemistry and Assay
Differences were assessed using Student’s two-tailed unpaired t-tests.
Results are expressed as mean + s.e.m.; p<0.05 was taken as significant.
73 3.6 MATERIALS USED
Acetylcholine chloride, Sigma Chemical Company
Adenosine, Sigma Chemical Company
Adenosine 5’-triphosphate, Sigma Chemical Company a,/?-methylene ATP (lithium salt), Sigma Chemical Company
Anti-CGRP antibodies, Cambridge Research Biochemicals, U.K.
Anti-NPY antibody, Immunodiagnostics Ltd.
Anti-SP antibody, Cambridge Research Biochemicals, U.K.
Anti-VIP antibody, Immunodiagnostics Ltd.
Ascorbic acid, Sigma Chemical Company
Betaxolol hydrochloride, Alcon Laboratories
Calcitonin gene-related peptide, Cambridge Research Biochemicals, U.K.
Calcium chloride solution, Sigma Chemical Company
DBH antibody, Eugene Tech International Inc. U.S.A.
Disodium hydrogen orthophosphate (Analar), BDH
D-Glucose (Analar), BDH
(-)-Epinephrine bitartrate, Sigma Chemical Company *
Feneterol hydrobromide, Boehringer Ingleheim
Goat anti-rabbit IgG conjugated to fluorescein isothiocyanate, Nordic, U.K.;
Goat anti-rabbit IgG conjugated to alkaline phosphatase Sigma Chemical Company
Guanethidine sulphate as Ismelin, Ciba-Geigy
Histamine, Sigma Chemical Company
6-Hydroxydopamine, Sigma Chemical Company
5-Hydroxytryptamine creatinine sulphate, Sigma Chemical Company
ICI 118,551, ICI Pharmaceuticals
(-)-Isoproterenol bitartrate, Sigma Chemical Company *
74 Magnesium sulphate heptahydrate (Analar), BDH
2-Methylthio adenosine 5’-triphosphate, Research Biochemicals Inc. U.S.A.
Neuropeptide Y, Cambridge Research Biochemicals, U.K.
(-)-Norepinephrine bitartrate, Sigma Chemical Company *
OCT compound, Tissue-Tek
PBS/glycerol as Citifluor, City University, London, U.K.
Phenoxybenzamine hydrochloride, Smith, Kline and French
8-Phenyltheophylline (8-PT), Sigma Chemical Company
Poly 1-lysine hydrobromide, Sigma Chemical Company
Pontamine sky blue, Sigma Chemical Company
Potassium chloride (Analar), BDH
P-Nitrophenyl phosphate, Sigma Chemical Company
Practolol hydrochloride, ICI Pharmaceuticals
Propranolol hydrochloride, ICI Pharmaceuticals
Salbutamol sulphate, Allen and Hanburys,U.K.
Sodium chloride (Analar), BDH Chemicals
Sodium dihydrogen orthophosphate (Analar), BDH
Sodium dihydrogen orthophosphate (Analar), BDH
Sodium hydrogen carbonate (Analar), BDH
Sodium nitroprusside, Sigma Chemical Company
Substance P, Cambridge Research Biochemicals, U.K.
Sylgard, Dow Corning, Seneffe, Belgium
Triton X-100, Aldrich Chemical Co., Dorset, U.K.
Vasoactive intestinal polypeptide, Cambridge Research Biochemicals, U.K. 8-PT was prepared as a stock solution by dissolving it in 80% methanol, 20% 1 M
NaOH vol/vol, and diluted as required using distilled water. Drugs marked * were dissolved in 0.1 mM ascorbic acid and were serially diluted in distilled water or
Krebs solution. The remainder of the drugs used were all prepared in distilled water.
3.7 PREPARATION OF SOLUTIONS
3.7.1 General
i Modified Krebs Solution
For 8 1 modified Krebs solution weigh out:
NaCl 62.40g
KC1 2.80 g
NaH2P04 1.68 g
NaHC03 10.96 g
MgS04.8H20 1.20 g
Glucose 11.20 g
Add to large container (with bottom tap) and make up to 5 1 with double distilled
H20. Shake/stir to dissolve.
Then add CaCl2 1 M 15.2 ml and mix.
The solution should be made up fresh as required, but will keep for up to 24 h at
4°C. Oxygenate with 95% oxygen/ 5% C02 to give final pH 1.2-1 A.
76 ii Phosphate Buffered Saline (TBS')
Phosphate Buffer (0.1 M):
Solution A (Na2HPC> 4 ): 42.6 g in 3000 ml distilled H20
Solution B (NaH2P04): 31.2 g in 2000 ml distilled H20
Add solution B to solution A until the pH is 7.4-7.6. (This generally takes
less than 1000 ml of solution B for 3000 ml of solution A).
For 1000 ml PBS, add 50 ml 0.2 M phosphate buffer, 0.2 g KC1 and 8.76 g NaCl to 950 ml distilled water (pH 7.4-7.5).
iii Svleard
1 pt curing agent
10 pts sylgard resin
Mix well and place under vacuum to remove bubbles (with care- solution foams).
Pour into plastic container to depth of 3-4 mm and allow to dry for 2 days.
(Drying can be accelerated at 37-40°C). Peel away from container and cut to size.
Do not use glass as sylgard adheres too strongly when drying.
3.7.2 Histochemistry
i 2% Glvoxvlic Acid Solution
Phosphate Buffer (0.1 M):
Solution A: NaH2PC>4 2.75 g in 100 ml
NaH2P 04.2H20 3.12 g in 100 ml
77 Solution B: Na2HP04 2.83 g in 100 ml
Na2HP04.2H20 3.56 g in 100 ml
For 25 ml buffer, add 3.5 ml solution A and 9 ml solution B to 12.5 ml distilled
H2O. For 2% wt/vol glyoxylic acid solution, add 0.5 g glyoxylic acid to 25 ml buffer; the pH of this solution is 3.4 and must beadjusted to 7.5 using 5 M NaOH
(approximately 30-35 drops). This solution may be stored for up to 24 h at 4°C or for 4 h at room temperature.
For pontamine sky blue counterstain, add 5-10 mg of pontamine sky blue and 100
/xl DMSO to 10 ml 2% glyoxylic acid solution.
ii Sucrose-phosphate-glvoxvlic acid (SPG) Solution
For 150 ml SPG solution:
Analar sucrose 0.2 g
KH2P 04 4.8 g
Glyoxyic acid 1.5 g
Dissolve analar sucrose and KH2P04 in 100 ml distilled water using a magnetic stirrer. Add glyoxylic acid crystals and stir until the solution is clear. Adjust pH tp 7.4 using 5 M NaOH, and make final solution up to 150 ml with distilled water.
This solution should be made up fresh when required.
iii Antibody Diluting Medium
To make 500 ml antibody diluting medium:
Sodium azide 0.5 g (0.1%)
Bovine serum albumin 0.05 g (0.01%)
78 Lysine 0.5 g (0.1%)
Triton X-100 0.5 ml (0.1%)
Make up to 500 ml with PBS. This solution is prone to microbial attack and should be replaced every six weeks.
iv 7% Sucrose/azide PBS
Analar sucrose 70 g
Sodium azide 1 g
PBS 1000 ml
v Polv 1-lvsine and gelatin subbed slides
Clean the slides by boiling in detergent such as Decon 90 for 30 min or leaving in detergent solution overnight. Wash under running tap water for one or more hours, rinse in distilled water and then in absolute alcohol; dry in oven at
37°C. Dip slides into subbing solution for few seconds, drain and allow to dry overnight in a dust-free atmosphere.
Poly 1-lysine subbing solution: Make stock solution of 4 mg/ml poly 1-lysine hydrobromide in distilled K^O. Dilute 1:200 when required.
Gelatin subbing solution: Add 0.5 g gelatin to 100 ml distilled and heat to
39°C. Cool, then add 0.05 g chrome alum and stir well and filter.
79 vi 4% Paraformaldhvde
Make up stock solution of 20% parafomaldehyde in aqueous solution:
Weigh out 200 mg paraformaldehyde in a fume cupboard.
Put into flask from refluxing equipment and add 700 ml distilled H2O.
Reflux in a fume cupboard for at least 5 h until nearly dissolved.
Allow to cool; add few drops NaOH until solution clears.
Top up to 1000 ml with distilled H2O, filter and store in glass bottle.
Dilute to 4% in PBS.
3.7.3 Assay
i 0.1 M Carbonate-Bicarbonate Buffer
Solution A: Na2CC>3 1.06 g and
Sodium azide 0.02 g in 100 ml distilled H2O
Solution B: NaHC03 0.84 g and
Sodium azide 0.02 g in 100 ml distilled H2O
Stir both solutions, add solution A to solution B until pH 9.6 (9-10 pipettes).
Store at 4°C.
ii Aorotonin Gelatin Tween PBS Azide (Sample Buffer)
PBS-Tween: 10 tablets PBS into 1 1 double distilled H2O
Add 500 Tween 20
Gelatin PBS-Tween azide: Add 0.1 g gelatin to 100 ml PBS-Tween
Stir on a hotplate. Allow to cool then add 0.02 g sodium azide.
80 May be stored at 4°C for one week.
For sample buffer: Add 1 mg aprotonin to 100 ml gelatin PBS-Tween azide solution. Make up on day of use.
iii Glvcine Buffer
For 200 ml glycine buffer (large amount):
Glycine 1.5 g
MgCl2 (1M) 200 /d
ZnCl2 0.0272 g (as co-factor)
Add above to 150 ml distilled H20 and adjust pH to 10.4 with 1 M NaOH, stirring constantly. Make up to 200 ml.
Store at room temperature.
81 to transducer
FIGURE 3.1
Schematic diagram illustrating the arrangement of wires used to support the ring preparations of coronary artery. The lower wire, bent into a rectangle, was threaded through a small length of silicone tubing. This fitted over a rigid
L-shaped support rod which was then lowered into the organ bath and clamped into place. The upper wire, bent into a triangle, was tied to a loop of thread which could be hooked onto the transducer. A micrometer moved the transducer to apply the desired resting tension. CHAPTER 4
ENDOTHELIAL RESPONSES OF THE RABBIT CORONARY ARTERY
TO BETA-ADRENERGIC AGONISTS AND ACETYLCHOLINE
4.1 SUMMARY
Isolated ring preparations of the epicardial coronary artery of the rabbit were studied to identify the role of the endothelium in mediating the responses to acetylcholine and to determine whether a- or jS-adrenergic stimulation may cause relaxation via endothelial receptors in this vessel.
In vessels with the tone raised by 30 mM KC1, concentrations of acetylcholine up to 10"** M produced dose-dependent relaxation of the preparations with endothelium intact, but no relaxation in preparations denuded of endothelium. At higher concentrations, a marked vasoconstrictor response was seen in all preparations regardless of the presence of endothelium. At basal tone, acetylcholine produced vasoconstriction which reached a maximum of 1.0 + 0.14 g tension in preparations with endothelium and 1.74 + 0.27 g tension in those without endothelium (p<0.05).
In coronary arteries pretreated with 50 /xM phenoxybenzamine to block a-adrenoceptors, noradrenaline, isoprenaline and salbutamol produced dose-dependent relaxation of the preparations; this was unaffected by the presence of endothelium. In vessels not pretreated with phenoxybenzamine, separate experiments were carried out in the presence of propranolol. The relaxation to
83 noradrenaline and isoprenaline was inhibited by propranolol but again there was no difference between vessels with and without endothelium.
These results suggest that acetylcholine in the rabbit epicardial coronary artery may produce an endothelium-dependent relaxant response over a limited concentration range; the smooth muscle vasoconstrictor responses dominate at higher concentrations. jS-adrenoceptors mediating relaxation are present on the smooth muscle but there is no evidence for functional a- or /?-adrenoceptors on the endothelium.
4.2 INTRODUCTION
The nature of the response of the coronary arteries to acetylcholine has been controversial for many years (Feigl, 1983; Young et al. 1987), but the discovery by Furchgott and Zawadski in 1980 (Furchgott & Zawadski, 1980) of the role of the endothelium in mediating the responses of blood vessels to
ACetylcholine has shed light on many of the discrepancies previously noted.
Similar to the responses found in the dog femoral artery and rabbit aorta (De Mey
& Vanhoutte, 1981; Furchgott, 1981), acetylcholine relaxes dog coronary arteries via production of endothelium-derived relaxant factor (EDRF) (Angus & Cocks,
1984). This is not, however, true for all species - no EDRF-mediated relaxation to acetylcholine has been found in pigs (Graser et al. 1986; Nagata et al. 1985), sheep (Miller et al. 1984), or cattle (Kalsner, 1979; Wali, 1986), while conflicting reports exist for rabbits (de la Lande et al. 1974; Saeed et al. 1986), primates
(Taira et al. 1983; Toda et al. 1985), and man (Bossaller et al. 1987; Forstermann et al. 1986; 1988; Thom et al. 1987).
84 Controversy also exists over the role of the endothelium in mediating the response of the coronary arteries to adrenergic agonists. It is well recognised that noradrenaline can produce relaxation of coronary arteries through activation of
0-adrenoceptors (Feigl, 1983; Ross, 1976; Young et al. 1987), but recent studies indicate that vasodilation may be mediated via both a- and /^-adrenoceptors on the endothelium (Rubanyi & Vanhoutte, 1985; Cohen et al. 1988b; Cocks & Angus,
1983; Angus et al. 1986a; 1986b).
Isolated rabbit heart preparations are widely used in physiological and pharmacological research to study coronary blood flow; clearly it is important in this species that the effects of acetylcholine and adrenergic agonists on the epicardial coronary arteries are known, to allow interpretation of responses in the intact coronary bed. In this Chapter, isolated epicardial coronary arteries from the rabbit were examined to determine the role of the smooth muscle and endothelium in mediating responses to adrenergic agonists and to acetylcholine.
4.3 METHODS See Chapter 3.1.2.
Where indicated, the preparations were initially exposed to 50 fiM phenoxybenzamine for 30 min followed by repeated washing to block a-adrenoceptors, and neuronal and extraneuronal uptake. In separate experiments, the vessels were not pretreated with phenoxybenzamine but, after a dose response curve to the agonist was obtained, they were rinsed several times over a minimum of 20 min and exposed to 1 /*M propranolol for 20 min before the relaxant responses were re-established. Parallel ring segments were examined without antagonists to check that the control responses were reproducible.
85 Constrictor responses at baseline tension were obtained to acetylcholine in intact preparations and in preparations denuded of endothelium. Cumulative addition of acetylcholine at baseline tension produced a degree of tachyphylaxis to the constrictor response, so the acetylcholine was added as single doses as described in Chapter 3.1.3.
4.3.1 Materials See Chapter 3.6
4.3.2 Statistics See Chapter 3.5
4.4 RESULTS
4.4.1 Responses to Acetylcholine
At raised tone, concentrations of acetylcholine up to approximately 10"^ M produced dose-dependent relaxation of the preparations with endothelium intact but purely vasoconstrictor responses, if any, were obtained in preparations from which the endothelium had been removed. The relaxant response could be abolished by haemoglobin, an inhibitor of endothelium-derived relaxant factor
(Figure 4.1). At concentrations of acetylcholine greater than 10“** M, all preparations constricted strongly regardless of their endothelial status (Figure 4.2).
Contraction to KC1 was not significantly different in preparation with intact endothelium and in those from which the endothelium had been removed.
At basal tone, preparations showed purely vasoconstrictor responses to acetylcholine which reached a maximum of 1.0 + 0.14 g tension (48% of the
86 contraction to 30 mM KC1) in preparations with endothelium but was significantly higher at 1.74 + 0.27 g tension (90% of the contraction to KC1) in preparations denuded of endothelium (p<0.05) (Figure 4.3). There was no difference in the sensitivity to acetylcholine of vessels with and without endothelium (Table 4.1). In two preparations, the relaxant and vasoconstrictor response could be abolished by atropine (data not shown).
4.4.2 Responses to Adrenergic Agonists
Dose-relaxation response curves of the rabbit coronary artery to noradrenaline (NA), isoprenaline (ISO) and salbutamol (SALB) were obtained in vessels which had been pretreated with phenoxybenzamine to block a-adrenoceptors. All of the agonists produced vasodilation of the preparations, although the responses to SALB were only seen at high concentrations and did not reach a maximum at the concentrations used. The pD2 values, slope and maximum responses to NA and ISO are shown in Table 4.1.
Responses to ISO, NA and SALB in preparations with functional n endothelium (which relaxed to 10 M acetylcholine) were compared to those m preparations denuded of endothelium to determine whether stimulation of
/?-adrenoceptors on the endothelial cells contributed to the relaxant responses through the release of an endothelium-derived relaxant factor. There were no significant differences between the vessels with and without endothelium (Table
4.1); the relaxation-response curves to all three agonists were superimposed (Figure
4.4 A, B, C). Thus 0-adrenoceptors on the endothelium do not appear to mediate a pharmacologically detectable relaxation in this vessel.
87 A further set of experiments was conducted in vessels which had not been pre-treated with phenoxybenzamine (see Chapter 4.3). Relaxant responses to NA were compared in preparations with and without endothelium after the addition of
10“^ M propranolol to examine whether there was any a-adrenoceptor response mediated via the endothelium in this vessel; relaxation to ISO was examined under the same conditions. Although the relaxant responses to both agonists were inhibited in the presence of propranolol in all preparations (Table 4.1), there were no differences between the responses of preparations with and without endothelium (Figure 4.5, Table 4.1) indicating that stimulation of endothelial a-adrenoceptors in this vessel does not cause release of a functional endothelium-derived relaxant factor.
4.5 DISCUSSION
These results demonstrate that in the isolated rabbit epicardial coronary artery, acetylcholine can cause relaxation via an endothelial dependent mechanism at low concentrations, but at higher concentrations a direct smooth muscle vasoconstrictor response dominates. In contrast, endothelial a- and
/3-adrenoceptors are absent or, if present, do not produce functional vasodilation in this vessel.
In 1974 de la Lande reported that the rabbit coronary artery perfused in a strip of myocardium constricted in response to acetylcholine, but noted that different arteries varied widely in sensitivity (de la Lande et al. 1974); a consistent vasoconstrictor response to acetylcholine was also reported in isolated rabbit extramural stem arteries by Nakayama and coworkers (Nakayama et al. 1978).
88 More recently, a study of rabbit coronary arteries in contact with the beating perfused heart demonstrated a rise in the internal vascular diameter and an increase in coronary flow in response to topical administration of a single concentration of 0.1 nM acetylcholine in preparations with intact endothelium, but a decrease in coronary flow and internal vascular diameter in preparations with the endothelium removed (Saeed et al. 1986). Another recent study which used isolated rabbit coronary arteries (which did not comment on the endothelium) also reported relaxation to acetylcholine (Han & Abel, 1987), but this was complicated by the use of a magnesium-free extracellular solution which is itself known to cause a powerful endothelial-dependent vasodilation (Ann & Ku, 1986).
In this study endothelium-dependent vasodilatation was found at low concentrations but vasoconstriction, regardless of the presence of endothelium, at high concentrations of acetylcholine. This resembles the response seen in rat hearts (perfused in vitro through their coronary circulation) (Sakai, 1980), and in anaesthetised baboons (Van Winkle et al. 1988), although these experiments did not demonstrate that the relaxant responses were endothelial-dependent. This biphasic effect of acetylcholine is intermediate between the almost exclusively vasodilator response of the dog (Angus & Cocks, 1984; Feigl, 1983; Furchgott & Zawadski,
1980; Kalsner, 1985) and the purely vasoconstrictor response of the pig (Graser et al. 1986; Nagata et al. 1985; Sakai et al. 1983) and sheep (Miller et al. 1984).
Conflicting reports exist of the effect of acetylcholine in human coronary arteries (Bossaller et al. 1987; Forstermann et al. 1988; Forstermann et al. 1986).
These results in the rabbit coronary artery suggest that the response of coronary arteries to a single dose or to a limited concentration range of acetylcholine may give a misleading indication of the physiological effects of acetylcholine in a given
89 species, and does not allow interpretation of changes which may occur under pathophysiological conditions; an increase in the smooth muscle vasoconstrictor response alone could give an apparent reduction in the relaxant response to acetylcholine without any change in endothelial function. The results of studies in man which demonstrate a selective reduction of acetylcholine-mediated relaxation in atherosclerotic human coronary arteries, rather than a reduction in response to purely endothelial-dependent vasodilators such as substance P, should thus not be interpreted as indicative of endothelial dysfunction without further evidence
(Werns et al. 1989; Fish et al. 1988; Takaoka et al. 1988).
Recent studies have suggested the presence of functional or
/^-adrenoceptors on the endothelium of coronary arteries in the dog and the pig
(Rubanyi & Vanhoutte, 1985; Cohen et al. 1988b; Cocks & Angus, 1983; Angus et al. 1986b). However, we found no difference in the relaxation to NA, ISO or
SALB in preparations with and without endothelium in preparations pretreated with phenoxybenzamine, nor was there evidence of any difference in the response with and without endothelium when the /3-adrenoceptors were blocked with propranolol, which suggests that in the rabbit coronary artery there are no functional a- or /3- endothelial adrenoceptors. Radioligand-binding studies have found ^-adrenoceptors in membrane preparations of coronary arteries (Schwartz
& Velly, 1983; Nakane et al. 1988), but these may represent presynaptic adrenoceptors and do not necessarily have a functional vasodilator role. In fact, in one of these studies, no functional response mediated by ^-adrenoceptors could be detected (Nakane et al. 1988).
It has been suggested that membrane depolarisation with KC1 may prevent the response to endothelium-derived relaxant factor or diminish the relaxations to
90 NA to an extent where they cannot be decreased further by removal of the endothelium (Rubanyi & Vanhoutte, 1985). Stable, reproducible contractions to agents such as PGF2a or histamine could not be obtained in the rabbit coronary artery unless the endothelium was damaged and the possibility that an effect of the endothelium would be revealed if other agents had been used cannot be excluded.
However acetylcholine caused relaxation of the KCl-constricted vessels in this study, so that at least one endothelium-derived relaxant factor is functional.
Furthermore, other workers using dog coronary arteries constricted with several different agents have not been able to confirm a 0-adrenoceptor-mediated endothelial-dependent relaxant response (Macdonald et al. 1987). One group have even reported enhanced relaxation to (3 agonists after removal of the endothelial cells (White et al. 1986).
This study has shown that functional receptors to acetylcholine, but not to adrenergic agonists, exist on the endothelium of the rabbit epicardial coronary artery. The importance of this distribution of receptors, and the reason why it varies between different vessels in the rabbit and between the coronary arteries of different species is not yet clear. Moreover, since hypoxia and atherosclerosis both may increase the importance of the larger vessels in determining total coronary resistance, the effect is likely to vary with pathophysiological conditions. But advances in our understanding of the interaction of nerves, smooth muscle and endothelium in vasomotor control emphasises the similarities rather than the differences between vessels and between species and may in the future allow interpretation of the changes found in disease states.
91 TABLE 4.1 Responses of rabbit coronary arteries with and without endothelium to acetylcholine (contractile responses only), noradrenaline and isoprenaline (relaxant responses)
With Without Agonist endothelium endothelium * *
ACh pE>2 5.26 (0.13) 5.62 (0.15) NS slope 0.82 (0.13) 1.33 (0.29) NS maximum (g) 1.00 (0.14) 1.74 (0.27) * n 8 8
NA + PBZ PD2 5.78 (0.27) 5.86 (0.23) NS slope 28.6 (4.5) 29.2 (3.0) NS maximum (%) 73.4 (5.2) 70.6 (2.6) NS n 6 6
NA + PROP pE>2 3.80 (0.07) 3.86 (0.14) NS slope 35.6 (4.1) 30.8 (5.6) NS maximum (%) 53.6 (6.4) 51.7 (10.6) NS n 7 6
ISO + PBZ pE>2 6.54 (0.04) 6.40 (0.44) NS slope 24.8 (1.9) 20.8 (3.5) NS maximum (%) 63.3 (4.8) 55.0 (5.5) NS n 6 4
ISO + PROP Pd 2 4.43 (0.11) 4.32 (0.12) NS slope 25.2 (3.5) 25.3 (4.2) NS maximum (%) 51.6 (3.0) 45.9 (4.2) NS n 5 5
ACh = acetylcholine; NA = noradrenaline; ISO = isoprenaline. PBZ = preparations pretreated with 50 /xM phenoxybenzamine; PROP = in the presence of 1 /iM propranolol.
Results are expressed as mean (s.e.m.); n refers to the number of animals studied. Maximum responses to ACh are contractile responses in grams tension, while those to NA and ISO are percent relaxation of the tone induced by 30 mM KC1. * = p<0.05 compared to preparations with endothelium; NS = not significant at the 5% level.
Relaxation to acetylcholine in preparations with intact endothelium were obscured by smooth muscle contraction at higher concentration and no true pD2 or maximum could be calculated; preparations devoid of endothelium did not relax to acetylcholine but responded by further contraction.
92 FIGURE 4.1
Relaxation of the rabbit coronary artery to 1 /*M acetylcholine (ACh) in preparations with endothelium (A and B) and denuded of endothelium (C). The tone of the preparations was raised with 30 mM KC1; W indicates washout. ACh causes sustained relaxation of the preparations with endothelium which is completely inhibited by the addition of 1.25 nM haemoglobin (Hb). No relaxation is seen in preparations denuded of endothelium and Hb does not produce a contraction confirming that the relaxation to ACh is endothelium-dependent. A
4min
A KCI Acetylcholine
AKCI B Acetylcholine 8 * 7 • 6 • 5 • 4 •
AKCI
FIGURE 4.2 A,B
Effect of acetylcholine (ACh), added in a cumulative fashion, on ring segments of the rabbit coronary artery (A) with the endothelium intact and (B) with the endothelium removed. The tone of the preparations was raised with 30mM
KC1. W = washout. Numbers refer to -log concentrations of ACh, • indicates half-log increments in dose. Values shown are means + s.e.m. of data from 10 rabbits, and are expressed as a percentage of the tone induced by 30mM KC1 in each preparation. c
% Contraction to KCI IUE . C 4.2 FIGURE 160 presence (solid line) and absence (dotted line) of endothelium. Values shown are are shown Values endothelium. of line) (dotted absence and line) (solid presence means + s.e.m. of data from 10 rabbits, and are expressed as a percentage of the the of percentage a as expressed are and rabbits, 10 from data of s.e.m. + means oe nue b 3m KI n ah preparation. each in KCI 30mM by induced tone n Log cumulative response curves of the coronary artery to ACh in the the in ACh to artery coronary the of curves response cumulative Log 95 FIGURE FIGURE 4.3 artery to acetylcholine in the presence (solid line) and absence (dotted line) of of line) (dotted absence and line) (solid presence the in acetylcholine to artery nohlu. aus hw ae h mas ... fdt fo svn rabbits. seven from data of s.e.m. + means the are shown Values endothelium. Contraction (g tension] 2.0 0 1.2 1.6 0.4- . - 0 8 -
Non-cumulative log contraction-response curves of the rabbit coronary coronary rabbit the of curves contraction-response log Non-cumulative i - * Lg (Acetylcholine] Log M - 96 FIGURE 4.4 A,B
Log cumulative relaxation-response curves to (A) noradrenaline and (B) isoprenaline in ring preparations of the rabbit coronary artery with the endothelium intact (solid lines) and removed (dotted lines). Preparations were pretreated with 50 fiM phenoxybenzamine. Results are expressed as percent relaxation of the KCl-induced tone and are the means + s.e.m. of data from four to six animals. There were no significant differences between preparations with and without endothelium. A 100 n
80 -
sp C o 60 - ■4— ■ 03X 03 < D 40 - CL
20 -
0 -
B 100 1
80 -
vP v_/ C 6 0 - o '■4—• 03X jCO CD 40 - CL
20 -
0 J c 100 n
80 -
60 -
40 -
i------1------1— :----- 1 7 6 5 4 - Log CSalbutamoO M
FIGURE 4.4 C
Log cumulative relaxation-response curves to salbutamol in ring preparations of the rabbit coronary artery with the endothelium intact (solid lines) and removed
(dotted lines). Preparations were pretreated with 50^M phenoxybenzamine. Results are expressed as percent relaxation of the KCl-induced tone and are the means + s.e.m. of data from four to six animals. There were no significant differences between preparations with and without endothelium.
98 FIGURE 4.5
Log cumulative relaxation-response curves to (A) noradrenaline and (B) isoprenaline in ring preparations of the rabbit coronary artery with the endothelium intact (solid lines) and removed (dotted lines). Experiments were carried out in the presence of 1 /xM propranolol. Results are expressed as percent relaxation of the KCl-induced tone and are the means ± s.e.m. of data from five to seven animals. There were no significant differences between preparations with and without endothelium. A 100 n CHAPTER 5
BETA-ADRENERGIC RELAXATION OF THE RABBIT CORONARY ARTERY
IS MEDIATED BY AN HOMOGENOUS POPULATION OF
BETAj-ADRENOCEPTORS
5.1 SUMMARY
In this chapter, the /3-adrenoceptor subtypes which mediate relaxation of the epicardial coronary artery of the rabbit are examined. Isolated ring preparations of the vessel were pretreated with 50 /xM phenoxybenzamine to block a-adrenoceptors, neuronal and extraneuronal uptake and were preconstricted with
30 mM KC1.
The rank order of potency of /3-agonists in causing relaxation of the preparations was isoprenaline (ISO) > noradrenaline (NA) = adrenaline (ADR) > fenoterol (FEN) > salbutamol (SALB). -Log Kg values were obtained for the
0- adrenoceptor blocking agents practolol, betaxolol and propranolol against these agonists and were compatible with their action at the /3j -adrenoceptor. Schild plots for the highly /^-selective /3-blocking agent ICI 118,551 using ISO, NA, and
ADR were superimposed, indicating the presence of a single population of receptors, and gave a mean pA2 value for ICI 118,551 of 7.01, characteristic of the action of this antagonist at the /3j- rather than the ^-adrenoceptor.
100 These results suggest that the /3-adrenoceptors mediating relaxation in the epicardial coronary artery are a homogeneous population of /3j-adrenoceptors.
^-Adrenoceptors, if present, do not appear to contribute to the vasorelaxant responses.
5.2 INTRODUCTION
It is well recognised that noradrenaline in the coronary arteries generally produces relaxation of the vessels through direct activation of the /3-adrenoceptors and not simply as a consequence of the increase in myocardial metabolic demand, although this latter effect certainly confused the results of much of the early published work (Ross, 1976; Feigl, 1983; Young et al. 1987). More recent studies conclude that vasodilation via activation of the /3-adrenoceptors is the predominant action of neural stimulation in the large coronaries (Hayashi & Toda, 1982; Toda &
Hayashi, 1982; Cohen et al. 1983). But, despite a wealth of data, the nature of the
/3-adrenoceptors receptors involved j32-, or a combination of the two) is still unresolved and may vary between species (Ross & Jorgensen, 1970; Cornish &
Miller, 1975; Parratt, 1980; Yatner et al. 1982; Feigl, 1983). The experimental data has even led some workers to propose that the coronary vascular /3-adrenoceptors might have properties intermediate between the two (Ross & Jorgensen, 1970;
Cornish & Miller, 1975; Feigl, 1983). Both a- and /?2-adrenoceptors mediating dilation via the endothelium have recently been reported in some species (Cocks &
Angus, 1983; Rubanyi & Vanhoutte, 1985; Angus et al. 1986b; Angus et al. 1986a;
Cohen et al. 1988b) but endothelial adrenoceptors do not mediate functional vasodilation in the epicardial coronary artery of the rabbit (see Chapter 4).
101 In this Chapter, the responses of rabbit isolated epicardial coronary arteries were examined to identify the nature of the jff-adrenoceptors mediating relaxation in this vessel. To investigate this, the more recently developed, potent, jS-selective agonists (e.g. fenoterol, / y and antagonists (e.g. betaxolol, 0j; and ICI 118,551,
^2) were used in addition to agents such as practolol, salbutamol and isoprenaline which have been employed in many previously published studies.
5.3 METHODS See Chapter 3.1.2
In these experiments the preparations were initially exposed to 50 /tM phenoxybenzamine for 30 min followed by repeated washing to block a-adrenoceptors and neuronal and extraneuronal uptake. In preconstricted vessels the 0- adrenoceptor agonists were added cumulatively in full-log increments to the
bath. Following washout, the vessels were rinsed several times over a minimum of
20 min and then, where indicated, were exposed to antagonists for 20 min before
the relaxation responses were re-established. Following initial exposure to the agonists, the preparations declined slightly in sensitivity; to avoid this the
preparations were exposed to a single maximal concentration of the agonist which
was then washed out before cumulative concentration response curves were
obtained. Parallel ring segments were examined without antagonists to check that
the control responses were subsequently reproducible.
5.3.1 Materials See Chapter 3.6
In view of the light-sensitivity of ICI 118,551, experiments with this drug
were carried out in foil-shielded organ baths in a darkened room.
102 5.3.2 Statistics See Chapter 3.5
PA2 values were determined from Schild plots (Arunlakshana & Schild,
1959), or were calculated according to the equation: pA2 = log (CR - 1) - log
[antagonist M] where preliminary experiments indicated that the Kg was consistent at different concentrations (Besse & Furchgott, 1976; MacKay, 1978).
5.4 RESULTS
The 0-adrenergic agonists caused relaxation of all the rabbit coronary artery preparations in a dose-dependent manner; dose-response curves to isoprenaline (ISO), noradrenaline (NA), adrenaline (ADR), fenoterol (FEN) and salbutamol (SALB) are shown in Figure 5.1 and the pD2 values, slope and relative potencies are shown in Table 5.1. Responses to FEN and SALB were only seen at relatively high concentrations and did not reach a maximum at the concentrations used, thus no EC^q could be calculated for these agonists. However the results show that the rank order of agonist potencies in this vessel is ISO > NA = ADR >
FEN > SALB, which is generally characteristic of the response of
-adrenoceptors.
A more secure characterisation of the 0-adrenoceptor may be made using highly selective 0-receptor antagonists. The powerful 02-antagonist ICI 118,551 and the 0j-selective antagonist betaxolol were used in this study in addition to the non-selective agent propranolol to analyse the receptors involved. The effect of the older 0j-selective agent practolol, which was often used in earlier studies, was also examined.
103 5.4.1 Effect of /^-Antagonists and Propranolol
Dose-response relationships to the agonists were established in the presence and absence of 10"** M betaxolol, 5 x 10"** M practolol, and 10”** M propranolol
(Figure 5.2 A, B, C, D). In the presence of these j3-blockers, responses to the
agonists were significantly inhibited and did not always reach their maxima, so the
-log EC^q was calculated as the concentration of agonist required in the presence
of the antagonist to produce 50% of the control maximal relaxation to that agonist;
these results were used to calculate the PA2 values from the equation described
under methods (Table 5.2). The PA2 values obtained are characteristic of the
action of these agents at the jSj adrenoceptor. Despite the higher concentration
used, practolol was markedly less effective at inhibiting the relaxation to all of the
agonists than betaxolol or propranolol and considerable relaxation persisted which
could subsequently be blocked by 10”** M propranolol (data not shown).
5.4.2 Effect of ICI 118,551
The results indicated that the /J-agonists were acting on j8j adrenoceptors,
but this did not exclude the existence of a small population of /^-adrenoceptors
since the effect of these might be obscured by a much greater population of /?j
adrenoceptors. To investigate this possibility, dose-response curves to all the
agonists were obtained in the presence and absence of ICI 118,551. Responses to
FEN and SALB in 4 preparations were not significantly affected by ICI 118,551 at
concentrations of 0.1 and 0.5 nM (data not shown) but as the responses did not
reach a maximum, Schild plots could not be constructed for these two agonists.
ICI 118,551 (at concentrations of 0.1 /iM and 1 /-iM) caused a dose-dependent shift
in the relaxation-response curves to ISO, NA and ADR. Schild plots for the
104 agonists against ICI 118,551 were superimposed (Figure 5.3) and their slopes did not differ significantly from unity (Table 5.3), suggesting action at a homogeneous population of 0-adrenoceptors. The pA2 values, calculated from the intercept of the regression line with the x-axis (Table 5.3), were characteristic of the pA2 values reported for the action of ICI 118,551 on the 0j-adrenoceptor.
5.5 DISCUSSION
These results demonstrate that in the isolated rabbit epicardial coronary artery vasodilation in response to adrenergic agonists is mediated via
0-adrenoceptors which are an homogeneous population of the 0j subtype.
02-adrenoceptors are absent or, if present, do not produce functional vasodilation in this vessel.
While it is clear that noradrenaline dilates coronary arteries by activation of
0-adrenergic receptors, the traditional view was that 0j -adrenoceptors were dominant in the myocardium and 02~adrenoceptors in the vasculature (Lands et al.
1967b). However, it is now clear that 0j -adrenoceptors may be present in certain arteries such as the dog renal artery (Taira et al. 1977) and the cat middle cerebral artery (Edvinsson & Owman, 1974), while 02-adrenoceptors have been demonstrated in the myocardium of the cat (Carlsson et al. 1977) and, more recently, in man (Hall et al. 1989).
A pattern has emerged for the response of coronary arteries to adrenergic agents. Studies of isolated vessels from several species including dog (O’Donnell &
Wanstall, 1984a; Nakane et al. 1988), pig (Drew & Levy, 1972; Johansson, 1973),
105 sheep (Miller et al. 1984) and cow (Purdy & Stupecky, 1986; Purdy et al. 1988) have concluded that predominantly /3j-adrenoceptors are involved. Only one previous study has attempted to characterise the /3-adrenoceptors in the rabbit: de la Lande, in a study of perfused coronary arteries within a myocardial slab, first suggested that the adrenoceptors in the rabbit might be of the /3j-subtype based on the potency of single equi-effective doses of isoprenaline and salbutamol and the effect of practolol (de la Lande et al. 1974) but the results were not further analysed due to a wide variability in the responses and interpretation was complicated by the presence of the surrounding myocardial tissue. The work in this chapter is in agreement with these reports on isolated vessels in other species and confirms that the predominant /3-adrenoceptor present in the large epicardial coronary arteries of the rabbit is 0j. This conclusion is based on 1) our finding of the rank order of potency of /3-adrenoceptor agonists - ISO > NA = ADR >
FEN > SALB - which is characteristic of the /3j-adrenoceptor and 2) inhibition of the responses to the same degree by equipotent /3-blocking concentrations of the
/3j-selective antagonist betaxolol and the non-selective antagonist propranolol, with
PA2 values for these antagonists characteristic for their action at the
/3j-adrenoceptor.
Despite agreement between these in vitro studies on isolated vessels, other work using pig coronary arteries in vitro (Bayer et al. 1974) and many studies on different species in vivo conclude that ^-adrenoceptors are involved (see Feigl,
1983 for references). In view of accompanying changes in the mechanical and metabolic activity of the myocardium, the in vivo studies do not all reflect a direct effect of /3-adrenoceptor agonists on the coronary arteries. However, Vatner and coworkers in 1982 demonstrated the presence of /3j- and ^-adrenoceptors in conscious dogs (Vatner et al. 1982), and Gross and Feigl have clearly shown in the
106 potassium arrested dog heart in situ that salbutamol was only 8 times less potent than isoprenaline and practolol was virtually inactive, suggesting the presence of
^-adrenoceptors (Gross & Feigl, 1975).
The study in isolated coronary arteries in pigs which characterised the adrenoceptor as ^ examined the relative affinity of practolol, propranolol and H
35/25 on the myocardium and coronary arteries (Bayer et al. 1974). It is worth noting that practolol, even at a concentration of 5 x 10“^M, produced a non-significant shift in the relaxant responses to the 0-agonists in our study and a significant propranolol-sensitive relaxation remained. Nevertheless, the mean pA2 obtained in the present study for practolol against the different agonists (6.2) is characteristic of the pA2 described for practolol at the 0j -adrenoceptor; at the
02-receptor the pA2 for practolol is reported to be approximately 4.7. These results emphasise that practolol is a weak and relatively ineffective 0j -antagonist in the coronary artery and previous studies purporting to demonstrate the existence of 02-adrenoceptors based on the ability to obtain relaxation to 0-agonists after administration of practolol may be inadequate for these conclusions to be drawn.
Despite this caveat, the evidence outlined above that 02-adrenoceptors are present and functional in the coronary arterial bed of certain species in vivo is strong. There are several possible explanations for the discrepancies between these findings and the studies of isolated vessels: firstly, 0j- and 02-adrenoceptors may both be present, but the ratio may vary between different species and the experiments in vitro have not been designed to identify a small subpopulation of
02~adrenoceptors; secondly, 02-adrenoceptors may be present on the endothelium which could be inadvertently removed in vitro; and thirdly, b2-adrenoceptors may
107 predominate on the small resistance vessels which are less accessible for study in vitro.
To assess whether there was a small subpopulation of /^-adrenoceptors in the rabbit coronary artery, the method of O’Donnell and Wanstall was used in the present study (O’Donnell & Wanstall, 1981); this involves determining whether
Schild plots for a selective ^-adrenoceptor antagonist using /3j- and /^-agonists are superimposed (homogeneous receptor population) or separated (heterogeneous receptor population). The lack of efficacy of the most selective ^-agonists in this vessel precluded their use, but the agents used - ISO, NA and ADR - do differ in their selectivity for the /?j- and /^-adrenoceptor. Schild plots for these agonists against the highly selective /^-antagonist ICI 118,551 were superimposed, with a mean pA2 value of 7.02. This value is characteristic for the antagonist at the j3j -adrenoceptor (O’Donnell & Wanstall, 1984a; Nyborg & Mikkelsen, 1985; Purdy et al. 1988) and confirms that an homogeneous population of jSj -adrenoceptors are present.
Functional and /^-adrenoceptors have been reported on the endothelium of epicardial coronary arteries in the dog and the pig by some workers (Cocks & Angus, 1983; Rubanyi & Vanhoutte, 1985; Angus et al. 1986b;
Cohen et al. 1988b). However, other workers could detect no functional postsynaptic response mediated by endothelial /^-adrenoceptors in the dog
(Nakane et al. 1988) and the rabbit epicardial coronary artery does not possess endothelial adrenoceptors (see Chapter 4). Thus inadvertent removal of endothelial
/^-adrenoceptors from epicardial coronary arteries is unlikely to account for the reported differences between studies in vitro and in vivo.
108 Overall, therefore, the evidence points to an homogeneous functional population of /3j-adrenoceptors in large coronary arteries while the small resistance vessels may have @2~ or a mixture of jSj- and ^-adrenoceptors. In keeping with this, Grover and coworkers have demonstrated a dose-dependent recruitment of coronary micro vessels in the rabbit with /^-adrenergic stimulation which was independent of myocardial blood flow (Grover et al. 1986). One group which reported a single population of /?j-adrenoceptors in the rat small intramural coronary arteries in fact studied distal left coronary artery segments with a mean diameter of approximately 240 /xM, which for the rat probably still represents a
"large" coronary artery (Nyborg & Mikkelsen, 1985).
The importance of the presence of an homogeneous population of
/3j -adrenoceptors in the epicardial coronary arteries remains obscure. With the
recent identification of /^-adrenoceptors in human myocardium, it is now evident
that the traditional assumptions of the distribution of the adrenoceptor subtypes in
the heart can no longer be upheld. In diseases such as atherosclerosis the larger
coronary vessels may become flow-limiting but clinically the use of non-selective or /^-selective 0-adrenoceptor blocking agents does not appear to reduce
myocardial blood flow. This indicates that the inhibition of heart rate and
reduction in myocardial oxygen demand induced by these agents are probably
more important than any inhibition of 0j-adrenoceptor-mediated relaxation of the
coronary arteries under these pathophysiological conditions. Indeed, a recent study
has shown that propranolol actually inhibits vasoconstriction of atherosclerotic
segments of coronary arteries on exercise, although the mechanism is unknown
(Bortone et al. 1990).
109 TABLE 5.1 Potency, slopes and maximum relaxation to isoprenaline,
noradrenaline, adrenaline and fenoterol in isolated rabbit
coronary artery.
AGONIST n -Log EC50 Slopea Maximum Relative
relaxation (%) potency”
Isoprenaline 6 6.58 (0.04) 24.8 (1.9) 63.3 (4.8) 20.4
Noradrenaline 6 5.27 (0.15) 25.4 (2.8) 75.0 (4.5) 1.0
Adrenaline 5 5.41 (0.17) 29.3 (4.7) 68.3 (5.2) 1.4
Fenoterolc 6 4.60 (0.11) 28.0 (3.9) N/R 0.2
Results are given as mean (s.e.m.) of data from a number of animals, given by n.
a Slopes are expressed as a negative value. k The potencies of the agonists are expressed relative to noradrenaline. c Control responses to fenoterol and salbutamol did not reach a maximum, so no
EC^o could be calculated. For assessment of relative potency for fenoterol, the value for -logEC^Q shown is the -log of the concentration of fenoterol which would produce 50% of the maximum relaxation to isoprenaline. The response to salbutamol did not reach this value.
110 TABLE 5.2 pA2 values for practolol, betaxolol and propranolol against
isoprenaline, noradrenaline and adrenaline in the rabbit coronary
artery.
AGONIST ANTAGONIST
Practolol Betaxolol Propranolol
[5 x 10_6M] [10‘ 6M] [10"6M]
Isoprenaline 6.20 ± 0.09 7.77 ± 0.17 8.16 ± 0.12
(n) (5) (5) (5)
Noradrenaline 5.62 ±0.19 7.61 ±0.19 7.42 ± 0.26
(n) (6) (4) (5)
Adrenaline 6.73 ± 0.73 7.66 ± 0.04 7.47 ± 0.13
(n) (3) (4) (4)
Values given are mean ± s.e.m. of data from a number of animals, denoted by (n).
Preparations were pretreated with phenoxybenzamine and constricted with 30 mM
KC1.
Ill TABLE 5.3 Antagonist pA2 values and slopes derived from Schild plots for
ICI 118,551 on rabbit coronary artery using isoprenaline,
noradrenaline and adrenaline as agonists.
AGONIST n Slope pA2
Isoprenaline 6 1.05 ± 0.06 7.05 ± 0.21
Noradrenaline 6 0.91 ± 0.09 6.95 ± 0.23
Adrenaline 6 1.05 ± 0.07 7.04 ± 0.13
Values given are mean + s.e.m. of data from a number of animals, denoted by n; slopes are expressed as a negative value. Two concentrations of ICI 118,551 covering a 10-fold concentration range were used. Preparations were pretreated with phenoxybenzamine and contracted with 30 mM KC1.
112 Relaxation C%D 100
80-
60-
40 -
20 -
0
FIGURE 5.1
Log cumulative relaxation-response curves of the rabbit coronary artery to
isoprenaline (circles), noradrenaline (closed triangles), adrenaline (open triangles),
fenoterol (squares) and salbutamol (diamonds). Results are expressed as a
percentage of the tone induced by 30 mM KC1, and are the means + s.e.m. of data
from at least six rabbits for each agonist.
113 FIGURE 5.2 A,B
Log cumulative relaxation-response curves to A) noradrenaline and B) adrenalinein ring preparations of the rabbit isolated left coronary artery in the absence (solid symbols) and presence (open symbols) of the /^-blocking agents betaxolol (10- ** M, dotted line), propranolol (10"** M, irregular dashed line) and practolol (5x10- ** M, regular dashed line). All preparations were pretreated with
50 fiM phenoxybenzamine, followed by washout, and were constricted with 30 mM
KC1. Results are expressed as percent relaxation of the KCl-induced tone and are the means + s.e.m. of data from four to six rabbits (see Table 5.2). Relaxation (%) 100 n
80 -
60 -
40 •
20 -
0 J
i i 'i "■ i ...... i i 8 7 6 5 4 3 -Log (Noradrenaline!) M
Relaxation (%) 100 i
80 -
60 -
40 -
20 •
0 J 8 7 6 5 4 3 -Log (Adrenaline) M FIGURE 5.2 C,D
Log cumulative relaxation-response curves to C) isoprenaline and D) fenoterol in ring preparations of the rabbit isolated left coronary artery in the absence (solid symbols) and presence (open symbols) of the /^-blocking agents betaxolol (10“°f \ M, dotted line), propranolol (10~° M, f \ irregular dashed line) and practolol (5xl0-^ M, regular dashed line). All preparations were pretreated with
50 (iM phenoxybenzamine, followed by washout, and were constricted with 30 mM
KC1. Results are expressed as percent relaxation of the KCl-induced tone and are the means ± s.e.m. of data from six rabbits (see Table 5.2). c Relaxation C%3
Relaxation C%)
i------1------1------1------1 8 7 6 5 4 - Log (Fenoterol) M Log CCR - 1)
2 n
1.5 -
1 -
0.5 -
0 -
-0.5 - - Log CICI 118,551) M
FIGURE 5.3
Schild plots for ICI 118,551 in the isolated rabbit coronary artery using
isoprenaline, noradrenaline and adrenaline as agonists. The pA2 values for all three
agonists were not significantly different from each other, and the slopes of the plots
were not different from -1 (see Table 5.3). CR = concentration ratio. CHAPTER 6
VASODILATOR RESPONSE OF THE SMOOTH MUSCLE OF
THE RABBIT CORONARY ARTERY TO THE SYMPATHETIC
COTRANSMITTERS NORADRENALINE AND ADENOSINE TRIPHOSPHATE
6.1 SUMMARY
Vasodilator and vasoconstrictor responses to noradrenaline (NA), adenosine and adenosine 5’-triphosphate (ATP) were examined in isolated ring segments of the left anterior descending coronary artery of the rabbit in the absence of endothelium.
NA caused dose-dependent relaxation of potassium-constricted vessels in the absence of j3-adrenergic blockade, with a PD 2 of 5.96 ± 0.21. Constrictor responses of vessels at baseline tension were only seen at concentrations of NA greater than 1 mM, and reached a maximum of 6% of the contraction to 30 mM
KC1. ATP relaxed the potassium-constricted ring segments in a dose-dependent manner, although a transient constriction often preceded the relaxation. Adenosine was equipotent with ATP in producing relaxation; this was significantly inhibited by the Pj-purinoceptor antagonist, 8-phenyltheophylline (8-PT). The responses to
ATP were little affected by 8-PT, indicating that ATP was not acting through breakdown to adenosine. At basal tone, ATP produced transient vasoconstriction only at concentrations greater than 0.1 mM, reaching a maximum of 38% of the contraction to 30 mM KC1.
117 It is concluded that in the rabbit coronary artery, both NA and ATP produce vasodilatation by a direct action on the smooth muscle; this is consistent with the general hypothesis that NA and ATP act as synergistic co-transmitters.
6.2 INTRODUCTION
Noradrenaline (NA) and adenosine 5’-triphosphate (ATP) are now well established as co-transmitters in sympathetic nerves supplying many blood vessels, where they have synergistic vasoconstrictor activity on the smooth muscle via c*l -adrenoceptors and P2X~pur*noceptors respectively (Burnstock, 1988b). While coronary arteries are supplied with both a- and jS-adrenoceptors (Yurchak et al.
1964; Lioy, 1967; Andersson et al. 1972; Mark et al. 1972), the contribution of each seems to vary, both between species and between vessels of different calibre in the same species (Zuberbuhler & Bohr, 1965; Vatner, 1985). The epicardial coronary artery of the rabbit responds to NA almost exclusively by vasodilatation
(see Chapter 4), with minimal vasoconstriction at doses up to 0.1 mM, even in the presence of jS-adrenoceptor blocking agents (Han & Abel, 1987); this relaxation is mediated exclusively by /?j-adrenoceptors on the smooth muscle (see Chapter 5).
The aim of this study, therefore, was to examine whether the sympathetic co-transmitter ATP has a similar vasodilator action on the smooth muscle of the rabbit coronary artery, or whether it produces a direct vasoconstrictor response as in most vessels. The endothelium was removed from the preparations in all the experiments unless otherwise indicated.
118 6.3 METHODS
6.3.1 Pharmacology
Ring preparations of rabbit coronary arteries, with and without
endothelium, were set up as described in Chapter 3.1.2. Confirmation of the
presence or absence of the endothelium was ascertained in each preparation by
pharmacological assay of the response to a single dose of 0.1 /*M acetylcholine (see
Chapter 4) and in some cases by histological examination of segments for the
presence or absence of endothelial cell nuclei.
The vasoconstrictor response to 30 mM KC1 in ring segments with
endothelium removed was compared with the response in segments in which the
endothelium had been left intact, to ensure that the smooth muscle had not been
damaged during the process. As a further control, relaxant responses to sodium
nitroprusside, a direct-acting dilator of vascular smooth muscle, were also
investigated in the presence and absence of endothelium.
At basal tone, the tissues rapidly desensitised to ATP which was therefore
added as single doses at 40-min intervals; the preparations were washed repeatedly
between each application. At raised tone, the responses did not show
desensitisation and the agonists were added to the bath in a cumulative fashion
(see Chapter 3.1.3).
6.3.2 Materials See Chapter 3.6.
6.3.3 Statistics See Chapter 3.5.1.
119 6.4 RESULTS
6.4.1 At Raised Tone
In all ring segments, KC1 (30 mM) produced reproducible, sustained contractions, with no significant difference in the mean tension generated in the presence and absence of endothelium (2.02 + 0.20 g tension with endothelium, 1.78
+ 0.12 g tension without endothelium). Acetylcholine (0.1 fiM) consistently produced relaxation of the ring segments in the presence of intact endothelium, which was abolished, or converted to a contraction, in preparations with the endothelium removed (Figure 6.1). Sodium nitroprusside, a direct-acting vasodilator of vascular smooth muscle, produced greater than 90% relaxation of all ring segments. The maximum relaxation was not reduced by removal of the endothelium (Figure 6.2), and in fact there was a small, significant increase in the potency of sodium nitroprusside in preparations with the endothelium removed
(Table 6.1).
NA (10 nM - 1 mM) caused reproducible, concentration-dependent, sustained relaxation in ring segments without endothelium (Figure 6.3 A, B), although responses at concentrations above 100 /*m were variable. The PD 2 for
NA was 5.96 + 0.21. In order to compare the potency of NA with adenosine and
ATP (which did not reach their maximum responses), the EC^q, defined as the concentration required to produce a 40% relaxation of the KCl-induced tone, is shown in Table 6.1.
Despite the absence of endothelium, ATP (10 nM - 1 mM) also caused
concentration-dependant relaxations of the coronary vascular smooth muscle
120 (Figure 6.4 B). The relaxation in response to high concentrations was often preceded by a small, transient constriction (Figure 6.4 A). Maximum relaxation was not reached and no true EC^q could be calculated, but the EC^q, as defined above, was 4.07 + 0.28 (Table 6.1).
Adenosine (10 nM - 1 mM) induced concentration-dependant relaxations which were significantly inhibited by the potent, competitive, Pj-purinoceptor antagonist 8-PT (Figure 6.5 A-C). The responses did not reach a maximum over
the range of concentrations examined; the EC^q, as defined above, is given in
Table 6.1.
Comparison of the -log [EC^q] for adenosine and ATP shows that they
were equipotent in causing direct relaxation of the smooth muscle in the rabbit
coronary artery, while NA was significantly more potent. To determine whether
ATP was acting via breakdown to adenosine, concentration-response curves to
ATP were also constructed in the presence of 10 fiM 8-PT, a concentration that
significantly antagonised the relaxation to adenosine. There was no shift in the
concentration-response curve (Figure 6.4 B), indicating that ATP was not acting
via the Pj-purinoceptor or through breakdown to adenosine 5’-phosphate or
adenosine. ATP is thus probably acting directly on P 2 -purinoceptors, located on
the smooth muscle.
6.4.2 At Basal tone
At basal tone, none of the agonists used caused contraction at doses less
than 0.3 mM. At higher concentrations, NA produced a weak vasoconstriction in
approximately 50% of the ring preparations examined, which reached a maximum
121 of 6% of the contraction to KC1 (Figure 6.6 B,C). ATP also produced a transient
vasoconstriction at high concentrations which reached a maximum of 38 + 10% of
the response to KC1 at a concentration of 3 mM (Figure 6.6 A,C). Adenosine did
not cause a contraction at any dose examined up to 3 mM. Since the contractile
responses to the agonists did not approach a maximum even at these
concentrations, no direct comparison of the EC^q values could be made.
6.5 DISCUSSION
This study confirms that the predominant response of the rabbit coronary
artery to exogenous NA in the absence of adrenoceptor blocking agents is a
relaxation of the smooth muscle, which suggests that there is a substantially greater
population of (3- than a-adrenoceptors in this vessel. The weak vasoconstriction
seen in some preparations with very high concentrations of NA may represent
stimulation of a small population of a-adrenoceptors. More importantly, this study
demonstrates that, unlike most blood vessels, the rabbit coronary artery smooth
muscle also responds to ATP by vasodilatation rather than vasoconstriction.
Relaxation in response to sodium nitroprusside was not impaired in arterial
segments without endothelium, indicating that the method of removal of the
endothelium did not, in itself, impair the vasodilator responses of the preparations.
In fact, although the maximum relaxation was unchanged, the potency of sodium
nitroprusside in this study was slightly, but significantly, augmented by removal of
the endothelium. Sodium nitroprusside is believed to act by giving rise to nitric
oxide, which directly dilates the smooth muscle (Murad et al. 1979; Vanhoutte &
Rimele, 1983). Similar potentiation of the response to sodium nitroprusside by
removal of the endothelium has been reported in the rat thoracic aorta (Shirasaki
122 et al. 1986) and the rabbit mesenteric artery (Warland, 1987) and may indicate release of an endothelium-derived contracting factor stimulated by this agent.
ATP was potent at causing relaxation in the rabbit coronary artery in the absence of endothelium. In preparations at basal tone it produced contractions only at concentrations around 3 mM; in raised tone preparations, the relaxation
responses were preceded by transient contractions at high concentrations.
Kennedy & Burnstock (Kennedy & Burnstock, 1985a) have reported that in the
isolated rabbit central ear artery ATP produces vasodilatation via Pj-purinoceptors
located on the smooth muscle, possibly through breakdown to adenosine or
adenosine 5*-phosphate. In this study, the selective Pj-purinoceptor antagonist
8-PT did not affect the relaxation response to ATP, although the responses to
adenosine were significantly impaired, indicating that in the rabbit coronary artery
ATP does not act on the Pj-purinoceptor and probably acts directly via
P2 -purinoceptors.
Many workers have suggested that ATP is a co-transmitter with NA in
sympathetic nerves supplying blood vessels (Su, 1975; Head et al. 1977; Katsuragi
& Su, 1981; Muramatsu et al. 1981; Sedaa et al. 1986; Burnstock, 1988b).
Sympathetic nerve stimulation has been shown to produce contraction mediated by
both NA and ATP in the rat tail artery (Sneddon & Burnstock, 1984b; Vidal et al.
1986), the mesenteric artery (Ishikawa, 1985; von-Kugelgen & Starke, 1985;
Muramatsu, 1986), the rabbit ear artery (Kennedy et al. 1986), the rabbit
saphenous artery (Burnstock & Warland, 1987) and the dog basilar artery
(Muramatsu & Kigoshi, 1987). In many vessels in which NA mediates contractile
responses, exogenous ATP can produce relaxation, but this has been shown to be
mediated via P 2 "purinoceptors located not on the smooth muscle, but on the
123 endothelium (De Mey & Vanhoutte, 1981; Furchgott, 1981; Furchgott, 1983;
Hardebo et al. 1983). After removal of the endothelium, the response to ATP is usually converted to contraction, in keeping with the hypothesis that ATP and NA in the sympathetic nerves supplying the smooth muscle are acting as co-transmitters (Kennedy et al. 1985). In the rabbit coronary artery in this study, exogenous NA and ATP both produce a predominantly vasodilator response despite removal of the endothelium. Thus the vasodilation in response to ATP in the rabbit coronary artery appears to be mediated largely via receptors located directly on the smooth muscle.
Although a vascular smooth muscle relaxant response to ATP is unusual, it is not unique to this vessel. The vasodilator response to ATP in the rabbit mesenteric artery (Mathieson & Burnstock, 1985) and the rabbit portal vein (Reilly et al. 1987) is mediated via P 2 Y- PurjnocePtors located directly on the smooth muscle, and not on the endothelium. However, NA in these arteries is vasoconstrictor, and not vasodilator, which indicates that NA and ATP are less likely to be acting as co-transmittors by direct postsynaptic action on the smooth muscle. In fact, Ramme and coworkers suggested recently that in the resistance vessels in the smaller branches of the rabbit mesenteric artery, ATP is the sole neurotransmitter acting postjunctionally upon stimulation of the sympathetic nerves, and that the NA released concomitantly acts entirely on prejunctional c^-adrenceptors to modulate transmitter release (Ramme et al. 1987). Furthermore, there is now substantial evidence that ATP is a sensory neurotransmitter in some primary afferent nerve fibres (Jahr & Jessel, 1983; Salt & Hill, 1983; Fyffe & Perl,
1984) and may be released antidromically upon stimulation of sensory nerves
(Holton & Holton, 1954; Holton, 1959; Burnstock, 1987). Thus, exogenous ATP in the rabbit mesenteric artery may be acting upon receptors which would be
124 stimulated physiologically by sensory rather than sympathetic nerves. The rabbit portal vein is innervated by sympathetic nerves, but it is also supplied with non-sympathetic (purinergic) nerves which also release ATP and mediate vasodilatation (Burnstock et al. 1979b; Reilly et al. 1987; Su, 1987). These non-adrenergic nerves are the source of approximately 50% of the purines released by electrical field stimulation (Levitt & Westfall, 1982) and may account for the presence of the P2Y"pur*noceptor on the smooth muscle in this vessel. However, no such non-adrenergic innervation has been described for the epicardial rabbit coronary arteries.
ATP and NA have been shown to be co-transmitters in vessels in which both have direct constrictor effects on the smooth muscle. This study shows that in the rabbit coronary artery, they both mediate vasodilatation by direct action on the vascular smooth muscle, consistent with the hypothesis that they may also be co-transmitters in this vessel.
125 TABLE 6.1 Responses of isolated ring preparations of the rabbit coronary artery.
Agonist Response -Log [EC4Q]a (n) Maximum (n) relaxation (%)
ATP -E Contraction, 4.07 (0.28) (7) Not reached then relaxation
Adenosine -E Relaxation 4.33 (0.09) (6) Not reached
NA -E Relaxation 5.53 (0.23) (6) 65.0 (3.0) (6)
SNPb +E Relaxation 7.96 (0.11) (6) * 93.5 (1.5) NS -E Relaxation 8.04 (0.06) (6) 94.3 (1.2) (6)
a EC4q is defined as the concentration required to produce a 40% relaxation of the KCl-induced tone and is used because responses for ATP and adenosine did not reach a maximum.
b Responses to SNP are shown in the presence and absence of endothelium (E) as a control to ensure that the method of removal of the endothelium had not damaged the smooth muscle. * = P<0.05, NS = no significant difference between preparations with and without endothelium.
Values given are mean (s.e.m.); n = number of rabbits.
Abbreviations: Endothelium (E); Adenosine 5’-triphosphate (ATP); Noradrenaline (NA); Sodium nitroprusside (SNP).
126 A ACh -7
AKCI B ACh -7 w
AKCI
FIGURE 6.1
n Effect of acetylcholine (10 M) on ring segments of rabbit left circumflex coronary artery with tone raised by KC1 30 mM. A) In ring segments with intact endothelium acetylcholine produces a relaxation. B) In segments with endothelium removed acetylcholine does not produce relaxation; the response is converted to a contraction. Relaxation C%) 100 n 80- 60- 40- 20 ooay rey o oim irpusd (. n - . m) i te rsne (solid presence the in mM), 0.1 - nM (0.1 nitroprusside sodium to artery coronary FIGURE 6.2 eaain f h Klidcd oe ad r te en ..en f aa rm six from data of s.e.mean ± mean the percent are as and expressed tone, are Results KCl-induced the of relaxation endothelium. of line) (dotted absence and line) rabbits. - Log cumulative relaxation-response curves of the rabbit left circumflex circumflex left rabbit the of curves relaxation-response cumulative Log 0 8 6 4 5 6 7 8 9 10 Lg Sdu Ntorsie M CSodium Log Nitroprusside) - FIGURE 6.3
_ Q O A) Effect of noradrenaline 10 - 10 M, added in a cumulative fashion,
on a ring segment of rabbit coronary artery with endothelium removed. The tone
was raised by the addition of 30 mM KC1 to the bath as indicated; W = washout.
B) Log cumulative relaxation-response curves of the rabbit coronary artery to
noradrenaline (10 nM - 1 mM). Results are expressed as percent relaxation of the
KCl-induced tone; mean + s.e.mean of six rabbits. B
Relaxation C%] 3 4 5 6 7 8 Noradrenaline 100 80- 60- 40- 20 0 - - -
7 5 3 4 5 6 7 8 i ------1 ------Lg Nrdeaie M [Noradrenaline] Log - 1 ------1 ------1------1 FIGURE 6.4
A) Effect of 10”^ - 10”^ M adenosine 5’-triphosphate (ATP), added in a cumulative fashion, on a ring segment of the rabbit coronary artery denuded of endothelium. The tone was raised by 30 mM KC1. A transient contraction precedes the relaxation at higher concentrations of ATP. W = washout. B) Log cumulative relaxation-response curves to ATP in the absence (closed symbols) and presence (open symbols) of 8-phenyltheophylline (8-PT). There was no significant difference in the response before and after 8-PT. Results are expressed as percent relaxation of the KCl-induced tone, and are the mean + s.e.mean of the data from seven rabbits. A ATP 8 7 6 5 4 3
A KCI
B 100 T FIGURE 6.5
_ O _A Effect of adenosine 10 - 10“ M, added in a cumulative fashion, on ring segments of the rabbit coronary artery with endothelium removed A) in the absence, and B) in the presence of 10“^M 8-phenyltheophylline (8-PT). The tone of the preparations was raised with 30 mM KC1. W = washout. C) Log cumulative relaxation-response curve of the coronary artery to 10 nM - 0.3 mM adenosine (closed symbols). The responses were significantly inhibited by 8-PT
10 jiM (open symbols), ** = P<0.01, *** = P<0.001. Results are expressed as a percentage of the KCL-induced tone, and are the mean + s.e.mean of data from six rabbits for all concentrations except 0.3 mM, where results are the mean of two rabbits. Adenosine 7 6 5 4 3
ig|_ 4min
i KCi
Adenosine 7 6
1g|_ 4 min A KCI
100 - i FIGURE 6.6
Response of the rabbit circumflex coronary artery at basal tone to A) adenosine 5’-triphosphate (ATP) and B) noradrenaline (NA) in ring segments with endothelium removed. 30 mM KC1 was used to contract rings as a control, W = washout of KC1. ATP and NA were added as single doses and were washed out as soon as maximal contraction was reached or after 2 minutes if there was no response to avoid desensitisation of the preparations. C) Non-cumulative log contraction-response curves to NA (squares) and ATP (circles) (both 10 /xM - 30 mM) in the absence of endothelium. Results are expressed as a percentage of the contraction to 30 mM KC1, and are the means ± s.e.mean of data from at least six rabbits. 1°0lC B Contraction C% KCO 60- 80- A KCI 40- 20 A KCI 0 - -
w 5 3 2 3 4 5 6 i ------10 10 A A 1 Lg Aoit M (Agonist] Log ------0. 00 3000 1000 100. 0 10 3000 1000 100 ______A A A A A A A pM NA ATP 1 ------jj M r\_ A 1 ------L 4min 4min 1 CHAPTER 7
PURINOCEPTOR SUBTYPES ON THE SMOOTH MUSCLE AND
ENDOTHELIUM OF THE RABBIT CORONARY ARTERY.
7.1 SUMMARY
The purinoceptor subtypes in the coronary artery of the rabbit were studied by comparing the effects of adenosine, ATP and its analogues, a,/3-methylene ATP and 2-methylthio ATP, on isolated vessels with intact endothelium and on vessels from which the endothelium had been removed.
For contraction, the rank order of agonist potency was a, /3-methylene ATP
> 2-methylthio ATP » ATP; adenosine did not produce contraction. Removal of the endothelium did not affect these responses but they were abolished by prior desensitisation with a,/3-methylene ATP. For relaxation, adenosine, ATP and
2-methylthio ATP were equipotent but a,/3-methylene ATP was without vasodilator effect and caused further contraction of the preparations. A transient contraction often preceeded the relaxations to ATP at higher concentrations. The dilator responses to adenosine, ATP and 2-methylthio ATP were significantly reduced, but not abolished, in preparations denuded of endothelium; prior desensitisation with a,/?-methylene ATP had no effect on the relaxation responses but abolished the initial transient contractions to ATP and 2-methylthio ATP.
133 These results indicate the presence of purinoceptor subtypes in the rabbit coronary artery; namely, a Pj-purinoceptor on the smooth muscle and to a lesser
extent on endothelial cells, a P 2 X~pur*noceptor located on the smooth muscle mediating vasoconstriction, and P 2 y _ purinoceptor mediating vasodilatation on both the smooth muscle and the endothelium.
7.2 INTRODUCTION
The receptors mediating responses to purine nucleotides and nucleosides have been designated Pj- and P 2 ~purinoceptors, with selectivity for adenosine and
ATP, respectively (Burnstock, 1978b). While Pj-purinoceptors on blood vessels generally mediate vasodilator responses, ATP acting via P 2 ~purinoceptors may cause relaxation or contraction of blood vessels (Burnstock & Kennedy, 1986c).
Burnstock and Kennedy proposed a subdivision of the P 2 ~purinoceptor into
^ 2 X anc* ** 2 Y subtypes, based on the rank order of agonist potency of structural analogues of ATP (Burnstock & Kennedy, 1985). At the P2X-pur*noceptor a,/3-methylene ATP is more potent than 2-methylthio ATP and ATP, while at the
P2 Y_Purinoceptor 2-methylthio ATP is most potent and a,j3-methylene ATP has no effect. In support of this division, the subtypes can be further distinguished by the selective action of a,/3-methylene ATP, which may act as an antagonist at the
^2X"pur^noceptor by desensitising the receptor (Kasakov & Burnstock, 1983), and which has no effect on the P 2 Y-Purinoceptor (Burnstock & Kennedy, 1985;
Mathieson & Burnstock, 1985). Reactive blue 2, an anthraquinone sulphonic acid derivative, has been shown to act as an antagonist at the P 2 Y"’ but not the p 2 x - ’ purinoceptor (Burnstock & Warland, 1987; Ralevic & Burnstock, 1988; Reilly et al.
1987). In general, p2X_pur*noceptors mediate excitatory responses in vascular
134 smooth muscle, while P 2 Y- Pur*nocePtors mediate inhibitory responses, usually via the endothelium (Burnstock, 1987). Thus the presence and distribution of the
P2 ~purinoceptor subtypes determines the response of a given vessel to ATP.
In Chapter 6 , ATP was shown to mediate relaxation in the rabbit epicardial by a direct action on the smooth muscle, in contrast to its mode of action in most other blood vessels. While the vasodilator actions of adenosine on coronary vessels have been known for many years (Berne, 1963; Boyaner, 1969), few studies have been reported on P 2 -purinoceptors in coronary arteries (Houston et al. 1987;
Hopwood & Burnstock, 1987). The experiments in this chapter were designed to determine the P 2 ~purinoceptor subtypes in the coronary artery of the rabbit by examination of the rank order of potency of the agonists a,/?-methylene ATP,
2-methylthio ATP and ATP, and the effects of selective antagonists. The distribution of the receptor subtypes was studied in preparations with and without endothelium.
7.3 METHODS
7.3.1 Pharmacology See Chapter 3.1.2
Acetylcholine 0.1 was used to confirm removal of the endothelium; at this concentration, preparations with intact endothelium relax, while preparations denuded of endothelium give a purely vasoconstrictor response (see Chapter 4).
Contractions in response to 30 mM KC1 and relaxations to 0.1 mM sodium nitroprusside were compared in preparations with and without endothelium, to ensure that the smooth muscle was not damaged during removal of the endothelium.
135 At basal tone, ATP, a,0-methylene ATP, and 2-methylthio ATP caused desensitisation of their own contractile response, so these drugs were added as single doses at each concentration and washed out as soon as the response reached a maximum. The preparations were then washed repeatedly for 40 min before the next dose of the agonist was added. At raised tone, the responses did not show desensitisation and the agonists were therefore added cumulatively to the bath.
The tension returned to baseline once the drugs were washed out, and the preparations were then washed repeatedly for 30 - 40 min before the next cumulative relaxation-responses were obtained.
In experiments when complete desensitisation of the P 2 X receptor was intended, a,0-methylene ATP was added as a single concentration of 10 /xM. Once the transient response to this had returned to baseline, subsequent additions of the
drug did not produce contraction unless the preparations were repeatedly washed.
Cumulative relaxation-responses and non-cumulative contraction-responses to the agonists were repeated in the same ring segments before and after desensitisation
to a,0-methylene ATP. Reactive blue 2 caused a profound loss of tone in the
vascular ring preparations and often initiated spontaneous activity, so this putative
P2 Y-PurinocePtor antagonist could not be used in this study.
7.3.2 Materials See Chapter 3.6
7.3.3 Statistics See Chapter 3.5.1
136 7.4 RESULTS
7.4.1 At Basal tone
ATP, 2-methylthio ATP and a,/3-methylene ATP each produced dose-dependant transient constriction of the rabbit coronary artery which reached a peak within 30 s and then rapidly returned to baseline, even in the continued presence of the drugs (Figure 7.1). The order of potency of the purines in producing contractions was a,j3-methylene ATP > 2-methylthio ATP > ATP
(Figure 7.2, Table 7.1); ATP was a weak agonist and produced contractions only at concentrations above 1 mM. At concentrations greater than 30 fiM the desensitising effect of a,/3-methylene ATP could not always be overcome and constrictor responses to subsequent higher doses of a,/3-methylene ATP were variable, a,/3-Methylene ATP was 10 times more potent than 2-methylthio ATP in producing contractions. Responses to ATP did not reach a maximum so a direct comparison of the -log[EC^Q] values could not be made, but comparison of the
ATP concentration required to produce a contraction equivalent to that seen with the EC^o of a,(3-methylene ATP indicates that a,/3-methylene ATP is >1000 times more potent than ATP in producing constrictor responses in this vessel (Table 7.1).
There were no significant differences in the slopes of the concentration-response curves for the three agonists (Table 7.1).
Following desensitisation of the P2X”pur*nocePtor with 10 fiM a,/3-methylene ATP, constrictor responses to all of the purine nucleotides were totally abolished even at the maximum concentrations examined (see Figure 7.1).
This was not due to a general depression of the contractility of the preparations, since the response of the preparations to KC1 was not affected (data not shown).
137 Removal of the endothelium had no effect on the constrictor responses to a,0-methylene ATP; the dose-responses curve to ATP was slightly shifted to the left, but this shift was not significant (Figure 7.2).
Adenosine did not produce a vasoconstrictor response in any of the preparations, whether the endothelium was present or not.
7.4.2 At Raised Tone
In preparations in which the tone was raised with 30 mM KC1, ATP and
2-methylthio ATP elicited dose-dependant relaxation of the preparations (Figure
7.3), preceded by a transient contraction at the higher concentrations (see Figure
7.5 A). The relaxation did not reach a maximum even at doses up to 3 mM, and thus no EC50 could be calculated, but comparison of the EC40, defined as the concentration required to produce a 40% relaxation of the KCl-induced tone, indicated that the two drugs were equipotent (Table 7.1). There was no difference in the slope of the curves. No concentration of a,0-methylene ATP caused
relaxation in any of the preparations; this analogue of ATP only elicited further contractions.
Relaxant responses to both 2-methylthio ATP and ATP persisted in
preparations denuded of endothelium, although at concentrations above 10 /xM
they were significantly reduced (Figure 7.4 A, B). Removal of the endothelium
appeared to have a greater effect on the relaxation to 2-methylthio ATP, but the
relaxation-response curves to the two agonists in vessels without endothelium were
not significantly different. The rapid, transient contraction preceding relaxation at
the higher concentrations was not affected by removal of the endothelium but
138 pretreatment with a,0-methylene ATP to desensitise the E2X“pur*noceptor abolished it, leaving a purely vasodilator response (Figure 7.5 A, B). The
vasodilation to both 2-methylthio ATP and ATP was not significantly affected by
desensitisation of the E2X-pur*noceptor with a,0-methylene ATP (Figure 7.5 C,
D).
Adenosine produced a purely vasodilator response in the preparations, with
an EC 4 Q (as defined above) equipotent with ATP (Table 7.1). The relaxation to
adenosine was significantly decreased in preparations denuded of the endothelium,
with a reduction in maximum relaxation and a shift in the EC^q (Figure 7.6,
Table 7.1).
7.5 DISCUSSION
This study demonstrates the presence of Pj- and P 2 -purinoceptors in the
rabbit isolated left coronary artery and identifies the subtypes of P 2 -purinoceptors
on the smooth muscle and endothelium which determines the nature of the
response to ATP in this vessel. In addition to the presence of P j- and
^2X"pur^noceptors’ coronary artery was found to be very unusual in that it
possesses E2 Y-pur*nocep*ors on the smooth muscle; this finding explains the
observed vaodilatation of coronary arteries to ATP in the absence of endothelium
described in Chapter 6 .
At the P2 ~purinoceptor that mediated vasoconstrictor responses, the agonist
order of potency was a,0-methylene ATP > 2-methylthio ATP > ATP, which
conforms to the pattern originally described by Burnstock and Kennedy (Burnstock
& Kennedy, 1985) for the P 2 x _Pur^nocePtor- Full anc* stable agonists of different
139 potencies acting on the same receptor should produce the same maximal response and the same slope of the concentration-response curves. In this study, ATP proved to be a weak vasoconstrictor agonist in this vessel and the maximal contraction could not be obtained despite the high concentrations used; the maximal contraction to a,j3-methylene ATP was less than that to 2-methylthio
ATP, probably due to desensitisation at high concentrations of a,j 8 -methylene
ATP. However, the slopes of the concentration-response curves were similar, and it is likely that the agonists were all acting on the same receptor. The abolition of all vasoconstrictor responses to the purine nucleotides by prior desensitisation with a,jS-methylene ATP confirms that this receptor is of the P 2 X subtype. It is likely that the density of these P2X“pur*noceptors in the coronary artery is relatively low since the contraction to ATP was weak despite the high concentrations used.
The p2X”pur*noceptors in the rabbit coronary artery appear to be located on the smooth muscle, since the responses were not diminished by removal of the endothelium; the non-significant increase in the constrictor responses to
2-methylthio ATP and ATP when the endothelium was removed may reflect the opposing vasodilator action of these nucleotides via the endothelium (see below).
Similar P2X“pur*noceptors have been found on the smooth muscle of many blood vessels, including the rat aorta and femoral artery (White et al. 1985; Kennedy et al. 1985), the rabbit mesenteric artery (Burnstock & Warland, 1987) and ear artery
(Kennedy & Burnstock, 1985a), the dog saphenous vein (Houston et al. 1987), and the perfused rat mesenteric vascular bed (Ralevic & Burnstock, 1988). The
^ 2 X“Pur^noceptor subtyPe d°es not appear to be found on the endothelium in any vessel studied so far.
140 Vasodilator responses to the purine nucleotides in this study of the isolated rabbit coronary artery were mediated by a purinoceptor subtype that was activated by ATP and 2-methylthio ATP, but not by a,/?-methylene ATP. Furthermore, the responses were not significantly affected by prior desensitisation with a,(3-methylene ATP, and thus the receptor can be clearly distinguished from the
P2 x subtype. Although the responses to high concentrations of the agonists were reduced, the relaxation persisted in vessels denuded of endothelium, demonstrating that the purinoceptor subtype(s) mediating vasodilator responses in this vessel are located directly on the smooth muscle as well as on the endothelium.
In most blood vessels, vasodilator responses to the purine nucleotides are mediated through their actions on the T2Y~Pur*nocePtor (Houston et al. 1987;
Ralevic & Burnstock, 1988) where 2-methylthio ATP is generally a much more potent agonist than ATP (Burnstock & Kennedy, 1985). In this study of the rabbit isolated coronary artery, 2-methylthio ATP and ATP were equipotent in vessels with an intact endothelium; in vessels denuded of endothelium, ATP appeared more potent than 2-methylthio ATP. One explanation of these findings is a
P2 Y_PurinocePtor located on the endothelium in the rabbit coronary artery which is activated preferentially by 2-methylthio ATP, and a second vasodilator purinoceptor on the smooth muscle at which ATP is the more potent agonist.
However, it seems more likely that the subtype mediating both these responses is the P2 Y"PurinocePtor- The maximal contraction to the agonists was not reached despite the high concentrations used, but the slope of the curves was the same,
indicating that they are probably acting on the same receptors.
A more secure characterisation of receptor subtypes may be made by the use of antagonists. Unfortunately, the best known antagonist at the
141 P2 Y-PurinocePt°r» reactive blue 2 , produced a profound loss of tone in this preparation and could not be used to examine this receptor further. Reactive blue
2 has been found by other workers to cause a similar loss of vascular tone (Ralevic
& Burnstock, 1988; Muramatsu & Kigoshi, 1987) and is relatively selective for the
P2 Y-purinoceptor only over a limited concentration range (Burnstock & Warland,
1987). The development of a more specific and less toxic antagonist at the
P2 Y“PurinocePtor would allow better characterisation of responses such as those found in this study.
This is the first study to suggest the presence of P 2 Y- PurinocePtors on the smooth muscle of the coronary artery. P 2 Y~PurinocePtors have been found on the smooth muscle a few specialised blood vessels such as the rabbit mesenteric artery
(Burnstock & Warland, 1987) and portal vein (Kennedy & Burnstock, 1985b; Reilly
et al. 1987) but in almost all other blood vessels, including the canine coronary artery (Houston et al. 1987), relaxant responses to ATP are mediated exclusively
via the endothelium (De Mey & Vanhoutte, 1981; Furchgott, 1981; Rapoport et al.
1984; Martin et al. 1985). The study shows that, as for acetylcholine, the response
of the dog coronary artery is not necessarily representative of other species. The
response to ATP after removal of or damage to the endothelium would be
qualitatively different in the dog and the rabbit; to date, no data exist for the
response in human coronary arteries.
It should be noted that the dog coronary artery shows appreciable
contraction to NA, unlike coronary arteries from many other species; as shown in
Chapter 6 , the vasodilator smooth muscle response to ATP in the coronary artery
of the rabbit parallels the response to noradrenaline. The differences between the
142 rabbit coronary artery and most other blood vessels is illustrated schematically in
Figure 7.7. The reasons for the variation in receptor distribution among different blood vessels, and even in the same vessel among different species, are not known but clearly will produce a divergence in the response to neuronally-released purine nucleotides. Moreover, in vessels with smooth muscle P2Y“Pur*nocePtors’ damage to the endothelial cells, for example by atherosclerosis, will not shift the ATP response from one of dilation to one of constriction; in the coronary artery this may represent a mechanism to protect the tissues subserved by these vessels from hypoxic damage.
Adenosine has been shown to relax the coronary arteries of various species,
including those in the dog, cow, and man, through its action on Pj-purinoceptors
(Belardinelli et al. 1989). In the present study, the relaxant responses to adenosine
seen in the rabbit coronary artery were significantly inhibited but not abolished by
removal of the endothelium, suggesting an endothelial-dependant component to the
response. Pj-purinoceptors appear to be located on the smooth muscle in most
blood vessels (Burnstock & Kennedy, 1986), although there are a few reports in
which adenosine has been shown to act via the endothelium (Frank & Bevan, 1983;
Gordon & Martin, 1983). In the coronary arteries of dogs, Rubanyi and
Vanhoutte found that the relaxation to adenosine was inhibited but not abolished
by removal of the endothelium, signifying an endothelial-dependant component to
the response (Rubanyi & Vanhoutte, 1985). However, White and Angus (White &
Angus, 1987) did not find any endothelial component of the response to adenosine
in rings of greyhound coronary arteries contracted with the thromboxane
A2 ~mimetic U46619. The reasons for this discrepancy are not obvious, although it
is possible that mongrel dogs have a different receptor distribution than
greyhounds. Support for an endothelial effect of adenosine is provided by Biro
143 and coworkers who found that haemoglobin, a known inhibitor of EDRF, abolished the vasodilator response to adenosine in the coronary vascular bed of the in-situ dog heart (Biro et al. 1986). Although the Pj-purinoceptors on the smooth muscle and endothelium may be of different subtypes, the nature of the response to adenosine is unlikely to be affected by diseases involving the endothelium since both subtypes mediate vasodilatation.
Adenine nucleotides are rapidly degraded to adenosine in the coronary circulation in vivo (Baer & Drummond, 1968; Paddle & Burnstock, 1974;
Ronca-Testoni & Borghini, 1982) but adenosine is not a more potent agonist than
ATP in this vessel. It was shown in coronary arteries denuded of endothelium in
Chapter 6 that the smooth muscle responses to ATP are little affected by the
Pj-purinoceptor antagonist 8 -phenyltheophylline at a concentration that significantly inhibited the relaxation to adenosine; the results presented here in vessels with the endothelium intact were similar.
This work has demonstrated the presence of purinoceptor subtypes in the isolated coronary artery of the rabbit. P 2 X~pur*noceptors mediate vasoconstriction and are located exclusively on the smooth muscle, while Pj- and
P2 Y~PurinocePtors mediating vasodilatation are present both on the smooth muscle and on the endothelium. The smooth muscle vasodilator responses dominate in this vessel and this may have important functional consequences in determining both the normal response of the vessel to neuronal and humorally released adenine nucleotides and nucleosides, and the effect of damage to the endothelium.
144 TABLE 7.1 Responses of the rabbit coronary artery to ATP and its analogues,
a) Basal tone
Agonist Response pE>2 Slope Relative potency
of,/J-mATP +E Contraction 5.59 (0.14) 0.40 (0.07) 1,047 -E Contraction 5.74 (0.24) 0.36 (0.07) 1,479
2-MT +E Contraction 4.56 (0.12) 0.53 (0.09) 98 -E Contraction 4.78 (0.13) 0.51 (0.06) 162
ATP +E Contraction 2.57 * 0.60 (0.24) 1 -E Contraction 3.03 * 0.57 (0.17) 3
Adenosine +E No response -E No response
b) Raised tone
Agonist Response -Log EC4 0 Slope Relative potency
a,j8 -mATP +E Contraction only -E Contraction only
2-MT +E Relaxation 4.72 (0.56) 26.6 (5.7) 2 -E Relaxation Not reached
ATP +E Relaxation, 4.95 (0.23) 27.0 (3.6) 1 -E or 4.18 (0.32) 27.3 (3.8) 6 contraction then relaxation
Adenosine +E Relaxation 5.02 (0.18) 27.4 (4.7) 1 -E Relaxation 4.31 (0.08) 27.1 (3.0) 1
a) * Maximal contraction to ATP was not reached and pD 2 values were estimated as the mean concentration required to produce 50% of the maximal contraction to a,jS-mATP. b) The tone of the preparations was raised with KC1. Maximal relaxations were not reached; the -Log EC^g is defined as the concentration required to produce 40% relaxation of the KCl-induced tone.
Values given are mean (standard error of the mean) of data from seven animals. Abbreviations: a,j(?-mATP = a,/J-methylene ATP; 2-MT = 2-methylthio ATP.
145 FIGURE 7.1
Response of the rabbit coronary artery at basal tone to A) a,/?-methylene
ATP, B) 2-methylthio ATP and C) ATP. The agonists were added as single doses increasing in half-log increments and produced a transient vasoconstrictor response. The preparations were washed repeatedly for 40 min between each addition to avoid desensitisation. Where indicated, 10“^ M a,/?-methylene ATP was added to deliberately desensitise the P 2 X~Pur*nocePtor; this abolished responses to maximal concentrations of all three agonists (* indicates a concentration of 100 jtiM for a,jS-methylene ATP, 300 for 2-methylthio ATP and 3000 /*M for ATP). A a,/3-m ATP t J k -L Jv. Jl -i_L a,)3-methylene ATP
B a./3-m ATP
2-methylthio ATP
C a,/3-rn ATP
4min i * _
ATP
kkkkkkkkkk k 0.1 0.3 1 3 10 30 100 300 1000 3000 * jjM Contraction (g tension!) 1.0 -i
0. 8-
0.6-
0.4-
0 . 2 -
0 - I------1------1------1------1------1------1------1------1------1 7 6.5 6 5.5 5 4.5 4 3.5' 3 2.5 - Log (Purine) M
FIGURE 7.2
Non-cumulative log contraction-response curves of the rabbit coronary
artery to a,0-methylene ATP (diamonds), 2-methylthio ATP (triangles) and ATP
(circles) in the presence (solid line) and absence (dotted line) of endothelium. The
agonists were added as single doses, followed by repeated washing for 40 min to
avoid desensitisation of the preparations. There were no significant differences
between preparations with and without endothelium. Results are the mean + s.e.m.
of data from seven rabbits. Relaxation (%]
100 T
FIGURE 7.3
Log cumulative relaxation-responses curves of ring segments of rabbit
coronary artery with intact endothelium to 2-methylthio ATP (triangles) and ATP
(circles). The responses to ATP were preceded by a transient contraction at higher
concentrations. a,j3-Methylene ATP elicited only further contractions. Results are
expressed as percent relaxation of the tone induced by KC1 and are the means +
s.e.m. of data forom seven rabbits. FIGURE 7.4 A,B
Relaxation-response curves to A) ATP and B) 2-methylthio ATP in rabbit coronary artery preparations with endothelium intact (solid line) or removed
(dotted line). Relaxation to these agonists persisted in preparations denuded of endothelium but was significantly reduced at higher concentrations; * = p<0.05.
Results are expressed as percent relaxation of the tone induced by 30 mM KC1 and are the means + s.e.m. of data from seven rabbits. Relaxation C%3 100-i A ATP -7 -6 -5 -4 -3
4min
B ATP -7 -6 -5 -4 -3 Y Y Y c------CQ 4min A KCI
FIGURE 7.5 A,B
Effect of ATP added in a cumulative fashion on ring segments of the rabbit coronary artery. The tone of the preparations was raised with 30 mM KC1.
Transient vasoconstriction which preceded the relaxation to higher concentrations of ATP in the control preparations (A) was abolished by prior desensitisation with a,j3-methylene ATP while the relaxation response was unaffected (B). Numbers refer to log molar concentrations of ATP; W indicates washout. FIGURE 7.5 C,D
Log cumulative relaxation-response curves to C) ATP and D)
2-methylthio ATP in preparations with endothelium intact (solid lines) and removed (dotted lines). Solid symbols represent control responses to the agonists, open symbols represent responses after desensitisation of the preparations with a, 0-methylene ATP. Results are expressed as percent relaxation of the
KCl-induced tone and are the means + s.e.m. of data from seven animals. Relaxation (%)
100 -i
i------1------1------1------1 8 7 6 5 4 -Log C2-methylthio ATP] M Relaxation (%) 100 T
- Log CAdenosine] M FIGURE 7.6
Log cumulative relaxation-response curves to adenosine in rabbit coronary artery preparations with the endothelium intact (solid line) or removed (dotted line). Relaxation to adenosine persisted in preparations without endothelium but was significantly reduced at higher concentrations; * = P,0.05, ** = p<0.01.
Results are expressed as percent relaxation of the tone induced by KC1 and are the means + s.e.m of data from seven rabbits, except at concentrations of 3x10“^ M adenosine, where data is from 2 animals. FIGURE 7.7
Schematic diagram illustrating the specialised postjunctional smooth muscle responses of the rabbit epicardial coronary artery to the sympathetic cotransmitters, noradrenaline (NA) and ATP. In most vessels, where NA is vasoconstrictor (+) by action on a-adrenoceptors, ATP also produces direct vasoconstriction of the smooth muscle via P 2 x -PurinocePtors- I *1 contrast, in the coronary artery which responds to NA by vasodilation via /J-adrenoceptors, ATP also acts to vasodilate the smooth muscle by acting on P 2 Y_PurinocePtors- MOST VESSELS CORONARY ARTERIES CHAPTER 8
RESPONSES OF CORONARY ARTERIES TO NEUROTRANSMITTERS: CHANGES WITH SEXUAL MATURITY IN THE FEMALE RABBIT.
8.1 SUMMARY
In isolated epicardial coronary arteries from female New Zealand white rabbits, vasodilator responses to substance P (SP) and acetylcholine (ACh) were found to be mediated exclusively via the endothelium, while noradrenaline (NA), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) caused direct relaxation of the smooth muscle. Changes associated with the development of sexual maturity in these vasodilator responses, as well as changes in vasoconstrictor responses to potassium (KC1), ACh, neuropeptide Y (NPY) and serotonin (5HT), have been assessed in rabbits aged 4, 6 , and 12 months. These animals become sexually mature between 4 and 6 months of age.
There was a significant reduction from 4 to 12 month old animals in both the direct smooth muscle vasodilator responses to CGRP (P<0.01) and VIP
(P<0.001) and in the endothelium-mediated response to SP (P<0.005). Vasodilator responses to concentrations of ACh greater than 0.1 /iM were virtually absent in the 6 - and 12-month-old animals. No change in maximal relaxation to NA was seen with maturation, although there was a small, significant increase in potency.
154 The contractile responses of the smooth muscle to 30 mM KC1 declined steadily as the animals matured, but the maximal contraction to ACh (P<0.05),
NPY (p<0.02) and 5HT (p<0.05) increased significantly between 4 and 12 months of age.
These results indicate that following sexual maturation in the female rabbit the epicardial coronary artery shows a significant increase in maximum responses to vasoconstrictor neurotransmitter agents and, at the same time, a marked decline
in responses to some vasodilator agents, both those acting directly on the smooth
muscle and those acting via the endothelium.
8.2 INTRODUCTION
Perivascular nerves contain many different neurotransmitter substances, such as adenosine 5’-triphosphate (ATP), serotonin (5HT) and various peptides, in addition to the "classical" neurotransmitters, noradrenaline (NA) and acetylcholine
(ACh) (Burnstock, 1988; Dhital & Burnstock, 1989). One nerve may contain more
than one neurotransmitter (Burnstock, 1976; 1990) and nerve populations supplying
some blood vessels and organs have been shown to change with development, aging
and disease (Crowe et al. 1983; Dhall et al. 1986; Dhital et al. 1988; Mione et al.
1988). The vasomotor response of a blood vessel in vivo will depend not only on
the nature and density of the perivascular nerves but also on the postjunctional
actions of these neurotransmitters on the vascular smooth muscle. Several studies
have found changes in the responsiveness of vascular smooth muscle associated
with aging (Fleisch, 1980; Duckies, 1983; Duckies & Banner, 1984) but most of
these have focused on the changes in responses to NA.
155 In some studies, differences have been noted between the changes in males and females (Fleisch et al. 1970). Oestrogens are known to affect the responsiveness of blood vessels to some vasoactive agents including NA and 5HT
(Altura & Altura, 1977; Altura & Altura, 1977); in this study changes in the vasodilator and vasoconstrictor responses of epicardial coronary arteries have been investigated in female rabbits around the age of development of sexual maturity.
In addition to the responses to NA and ACh, we examined changes in the responses to 5HT, neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (YIP) and substance P (SP). All these substances are known to be present in sympathetic, parasympathetic or sensory perivascular nerves and to alter vasomotor tone by acting directly on the smooth muscle, or via the production of endothelium-derived relaxant factor (EDRF) when released from subpopulations of endothelial cells (Lincoln & Burnstock, 1990).
8.3 METHODS
Isolated epicardial coronary arteries from female New Zealand white rabbits aged 4, 6 and 12 months were examined as described in Chapter 3.1.2.
These animals become sexually mature between 4 and 6 months of age; no attempt was made in these studies to ascertain the phase of the oestrus cycle of the animals at the time of experimentation.
NA and ACh were added cumulatively to the organ bath, but responses to the peptides and 5HT demonstrated tachyphylaxis, so single maximal concentrations of these compounds were used (see Chapter 3.1.2).
156 8.3.1 Materials See Chapter 3.6
8.3.2 Statistics See Chapter 3.5.1
8.4 RESULTS
There was a small, steady rise in the mean body weight of the animals between the age groups studied, from 3.38 ± 0.12 kg at 4 months, to 3.54 + 0.09 kg at 6 months (p<0.05) and 4.52 ± 0.04 kg at 12 months of age (p<0.001).
8.4.1 Contractile Responses
KC1 (30 mM), NPY (0.1 /*M), 5HT (10 fiM) and ACh (0.1 /*M - 1 mM) all produced contractile responses in the preparations at basal tone. The responses to
KC1, ACh and 5HT were rapid, while those to NPY were slower in onset. NA a produced no contraction of the preparations at concentrations less than 10 M; no contractile responses to SP, CGRP or VIP were seen.
The contractility of the smooth muscle to KC1 was greatest in the younger animals and declined with age; by 12 months it was significantly lower than in the
4-month-old animals. In spite of this, NPY and 5HT produced only small contractions in the 4-month-old and 6-month-old animals and significantly greater responses in the 12-month-old animals (p<0.02 for NPY, p<0.05 for 5HT; Figure
8.1). When expressed as a percentage of the contraction to KC1 in each ring preparation, the contraction to NPY rose from a mean of 6.5% at 4 months to 88% in the 12-month-old animals while the contraction to 5HT rose from 13.5% to
157 56%. There was, however, no clear relationship between the magnitude of the contractions to KC1 and those to NPY and 5HT in individual ring preparations even in animals of the same age.
The contractile responses to ACh were more variable than the responses to the other vasoconstrictor substances examined and this is reflected in the larger standard errors of the means (Figure 8.2). There was, however, a marked and significant increase in the maximal response to ACh between the 4- and
6-month-old animals which persisted at 12 months of age (Figure 8.2, Table 8.1).
The vT>2 °f the responses also showed a small, significant increase with age
(Table 8.1).
8.4.2 Relaxant Responses
In preparations with the tone raised by KC1, relaxant responses were obtained to NA (1 nM - 1 mM), CGRP (0.3 /xM), SP (0.1 /xM), VIP (0.1 /xM) and
ACh (10 nM - 10 /xM). These responses were examined in the 4-month-old animals in preparations with the endothelium intact and with the endothelium removed to determine whether the agonists were acting directly on the smooth muscle or via release of EDRF (Figure 8.3). The responses to NA, CGRP and VIP were unchanged in preparations denuded of endothelium but the relaxant responses to SP and ACh were entirely endothelium-dependent and were abolished when the endothelium was removed.
The maximal responses to NA were unchanged as the animals matured
(Figure 8.4, Table 8.1), although the pD2 of the responses to NA showed a small, significant increase from 4- to 6-month-old rabbits. The relaxant responses to the
158 vasodilator neuropeptides all declined with age; this was most marked between 4 and 6 months and then continued more gradually up to 12 months (Figure 8.5).
By 12 months of age, the relaxation was very significantly lower than in the
4-month-old animals for CGRP (p<0.01), VIP (pcO.OOl) and SP (p<0.005). The decline was roughly parallel for both the endothelium-independent vasodilators,
CGRP and VIP, and the purely endothelium-dependent vasodilator SP (Figure 8.5).
Relaxant responses to concentrations of ACh greater than 10 M were rarely seen in preparations from the 6- and 12-month-old animals (Figure 8.6) but it should be noted that a marked increase in constrictor responses to ACh also occurred in these 6- and 12-month-old animals at concentrations of ACh greater than 10 M
(see Figure 8.2).
8.5 DISCUSSION
The results of this study indicate that vasoconstrictor responses of the coronary artery to NPY and 5HT increase while responses to the vasodilator neuropeptides CGRP, SP and VIP decline after the development of sexual maturity in female rabbits. The vasodilator response to the classical sympathetic neurotransmitter, NA, did not show a corresponding decline over the period of study. Changes in endothelial vasodilator responses to ACh were obscured by a pronounced increase in the vasoconstrictor responses to ACh as the rabbits matured.
Age-related changes in the responsiveness of vascular smooth muscle to several agents have been examined in previous studies (Fleisch, 1980; Duckies &
Banner, 1984). The findings vary from one artery to another, e.g. the aorta, pulmonary artery and mesenteric artery in the rat (Cohen & Berkowitz, 1974;
159 Ljung & Stage, 1975; Fleisch & Hooker, 1976) and ear artery of the rabbit (Owen,
1986), and may differ from one species or even one strain to another (Fleisch,
1971; Fleisch & Hooker, 1976). The direction of change for both vasodilator and vasoconstrictor responses depends on the vasoactive agonist examined.
8.5.1 Vasoconstrictor Responses
In this study, there was an age-related fall in the contractile responses of the rabbit coronary arteries to KC1 but contraction in response to stimulation by
ACh, NPY and 5HT increased over the same period. The lower contraction to
KC1 in the older animals might be related to changes in the structure and mechanics of the vessel wall - previous studies have shown changes in systemic arteries, such as an increase in the collagen content and collagen-to-elastin ratio during development and an increase in wall stiffness with age (Cox et al. 1976;
Dobrin, 1978) - but from this study in vitro it cannot be shown to reflect a true change in the maximal contractility of the vessels in vivo. However, the fall in response to KC1 demonstrates that the increase in vasoconstrictor responses to
ACh, NPY and 5HT in the study cannot be attributed to a non-specific increase in the contractility of the coronary arteries with maturation and are more likely to be associated with changes in receptor expression.
The increases in vasoconstriction to ACh, NPY and 5HT with age did not occur in parallel; the change in response to ACh occurred during the period between 4 and 6 months when the rabbits become sexually mature, while the responses to NPY and 5HT showed no change during this stage but a marked increase 6 months later. In view of the different time course of the changes to
NPY and 5HT compared to ACh, more than one mechanism may be operating. In
160 beagle coronary arteries, Toda has reported that the response to NA changed from constriction to relaxation as the animals matured (Toda et al. 1986b) but the rabbit coronary arteries in this study showed no constriction to noradrenaline at concentrations less than 10 M even in the young animals.
Age-related changes in vasoconstrictor innervation and responses have been studied in most detail in cerebral blood vessels. Differential changes in cerebrovascular innervation with vasoconstrictor NA-, NPY- and 5HT-containing nerves have been demonstrated in the rat: the density of NPY-containing nerves supplying the basilar artery increased between 1 and 4 months of age, while 5HT-containing nerves markedly declined and noradrenergic nerves either rose or showed no change (Dhital et al. 1988; Mione et al. 1988). In a study of the smooth muscle responses of the rabbit basilar artery the vasoconstriction to KC1, NA and 5HT showed a marked increase between 3 and 6 months of age, but the response to 5HT then declined in 12-month-old rabbits (Toda & Hayashi, 1979). This contrasts with a study of the beagle cerebral artery by the same group where the maximum contraction to NA fell between 3 months and 3 years of age but the maximum response to 5HT was unchanged (Toda et al. 1986a).
8.5.2 Vasodilator Responses
8.5.2.i Responses to Noradrenaline
Vasorelaxant responses to NA are generally mediated via ^-adrenoceptors. A decrease in the relaxation to /3-adrenoceptor stimulation with age has been observed in several blood vessels (Fleisch et al. 1970; Fleisch & Hooker, 1976; Fleisch, 1980; Duckies & Banner, 1984) although some workers have found an
161 age-related increase in relaxation (Park et al. 1976). The response of the rabbit epicardial coronary artery to noradrenaline was shown in Chapter 5 to be mediated by jSj- and not by /^-adrenoceptors; in the present study, there was no change in the maximum relaxation to noradrenaline at any age, although there was a slight increase in the sensitivity of the coronary vascular smooth muscle to noradrenaline between 4 and 6 months of age. O’Donnell and Wanstall reported a decrease in relaxation to isoprenaline in aged rats and suggested from their study that the effect may be greater on the /^-adrenoceptor than on the /?j -adrenoceptor (O’Donnell & Wanstall, 1984b). However, the magnitude of drug-induced relaxation has been shown to decrease when the level of initial smooth muscle tone rises (Cohen & Berkowitz, 1974), so it is possible that a decrease in the maximum
relaxation of the coronary artery to noradrenaline would have been observed if the contraction of the vessels to KC1 in the older animals had been equal to that in the
young animals.
8.5.2.ii Responses to Vasodilator Peptides
To my knowledge, there are no previous reports of changes in vascular smooth muscle responses to the peptidergic neurotransmitters during sexual
maturation. Perivascular peptide-containing and noradrenergic nerves are known
to show different patterns of change with development; in the guinea-pig between
4 weeks and 4 months of age, there was either no change or a fall in the density of perivascular innervation with NA-, CGRP-, VIP- and SP-containing nerves,
depending on the vessel examined (Dhall et al. 1986), while the density of
innervation of the cerebrovascular nerves in male rats was unchanged (NA) or fell
(CGRP, VIP) from 4 weeks to 4 months of age (Mione et al. 1988). In the present study, a decrease in the postjunctional vasodilation of the coronary artery to
162 CGRP, VIP and SP at the age associated with sexual maturation in female rabbits has been demonstrated. No decrease in the vasodilator response to NA was found in the same animals which indicates that the changes in response to the neuropeptides are selective. Furthermore, the decrease is unlikely to be explained by changes in the tone of the preparations with age since there was an age-related fall in the tension induced by KC1: as outlined above (Cohen & Berkowitz, 1974), drug-induced relaxation should be greater under these circumstances rather than reduced. The decline in relaxation to CGRP and VIP was at least as great as that to SP, which suggests that the effect is more likely to be due to changes in the smooth muscle response rather than in the production or release of EDRF from the endothelium.
The relaxant responses to ACh in coronary vessels from the 4-month-old females in this study were smaller than the responses in the male New Zealand white rabbits in Chapter 4, which were of the same age and were examined under the same conditions. The responses to SP were, however, comparable to those which I observed in several ring preparations from these male animals (not shown), which suggests that the difference is not due to inadvertent damage to the endothelium. In Chapter 4, ACh was shown to cause endothelium-dependent vasodilation at concentrations up to 10-^ M but a dominant smooth muscle vasoconstriction at higher concentrations; this same pattern of response was observed in the 4-month-old female animals in this study. Changes in endothelium-mediated relaxation to ACh with maturation were obscured by a dominant increase in the contractile response of the smooth muscle to ACh over the same period.
163 8.5.3 The Role of Sexual Maturation
Most studies of aging or maturation have not distinguished between male and female animals but Fleisch and coworkers noted that age-related decreases in /?-adrenoceptor activity were slower in female than in male rabbit thoracic aortas
(Fleisch et al. 1970). In a study by Hayashi and Toda (Hayashi & Toda, 1978), which covered the development from immature to mature rabbits but did not distinguish between the sexes, the responses of the aortae showed a marked change around the age of maturity. They found the constriction to KC1, histamine and noradrenaline increased in the immature animals but stabilised at maturity, while constriction to 5HT was stable in the immature animals and showed a marked decline beginning when the animals reached maturity. Relaxation responses to isoprenaline and adenosine increased in the immature animals, but then began a marked decline after maturity. These studies suggest that the onset of sexual maturity may be associated with profound changes in the control mechanisms regulating vascular smooth muscle tone.
Vascular smooth muscle is known to possess specific oestrogen receptors
(Harder & Coulson, 1979) and the presence of oestrogen has been shown to influence blood flow and vascular tone both directly (Harder & Coulson, 1979) and by indirect mechanisms involving circulating or local levels of histamine, acetylcholine, kinins and catecholamines (Altura & Altura, 1977). It has also been shown that oestrogens enhance the responsiveness of blood vessels to angiotensin, epinephrine and serotonin (Altura & Altura, 1977). Untreated female rats as well as male rats treated with oestrogen have been found to have significantly higher arterial blood pressures after experimental trauma than do untreated male rats
(Altura, 1976). Some early studies in the 1940s also found evidence of a
164 relationship between ACh and oestrogens; it was shown that oestrogens increased the ACh-like substance in the nasal mucosa of cats and caused a temporary increase in the ACh content of the uterus of ovariectamised rabbits and cats, while atropine blocked oestrogen-induced vasodilation in the ears of ovariectamised rabbits (for references see Haigh et al. 1965). However a link between these effects and changes in the response of vascular smooth muscle to ACh remains obscure. A rise in endogenous oestrogens associated with sexual maturation in the rabbits in this study may be implicated in the altered vasomotor responses of the coronary vascular smooth muscle. If this is so, the effect cannot be due to an acute interaction with circulating oestrogens since we examined the vessels in vitro but it could, for example, be due a trophic change in receptor expression, density or coupling to the second messenger system.
From the results presented in this chapter, it is evident that changes in the responses of the coronary artery to the peptide neurotransmitters occur at the age of sexual maturation in female rabbits and may be independent of changes in the responses to the classical neurotransmitters. Coupled with differential development in the innervation of these vessels, this suggests complex and subtle changes may occur in the regional control of blood vessel tone in vivo. More detailed studies on the relationship to endogenous levels of oestrogens may help to clarify the mechanisms involved.
165 TABLE 8.1. Contractile responses to acetylcholine (ACh) and relaxant responses to noradrenaline (NA) in ring preparations of circumflex coronary arteries from sexually maturing female rabbits.
Age of Animals (Months) 4 6 12
Acetylcholine
Pd2 5.53 (0.12) 5.85 (0.18) 6.08 (0.13) * Maximal Contraction 0.69 (0.13) 1.30 (0.20) * 1.29 (0.22)
Slope 0.51 (0.05) 0.71 (0.09) 0.67 (0.12)
n 5 5 7
Noradrenaline pD2 5.86 (0.17) 6.38 (0.04) * 6.70 (0.13) **
Maximal Relaxation (%) 71.8 (6.1) 68.3 (5.0) 69.9 (3.6) Slope 33.8 (7.0) 33.2 (3.9) 27.1 (1.4)
n 6 5 7
Maximal contraction to ACh is expressed as g tension generated; maximal relaxation to NA is expressed as percent relaxation of the tone induced by 30 mM
KC1. Responses are expressed as mean (s.e.m.) of data from a number of animals in each group, denoted by n. * = p<0.05, ** = p<0.01 compared to 4-month-old animals.
166 2.00 n
1.75-
1.50-
c o 1.25- ’(Dc CD D) C 1.0 0 - o o Ctf l _ 4—• C 0.75 - o O
0.50-
0.25-
0 -
FIGURE 8.1
Contractile responses of coronary arteries from 4-, 6- and 12-month-old rabbits to 30 m M KC1 (squares), 0.1 /xM neuropeptide Y (circles) and 10 jtM 5-hydroxytryptamine. Results are expressed as grams of tension generated and are the means + s.e.m. of data from six to eight rabbits at each age. There was a significant rise in the contraction to neuropeptide Y (P<0.02) and 5-hydroxytryptamine (P<0.05) between 4 and 12 months, despite a fall in the
contraction to KC1 (P<0.005). 1.6 n
1.4 -
1.2 - /—> c o g 1.0 - 4-» 0.4 - 0.2 - 0 - l------1------1------1 l i 8 7 6 5 4 3 - Log (Acetylcholine!) M FIGURE 8.2 Cumulative contractile-response curves for acetylcholine (ACh) in 4- (circles), 6- (triangles) and 12-month-old rabbits (squares). Results are expressed as grams of tension generated and are the means + s.e.m. of data from five to eight rabbits. There was a significant increase in the maximal contraction to ACh between 4 and 6 months of age (P<0.05), but no further change at 12 months of age. ACh NA SP VIP CGRP FIGURE 8.3 Relaxant responses of coronary arteries from 4-month-old rabbits using preparations with the endothelium intact (open bars) or denuded (hatched bars). Removal of the endothelium did not affect the maximal relaxation to 100 /iM noradrenaline (NA), 3 /tM vasoactive intestinal polypeptide (VIP) or 2.6 n M calcitonin gene-related peptide (CGRP) but the responses to 0.1 /iM acetylcholine (ACh) and 0.1 n M substance P (SP) were abolished. Results are expressed as percent relaxation of the tone induced by 30 mM KC1 and are the means + s.e.m. of data from five to eight animals. 100 n FIGURE 8.4 Cumulative relaxation-response curves to noradrenaline in coronary arteries from 4- (circles), 6- (triangles) and 12-month-old rabbits (squares). Results are expressed as percent relaxation of the tone induced by KC1 and are the means + s.e.m. of data from six to eight rabbits at each age. 100-i 8 0 - ^_j c 60 - o cd x _cd (D 40 - a: 2 0 - 0 J i i ” i 4 6 12 Age [months) FIGURE 8.5 Relaxant responses of coronary arteries from 4-, 6- and 12-month-old rabbits to 0.1 f x M substance P (SP; squares), 3 /iM vasoactive intestinal polypeptide (VIP; circles) and 2.6 jxM calcitonin gene-related peptide (CGRP; triangles). Results are expressed as percent relaxation of the KCl-induced tone and are the means + s.e.m. of data from six to eight rabbits at each age. The relaxant responses all fell significantly between 4 and 6 months of age (P<0.005 for SP; P<0.001 for VIP and P<0.01 for CGRP). 8 7 6 5 - Log (Acetylcholine) M FIGURE 8.6 Cumulative relaxation-responses to acetylcholine in coronary arteries from 4- (circles), 6- (triangles) and 12-month-old rabbits (squares). Results are expressed as percent relaxation of the tone induced by 30 m M KC1 and are the means + of data from five to seven rabbits at each age. CHAPTER 9 RECOVERY OF IMPAIRED ENDOTHELIUM-MEDIATED RELAXATION IN CORONARY ARTERIES DURING EARLY DEVELOPMENT OF ATHEROSCLEROSIS. 9.1 SUMMARY Long-term changes in responses to endothelium-dependent and endothelium-independent vasoactive agents have been examined in coronary arteries from Watanabe Hereditable Hyperlipidaemic (WHHL) rabbits, which are known to have elevated plasma cholesterol and to develop coronary atherosclerotic lesions by around 12 months of age. In W H H L rabbits at 4 months of age, relaxations to the endothelium-dependant vasodilators substance P (SP) and acetylcholine (ACh) were significantly reduced compared to age-matched New Zealand White (NZW) rabbits but endothelium-independent relaxations to noradrenaline (NA), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were unchanged. Contractions to serotonin (5HT) and neuropeptide Y (NPY) in WHHL rabbits were increased compared to the NZW while maximal contraction to histamine was smaller. Age-related changes in the responses of WHHL rabbits to direct smooth muscle vasoconstrictors (KC1, histamine, 5HT and NPY) and vasodilators (NA, 173 CGRP and VIP) were very similar to those seen in NZW rabbits, suggesting that they were normal responses to maturation. However, endothelial-mediated relaxation to SP actually increased in the WHHL rabbits while the responses in the NZW animals declined; by 12 months the WHHL responses had recovered to equal those in NZW controls. Relaxation to ATP, which acts on both the smooth muscle and the endothelium in this vessel, was also higher in the older WHHL animals than in age-matched NZW rabbits. These results suggest that endothelium-mediated relaxation is impaired in hypercholesterolaemic WHHL rabbits but may actually show a compensatory recovery in the early stages of the development of atherosclerosis. 9.2 INTRODUCTION In recent years autonomic innervation and vasomotor responses have been shown to alter not only with physiological changes such as aging but also with diseases such as atherosclerosis (Burnstock, 1990b). Early studies in atherosclerotic vessels indicated that vasoconstrictor responses to noradrenaline (Rosendorff et al. 1981), ergonovine (Henry & Yokoyama, 1980; Yokoyama et al. 1983; Kawachi et al. 1984) and serotonin (Henry & Yokoyama, 1980; Heistad et al. 1984) were enhanced. Since the discovery by Furchgott and Zawadski of the role of the endothelium in vasomotor tone (Furchgott & Zawadski, 1980), a growing number of studies have indicated that endothelium-dependant relaxation to ACh is impaired by the induction of hypercholesterolaemia (Jayakody et al. 1985; Aksulu et al. 1986; Verbeuren et al. 1986; Chappell et al. 1987; Andrews et al. 1987) or atherosclerosis (Freiman et al. 1986). 174 For the most part, these studies were carried out in animal models using a relatively short period of cholesterol feeding with or without prior disruption of the vascular endothelium, a procedure known to promote the rapid formation of gross atherosclerotic lesions at the denuded site. However, atherosclerosis in man is a chronic disease which progresses slowly with age and has a characteristic prediliction for certain vessels such as the aorta and coronary arteries. In this study, changes in the responses of coronary arteries from Watanabe Hereditable Hyperlipidaemic rabbits (WHHL) were examined during the development of early atherosclerotic lesions to determine the long-term effects of the disease on vasomotor responses. This strain of rabbit lacks low density lipoprotein (LDL) receptors and is a unique model of human familial hypercholesterolaemia (Watanabe, 1980; Ishikawa et al. 1987; Rosenfeld et al. 1987). The rabbits have plasma cholesterol levels around 20 mmol/1 (90% contained in LDL) and gradually develop atherosclerotic disease very similar in its cellular pathology and distribution to the disease in man (Buja et al. 1983). In addition to the effects of noradrenaline (NA), histamine, serotonin (5HT) and acetylcholine (ACh), changes in the responses to the purinergic neurotransmitter adenosine triphosphate (ATP) and to the more recently recognised peptidergic neurotransmitters neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and substance P (SP) were also examined. As shown in Chapters 4 and 8 of this thesis, relaxations to SP and ACh in the rabbit epicardial coronary artery are entirely endothelium-dependent, while relaxation to ATP is partially endothelium-dependent. Relaxant responses to NA (see Chapter 4), CGRP and VIP (see Chapter 8) are independent of the endothelium. There are no previous reports in the literature of the long-term effects of atherosclerosis on responses to these agonists. 175 9.3 METHODS Using in-vitro pharmacological techniques as described in Chapter 3.1.2, responses of isolated epicardial coronary arteries from female Watanabe Hereditable Hyperlipidaemic (WHHL) rabbits aged 4, 6 and 12 months were compared with the responses of the female New Zealand white (NZW) rabbits of the same ages. Many of the normal age-related changes in the NZW rabbit coronary arteries have been reported and discussed in Chapter 8; these data are shown here in the figures to allow comparison with the changes in the WHHL animals but are not discussed further. NA, histamine, ATP and ACh were added cumulatively to the organ bath, but the responses to the peptides and 5HT demonstrated tachyphylaxis so a single maximal concentration of these compounds was used. 9.3.1 Materials See Chapter 3.6 9.3.2 Statistics See Chapter 3.5 9.4. RESULTS The extent of disease in vessels from the WHHL animals was assessed by transmission electron microscopy, performed by Dr. Annette Tomlinson. This demonstrated atheromatous lesions in several vessels such as the mesenteric, ear and saphenous arteries, largely confined to branch points and representing less than 5% of the total intimal surface; smooth muscle cells crossing the elastic lamina 176 to penetrate the intima were shown by 6 months of age but no lesions were seen in either endothelial or smooth muscle cells until 12 months, when damaged cells were clearly apparent. The aorta was more severly affected with more extensive damage present at an earlier age; less than 20-30% of the endothelial cells appeared normal by 12 months. No atheromatous lesions were visible in the coronary arteries from the 4-month-old animals but by 12 months of age macroscopic lesions were visible; a larger area of intima was affected than in other vessels, with the exception of aorta. Figure 9.1 shows the weights of the two strains of rabbits during the period of maturation under study. The WHHL rabbits tended to be slightly smaller than the NZW, although this difference only reached statistical significance (P<0.05) at six months of age. The rate of growth of the two strains was similar. 9.4.1 Effects of Hypercholesterolaemia At the age of 4 months, the WHHL rabbits are known to have high serum cholesterol levels even though they have not yet developed atherosclerotic lesions in the coronary arteries. The constrictor responses to KC1 and ACh were no different in the 4-month-old WHHL and NZW rabbits but contractions to 5HT and NPY were significantly greater in the hypercholesterolaemic animals (P<0.05) (Figure 9.2). The maximal contraction to histamine was significantly lower in the WHHL rabbits (P<0.02), but there was no difference in sensitivity to histamine compared to the NZW animals (see Table 9.1). 177 Maximal relaxation to the endothelium-dependent vasodilator SP was very significantly reduced in the WHHL rabbits (P<0.001) and relaxation to ACh was also smaller than in the NZW rabbits (Figure 9.1). Maximal relaxation to ATP was unchanged (Figure 9.3) but the sensitivity to ATP was reduced (see Table 9.1). However, maximal relaxations of the coronary arteries from WHHL rabbits to the direct smooth muscle vasodilators NA, CGRP and VIP were no different from those in NZW rabbits (Figure 9.3), although the vessels from the WHHL animals showed slightly greater sensitivity to NA (Table 9.1). 9.4.2 Changes with Development of Atherosclerosis Responses of coronary arteries from the WHHL rabbits to the agonists showed marked changes with age. To assess the extent to which these changes were due to normal maturation or to the development of atherosclerosis, the data from the WHHL animals were compared with responses obtained from the NZW rabbits of the same ages. 9.4.2.i Contractile Responses The pattern of changes with age in the contractile responses were remarkably similar in the NZW and WHHL rabbits. Under the conditions used, the contraction of the coronary arteries from WHHL rabbits to 30 mM KC1 fell as the animals matured, but the fall was in parallel with that seen in normal NZW rabbits and could not be attributed to progressive damage to the smooth muscle by atherosclerosis (Figure 9.4 A). The contraction to NPY and 5HT in the WHHL rabbits fell between 4 and 6 months (P<0.01 for 5HT) and then increased by 12 months of age (P<0.01 for NPY) (Figure 9.4 B,C). Responses to histamine in both 178 the WHHL and the NZW rabbits showed a slight fall in the maximal contraction between 4 and 6 months of age with no change in the pD2 (Figure 9.5; Table 9.1); histamine responses were not examined in the 12-month-old animals. The differences in contraction to KC1, NPY and histamine which were observed between the young WHHL and NZW rabbits remained constant during development; the pattern of change to 5HT with age was also similar in the two strains of rabbits, although the increase in contraction to 5HT in the 12-month-old WHHL rabbits was lower than in the NZW animals, resulting in loss of the difference between the two groups at this age. As shown in Chapter 4, ACh in the rabbit coronary artery causes some endothelial-mediated relaxation, and the contraction observed in response to this agonist is the net response of direct smooth muscle contraction and the endothelium-mediated relaxation. Net contractile responses to ACh in the WHHL rabbits did not show the marked increase which was seen in the NZW animals between 4 and 6 months of age, so that in the 6-month-old animals the maximal contraction to ACh was significantly lower in the WHHL than in the NZW animals (P<0.02). However, by 12 months the contractile responses to ACh in the WHHL rabbits had shown the same increase seen in the younger NZW animals and there was no longer any difference between them (Figure 9.6, Table 9.1). 9.4.2.ii Relaxant Responses The pattern of changes in the smooth muscle relaxant responses with age in the WHHL rabbits were similar to those in the NZW but changes in the endothelium-dependent responses were different in the two groups. As the WHHL animals matured, there was no significant change in the maximum relaxation or 179 pD2 for NA (Figure 9.7). Relaxation to both CGRP and VIP showed a progressive fall (Figure 9.8 A,B); although this was less pronounced in the WHHL than in normal NZW rabbits at the same ages, the pattern of changes in the responses to these agonists was similar in the two groups. However, the endothelium-dependent responses to SP did not follow those seen in the NZW but showed the opposite trend. While there was a steady decline in the maximal relaxation to SP with maturity in normal NZW animals, the relaxation to SP in WHHL rabbits steadily increased with time (P<0.01 at 12 months compared to 4-month-old animals). By 12 months of age, the responses to SP had recovered to equal those in the normal NZW animals (Figure 9.9). Furthermore relaxation to ATP, which is mediated partly through the endothelium, showed no decline between 4 and 6 months of age in the WHHL animals despite a significant fall during this period in the NZW controls and was significantly greater in the WHHL animals at 6 months of age (P<0.05) (Figure 9.10, Table 9.1). No increase in endothelium-dependent relaxation to ACh was seen in this study (Figure 9.9), although this may be obscured by the powerful direct smooth muscle constriction to ACh in the rabbit coronary artery. 9.5 DISCUSSION The results of this study lead to the conclusion that endothelium-dependent responses of the coronary vessels are reduced in the presence of elevated plasma cholesterol but show a compensatory recovery early in the development of atherosclerosis despite persistence of the hypercholesterolaemia. In contrast, responses mediated directly via the smooth muscle in atherosclerosis-prone animals may be altered by the presence of hyperlipidaemia but showed a normal pattern of 180 change with development and no effects attributable to the development of atherosclerosis. 9.5.1 Effects of Hypercholesterolaemia At 4 months of age, the coronary vessels in WHHL rabbits do not show atheromatous lesions (Buja et al. 1983) although the rabbits are beginning to develop lesions in other vessels such as the aorta and the plasma lipids are consistently elevated (Watanabe, 1980). At this age the rabbits are comparable to many of the rabbit models of hyperlipidaemia which have been used by others (Jayakody et al. 1985; Verbeuren et al. 1986; Chappell et al. 1987) and, in agreement with these authors, we found that the relaxation to the endothelium-dependent vasodilators SP and low-dose ACh in these animals was significantly reduced while relaxation to other agonists mediated predominantly by direct action on the smooth muscle was unimpaired. Several studies of hypercholesterolaemia in other animals including pigs (Cohen et al. 1988a; Shimokawa & Vanhoutte, 1988), chickens (Aksulu et al. 1986) and primates (Freiman et a L 1986) have also reported selective reductions in endothelium-mediated relaxation in various blood vessels. It is unlikely that this is a simple consequence of endothelial denudation or non-specific damage to the endothelial cells per se, since endothelium-dependant responses to noradrenaline or the calcium ionophore A23187 have been found to be unaffected in the same preparations (Bossaller et al. 1987; Cohen et al. 1988a) and production and release of endothelium-derived relaxant factor to some agonists is unimpaired (Verbeuren et al. 1986; Bossaller et al. 1987). Andrews and coworkers recently reported that acute exposure to pathophysiological concentrations of low density lipoprotein (LDL) directly inhibited the endothelium-dependent relaxation to ACh in rabbit 181 aorta via a receptor-dependent mechanism (Andrews et al. 1987) and this may be one of the mechanisms operating in these models. The constrictor responses to NPY and 5HT were increased in the 4-month-old WHHL rabbits despite no difference in the smooth muscle contraction to KC1 and a reduction in contraction to histamine. Enhanced constrictor responses to noradrenaline (Rosendorff et al. 1981), ergonovine (Yokoyama et al. 1983; Kawachi et al. 1984) and serotonin (Henry & Yokoyama, 1980; Heistad et al. 1984) have been reported in previous studies, although the data is not always consistent (Chappell et al. 1987). It is possible that the enhanced constrictor responses to these agonists might also be a consequence of dysfunction of endothelium-dependent relaxant responses, since studies in the past few years have shown that both noradrenaline and serotonin may mediate relaxation via endothelial receptors in addition to producing contraction via smooth muscle (Cocks & Angus, 1983; Furchgott, 1983; Furchgott, 1984) and the supersensitivity to ergonovine has been reported to be mediated by a serotoninergic mechanism (Henry & Yokoyama, 1980; Heistad et al. 1984). However, in the rabbit coronary artery I found vasoconstrictor responses to NPY were unaffected by removal of the endothelium (data not shown) and noradrenaline does not cause endothelium-dependent relaxation (see Chapter 4) so this mechanism is unlikely to account for all the changes observed in these constrictor responses. Alternative mechanisms could involve incorporation of cholesterol into the membrane of the arterial smooth muscle, which may underlie increased susceptibility to contractile stimulation (Yokoyama & Henry, 1979), or a change in the number or coupling of receptors on the smooth muscle. In a preliminary report, an increase in serotonergic and alpha-adrenergic receptors was noted in the aorta of 182 atherosclerotic rabbits (Nanda & Henry, 1982) but further studies of changes in receptor density in atherosclerosis are still awaited. 9.5.2 Effect of Development of Early Atherosclerosis It is evident that changes in vasomotor responses with diet-induced hypercholesterolaemia are not necessarily the same as those found in the presence of established atherosclerosis (Freiman et al. 1986; Shimokawa & Vanhoutte, 1989). This is not surprising, since even the nature of the lipid abnormality may differ - cholesterol-fed animals have elevated plasma beta subfractions of very low density lipoproteins (/J-VLDL) while in human hypercholesterolaemia and in WHHL rabbits the hypercholesterolaemia is mostly due to an increase in LDL (Steinberg, 1987). The atherosclerotic process is slow and complex and involves the formation of fibrous plaques, made up of increased intimal smooth-muscle cells surrounded by a connective tissue matrix containing intracellular and extracellular lipid and covered by a fibrous cap (Ross, 1986). The vasomotor responses of the coronary arteries from WHHL rabbits in this study underwent profound changes with maturation during the period preceding the development of complex atherosclerotic lesions; the extent to which these changes are related to normal maturation or to the development of atherosclerosis was identified by comparing the changes over time with those of age-matched normal rabbits. 9.5.2.i Contractile Responses Constrictor responses to KC1, NPY and histamine in the WHHL rabbits showed changes with age very similar to those seen in normal NZW rabbits, which implies that they are a consequence of normal maturation rather than secondary to 183 the development of progressive atherosclerotic lesions in these animals. The pattern of changes in the response to 5HT was also very similar in the WHHL and NZW rabbits, although the increase in contraction to 5HT at 12 months was greater in the NZW; it is tempting to speculate that this difference might be due to a concomitant increase in endothelium-mediated relaxation to 5HT in the WHHL animals, but this cannot be confirmed from this study. At 6 months of age, maximum contraction to ACh was significantly greater in the NZW rabbits than in the WHHL animals. As described above, the contractile response to ACh is the net result of a powerful direct smooth muscle contraction and an endothelium-mediated relaxation; it is therefore possible that this difference in response to ACh was due to an increase in endothelium-mediated relaxation to ACh in the 6-month-old WHHL rabbits alone, while contractile responses to ACh were increasing in all the animals. However, the differences were lost by 12 months of age, and a more likely explanation is that the increase in contraction to ACh was simply delayed in the WHHL rabbits compared to the NZW. Changes in response to ACh in the normal rabbits occured around the time when the animals became sexually mature; the slightly later development of sexual maturity in WHHL rabbits may thus account for differences seen at 6 months of age which are then lost in the mature animals. Further studies of the time course of these changes and their relation to the menarche may help to clarify this. 9.5.2.H Relaxant Responses CGRP, VIP and NA mediate direct smooth muscle relaxation and have no effect on the endothelium in the rabbit coronary artery. Relaxant responses to NA 184 showed no change with age in the WHHL rabbits over the period of development under study; similarly little change was seen in the NZW rabbits. The pattern of age-related changes in relaxation to CGRP and VIP was also similar in the WHHL rabbits to that seen in the NZW animals. This suggests that the changes are related to normal maturation and are not due to the development of atherosclerosis. Unexpectedly, endothelium-dependent relaxation to SP in the WHHL rabbits did not follow the changes seen in the normal NZW rabbits but showed a marked increase as the animals matured. By the age when early atherosclerotic lesions were present in the coronary arteries of the WHHL rabbits, the responses to SP had recovered to equal the normal NZW controls. Relaxation to ATP, which acts both directly on the smooth muscle and via the endothelium in the rabbit coronary artery, was also higher in the older WHHL rabbits relative to the NZW. The responses to ATP in the coronary arteries of NZW and WHHL rabbits in our study agree closely with a recent study by Ragazzi and coworkers in which the relaxation to ATP in the aorta of WHHL rabbits aged 11-14 months was found to be unimpaired and was much higher than in age-matched controls (Ragazzi et al. 1989). The mechanism was unexplained. In contrast, results obtained in studies of cholesterol-fed rabbits by the same author (Ragazzi et al. 1988) and by Verbeuren (Verbeuren et al. 1986) showed ATP-mediated relaxation was markedly diminished. Clearly, extrapolation of results from cholesterol-fed animals to endogenous hyperlipidemia or atherosclerosis should thus be done with caution. Relaxation to ACh did not show a similar increase between 6 and 12 months of age. However, the possibility exists that an increase in endothelium-dependent relaxation to ACh was obscured by the increase in direct smooth muscle contraction to ACh, as described above. This study emphasises that 185 relaxation to ACh may be less useful than relaxation to a purely vasodilator endothelium-dependent agent, such as SP, as an indicator of the endothelium-dependent responses in patients with and without atherosclerosis. The mechanism of the increase in endothelium-dependent relaxation cannot be determined from this study but in the past few years it has emerged that vascular innervation and receptor mechanisms are much more flexible than previously thought and may profoundly alter in response to physiological and pathophysiological events. Changes may occur in the both the density of autonomic innervation and the neurotransmitters expressed in perivascular nerves (Burnstock, 1990b); there may be selective up- or down-regulation of receptors expressed on vascular smooth muscle (Fleisch & Hooker, 1976; Duckies, 1983; Hyland et al. 1987); or second messenger systems may be affected (Cohen & Berkowitz, 1974; Docherty, 1988). Little is known at present about the normal and abnormal function of endothelial cells, but it would seem that they, too, have the potential for compensatory changes. The production of endothelium-derived relaxant and contracting factors may be altered under conditions such as shear stress or hypoxia. Furthermore,it has been shown that endothelial cells are capable not only of responding to agonists such as SP, 5HT and ACh, but also of their synthesis (Parnavelas et al. 1985; Loesch & Burnstock, 1988; Milner et al. 1989); their release by endothelial cells of coronary arteries has been demonstrated under conditions of hypoxia (Burnstock et al. 1988a). The present study concentrated on the period associated with the early development of atherosclerosis in the rabbit coronary arteries and it is not clear whether the recovery in endothelium-dependent relaxation would be sustained as the disease progressed. Indeed, it seems likely that more extensive damage to 186 endothelial cells, coupled with thickening and infiltration of the intima which occurs with progressive atherosclerotic disease, would overcome these compensatory changes, and that endothelium-dependent relaxant responses would later decline. Work by my colleagues, Antonia Brizzolara and Anne Stewart-Lee has demonstrated that there is no increase in endothelium-dependent relaxation in the aortae of WHHL rabbits, which develops gross atherosclerotic lesions much earlier than the coronary arteries, while endothelium-dependent responses to SP and ACh are increased in other arteries even when no atheroma is present on ultrastructural examination (Burnstock et al. 1990a). Whatever the longer term effects, these studies suggest that the endothelium is capable of compensatory changes during the development of early atherosclerosis. In conclusion, the work presented in this chapter shows that endothelium-dependent responses to ACh and SP are depressed and contractions to 5HT and NPY are increased in coronary arteries from young hyperlipidaemic WHHL rabbits before the development of gross atherosclerotic lesions. Moreover, while direct smooth muscle constrictor and dilator responses in the atherosclerosis-prone W H H L animals change normally with age, endothelium-dependent relaxation to SP shows the opposite trend to that observed in normal female rabbits and actually recovers during the development of early atherosclerosis of the coronary arteries. These findings may reflect a compensatory change which would maximise myocardial blood flow through an atherosclerotic vessel to ischaemic myocardium. Further studies of the long-term effects of atherosclerosis on smooth muscle and endothelial cell function are necessary to elucidate the mechanisms involved. 187 TABLE 9.1. pD2 values and maximum responses to noradrenaline, ATP, acetylcholine and histamine in rabbits developing atherosclerosis (WHHL) and age-matched controls (NZW). Age (months') 4 6 12 pD2 Maximum pD2 Maximum pD2 Maximum Response Response Response Noradrenaline WHHL 6.42* 78.2 (8) 6.17 81.4* (7) 6.66 81.5* (6) (0.10) (3.4) (0.09) (2.3) (0.30) (2.3) NZW 5.86 71.8 (6) 6.38+ 68.3 (5) 6.70++ 69.9 (7) (0.17) (6.1) (0.04) (5.0) (0.13) (3.6) ATP WHHL 5.05* 69.8 (8) 5.55 66.4* (7) - - (0.17) (5.3) (0.15) (4.7) NZW 5.67 68.9 (6) 5.29 46.5+ (5) - - (0.18) (2.7) (0.17) (7.6) Acetvlcholine WHHL 5.81 0.76 (8) 5.71 0.75* (7) 5.93 1.18+ (5) (0.10) (0.12) (0.09) (0.09) (0.08) (0.16) NZW 5.53 0.69 (5) 5.85 1.30+ (5) 6.08 1.29 (7) (0.12) (0.13) (0.18) (0.20) (0.13) (0.22) Histamine WHHL 5.26 2.79* (8) 5.11 2.40* (7) - - (0.17) (0.21) (0.25) (0.30) NZW 5.11 3.77 (6) 5.36 3.35 (5) - - (0.13) (0.31) (0.04) (0.06) Data is given as mean (s.e.m.). Maximum response is expressed as percent relaxation of the tone induced by KC1 for noradrenaline and ATP, and grams contractile tension for acetylcholine and histamine. * = p<0.05 compared to NZW + = p<0.05, ++ = p>0.01 compared to previous age group. Numbers in brackets refer to number of animals studied. 188 IUE 9.1FIGURE a n d 1 2 m o n t h s o f a g eVa . l u e s a r e m e a n s + e a c h a g eEr . r o r b a r s f a l l i n g e n t i r e l y w i t h i n t h e s y m b o l s h a v e b e e n o m i t t e dTh . e 4 a n d 1 2 m o n t h s w e r e n o t r s a i t g e n o i f f i i n c c a r n e t a . s e i n w e i g h t w a s t h e s a m e i n b o t h g r o u p sAt . 6 m o n t h s o f a g e , t h e WHHL aWHHL n i m a l s w e r e s l i g h t l y l i g h t e r t h a n t h e ( NZW * = Weight CKg) 1.0 W e i g h t s ( K g ) o f ( WHHL s o l i d l i n e ) a n d r NZW a b b i t s ( d o t t e d l i n e ) a t 4 ,6 - 0 - 6 12 6 4 ii ■ 'i g (months] Age s.e.m. o f d a t a f r o m 1 2 - 1 4 r a b b i t s a t P<0.05); t h e d i f f e r e n c e s a t 100 -I 80 - v p 'c 60 o *-t—*CT3 X * o 40 0C * * 20 - 0 J i i NA ATP CGRP VIP SP A C h FIGURE 9.2 Maximal vasodilation in coronary arteries of WHHL rabbits (hatched bars, n = 8) compared to NZW rabbits (open bars, n = 6) at 4 months of age. Results are expressed as percent relaxation of the KCl-induced tone and are given as the means + s.e.m. There were no differences between the groups in the relaxation to noradrenaline (NA, 10”^M), ATP (10~^M), calcitonin gene-related peptide (CGRP, 2.6x10 M) or vasoactive intestinal polypeptide (VIP, 3x10 M) but the relaxation to substance P (SP, 10“'M) and acetylcholine (ACh, 10-oM) was lower in the WHHL than in the NZW rabbits (*** = P<0.001). ContractionCg tension] FIGURE9.3 t h e m a x i m a l c o n t r a c t i o n t o K C 1 ( 3 0 mM) a n d a c e t y l c h o l i n e ( A C h , 1M) 0 b e t w e e n r a b b i t s ( h a t c h e d m o n t h s o f a g e .Re s u l t s a r e s h o w n a s m e a n s ± 5 - h y d r o x y t r y pl t o a m w i e n r ei nt hw e hWHHL i t l h e e c a o n WHHL d n t t r h a e c a t NZW n i i o m n at l o sn ; e u m r a x o i p m e a l p t c i o d n e (Y t N10 PM) ra Y n a , d c t i o n t o h i s t a m i n e (M) 1 w 0 a s r a b b i t s ( * = M a x i m a lc o n t r a c t i o n( g r a m st e n s i o n )o fc o r o n a r ya r t e r i e sf r o mWHHL P<0.05). bars, n = 8 ) c o m p a r e d t o r NZW a b b i t s ( o p e n (5HT, 1 0 ” ^ M ) w a s g r e a t e r i n t h e t h WHHL a n i n t h e NZW s.e.m. Th e r e w e r e n o d i f f e r e n c e s i n bars, n = 6 ) a t 4 FIGURE 9.4 A,B Contraction (grams tension) of coronary arteries to A) KC1 (30 mM) and B) neuropeptide Y (10"^M) in WHHL (solid lines) and NZW rabbits (dotted lines) at 4, 6 and 12 months of age. Each point represents the means + s.e.m of data from six to eight animals. * = P<0.05 for WHHL compared to age-matched NZW rabbits. > Contraction (g tension) Contraction (g tension) 0.75 o o H 0.5 ro cn - j. -x [>j ro o Ol O o a i o cn ______i______i______i ______i c 1.00 n c o ’co 0.75 - c CD * CD V___> C 0.50 - o o CO c 0.25 - o o 0 J Age [month's} FIGURE 9.4 C Contraction (grams tension) of coronary arteries to 5-hydroxytryptamine (10"^M) in WHHL (solid lines) and NZW rabbits (dotted lines) at 4, 6 and 12 months of age. Each point represents the means + s.e.m of data from six to eight animals. * = P<0.05 for WHHL compared to age-matched NZW rabbits. A 4.0 n 3.5 - - Log (Histamine) M FIGURE 9.5 A Cumulative contraction-response curves of coronary arteries to histamine in WIIHL rabbits at 4 months (circles) and 6 months of age (triangles). Each point represents the mean + s.e.m. of data from six to eight rabbits. 1 ’ 'I "' ' 1 ...... '1------1 I 8 7 6 5 4 3 - Log (Histamine] M FIGURE 9.5 B Cumulative contraction-response curves of coronary arteries to histamine in NZW rabbits at 4 months (circles) and 6 months of age (triangles). Each point represents the mean + s.e.m. of data from five to eight rabbits. FIGURE 9.6 Cumulative contraction-response curves of coronary arteries to acetylcholine (ACh) in WHHL (solid lines and symbols) and NZW rabbits (dotted lines, open symbols) at 4 (circles), 6 (triangles) and 12 months of age (squares). Each point represents the mean + s.e.m of data from five to eight animals. There was a significant increase in the maximal contraction to ACh in NZW rabbits between 4 and 6 months, and in WHHL rabbits between 6 and 12 months of age (P<0.05 for both). At 6 months, the contraction in WHHL animals was less than the NZW (P<0.02) but the differences between the groups at 4 and 12 months were not significant. Contraction [g tension] 0.6 . - 0.4 0.8 0.2 1.2 1.0 - 1.4 1.6 - 0 - - - - - 7 5 3 4 5 6 7 8 i i i i Lg (Acetylcholine] M -Log ------i i 'i r IUE 9.7 FIGURE eut ae xrse a pret eaain f h Klidcd oe n each and tone KCl-induced (squares). the age of of relaxation months 12 percent and as expressed (triangles) 6 are Results (circles), 4 at rabbits WHHL in on rpeet te en ... fdt fo sx o ih animals. eight to six from data of s.e.m. + mean the represents point Relaxation (%) 100 n 100 Cumulative relaxation-response curves of coronary arteries to noradrenaline noradrenaline to arteries coronary of curves relaxation-response Cumulative FIGURE 9.8 A,B Relaxation of coronary arteries to A) calcitonin gene-related peptide (2.6x10”^ M) and B) vasoactive intestinal polypeptide (3xl0- ^ M) in WHHL (solid lines) and NZW (dotted lines) at 4, 6 and 12 months of age. Results are expressed as percent relaxation of the tone induced by KC1 and are the means + s.e.m. of data from six to eight animals in each group. ** = P<0.01 for WHHL compared to age-matched NZW rabbits. 4 12 Age (months} FIGURE 9.9 A,B Relaxation of coronary arteries to A) acetylcholine (10-^ M) abd B) substance P (10- ^ M) in WHHL (solid lines) and NZW (dotted lines) at 4, 6 and 12 months of age. Results are expressed as percent relaxation of the tone induced by KC1 and are the means ± s.e.m. of data from six to eight animals in each group. *** = P<0.001, ** = 0.01 for WHHL compared to age-matched NZW rabbits. A 50 n 40 - § | 30 - CO X CO 10 - 0 J 40 - cN\P C o 30 - CO X _C0 CD oc 20 - 10 - 0 J Age (months] 100 n « * i i i i i l 8 7 6 5 4 3 - Log CATP] M FIGURE 9.10 A Cumulative relaxation-response curves of coronary arteries to ATP in NZW rabbits at 4 months (circles) and six months of age (triangles). Results are expressed as percent relaxation of the KCl-induced tone and each point represents the mean + s.e.m. of data from five to eight animals. There was a fall in the relaxation to ATP in the NZW rabbits between 4 and 6 months of age. ** = P<0.05, *** = P<0.01 compared with 6-month-old NZW rabbits. t as —% IUE .0 B 9.10 FIGURE WHHL rabbits at 4 months (circles) and six months of age (triangles). Results are are Results (triangles). age of months six and (circles) months 4 at rabbits WHHL expressed as percent relaxation of the KCl-induced tone and each point represents represents point each and tone KCl-induced the of relaxation percent as expressed h ma + ... f aa rm ie o ih aias Tee a n fl in fall no was There animals. eight to five from data of s.e.m. + mean the eaain n h WH animals. WHHL the in relaxation Relaxation 100 0 - 80 0 - 60 0 - 40 20 0 uuaie eaainrsos cre o crnr atre t AP in ATP to arteries coronary of curves relaxation-response Cumulative - 7 5 3 4 5 6 7 8 I— " ..... I ...... " " ' T — '' I1 , 1 Lg AP M (ATP) Log - ""1" \ CHAPTER 10 MARKED INCREASES IN CALCITONIN GENE-RELATED PEPTIDE-CONTAINING NERVES IN THE DEVELOPING RAT FOLLOWING LONG-TERM SYMPATHECTOMY WITH GUANETHIDINE 10.1 SUMMARY Changes in the peptidergic innervation of the cardiovascular system, urogenital tract and ganglia have been examined following long-term chemical sympathectomy in the rat. High doses of guanethidine (50 mg/kg) were given daily for 3 weeks to 8-day-old rat pups; these were then sacrificed at 6 or 20 weeks of age. In rats of both ages, noradrenergic nerves were severely depleted or absent compared to control rats of the same age, but in some regions dramatic increases of calcitonin gene-related peptide (CGRP) were demonstrated. There was an increase in the density of nerve fibres and in the CGRP content (up to 18-fold), most notably in the right atrium and the superior cervical ganglion. No changes in substance P- or vasointestinal polypeptide-immunoreactive nerves were seen. Conversely, short term sympathectomy by 6-hydroxydopamine treatment caused a depletion of noradrenaline which was not accompanied by an increase in the number or content of CGRP-immunolabelled nerves. The possibility that nerve growth factor is involved in the mechanism of hyperinnervation by CGRP-containing sensory nerves following long-term sympathectomy is discussed. 202 10.2 INTRODUCTION It has been known for some time that, although sympathetic nerves are essential for maintaining homeostasis, they are not an absolute necessity for survival (Cannon et al. 1929b). This may be due to compensatory changes in innervation by other nerves, a view supported by studies showing growth of non-sympathetic nerves after sympathetic denervation (Malmfors et al. 1971; Evans et al. 1979a; Gibbins & Morris, 1988; Rosier & Waterson, 1988). Autonomic nerves are "attracted" to target cells by trophic factors, such as nerve growth factor (Levi-Montalcini & Angeletti, 1963) which is present in higher concentrations in tissues that are normally densely innervated (Chamlet et al. 1973; Burnstock, 1981). Tissue culture and transplant studies have shown that the nerves subsequently contact the target cells, e.g. muscle cells, at "recognition" sites which do not distinguish between adrenergic and non-adrenergic nerves (Unsicker et al. 1977; Campbell et al. 1978). After denervation, nerve fibres will regrow to restore a normal fibre density for the tissue (Burnstock et al. 1978a), but not necessarily with the same ratio of fibre types (Evans et al. 1979a). To investigate some of the fibre types which might compensate for the loss of sympathetic nerves, long-term selective denervation of neonatal rats was obtained by chronic administration of high doses of guanethidine (Burnstock et al. 1971; Eranko & Eranko, 1971; Heath & Burnstock, 1977); this treatment is known to be followed by substantial reinnervation of tissues but with nerve fibre types which are so far unidentified (Evans et al. 1979a). In this study, immunohistochemical and neurochemical methods were used to characterise these nerve fibres and to quantify the changes in innervation which occur in different 203 tissues after chronic removal of the sympathetic nerves. These results were compared with those obtained after acute denervation with 6-hydroxydopamine (6-OHDA), to establish whether the changes seen after chronic denervation are indeed compensatory. 10.3 METHODS 10.3.1 Long-Term Guanethidine Sympathectomy Neonatal rats were injected with high doses of guanethidine sulphate as described in Chapter 3.4.1; age-matched controls were injected with the same volume of saline. The animals were sacrificed at approximately 6 weeks (36-51 days) or 20 weeks of age (126-136 days) by an overdose of CO 2 gas. 10.3.2 Acute 6-Hydroxydopamine Sympathectomy Rats were treated with 6-OHDA at 6 or 20 weeks of age to cause acute destruction of the sympathetic nerves as described in Chapter 3.4.2. 10.3.3 Selection of Tissues for Study The following tissues were studied: superior cervical ganglion (SCG), inferior vagal ganglion (nodose), superior mesenteric artery and vein, femoral artery and vein, vas deferens (epididymal portion), bladder, and heart. The heart was divided into four regions consisting of the free walls of the right atrium, left atrium, right ventricle and left ventricle. The inter-atrial and inter-ventricular 204 septa were not included. These tissues were chosen to represent ganglia (sympathetic and sensory), the cardiovascular system, and the urogenital tract. 10.3.4 Fluorescence Histochemistry See Chapter 3.2.1 Blood vessels were examined as whole mounts, ganglia and other organs were examined as cut sections. 10.3.5 Immunohistochemistry See Chapter 3.2.2 Innervation by nerve fibres containing calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and substance P (SP) was studied. Blood vessels were examined as whole mounts using antibodies at a concentration of 1:200. All other tissues were sectioned and processed for immunolabelling of CGRP, VIP and SP using antibodies at a dilution of 1:400 (anti-CGRP and anti VIP) and 1:200 (anti-SP). A few sections from the ganglia were stained with toludine blue for routine morphology. Histochemical preparations were coded to enable them to be assessed by two independent investigators without knowledge of treatment. The immunolabelling seen was rated on a scale of + to ++++, where + = one or two fibres seen and ++++ = very densely packed fibres. On some slides, this assessment was repeated on whole-mount preparations using a computerised image analysis system (Seescan) that produced a measure of fibre density. 205 10.3.6 Assay The same tissues from a separate set of control and treated animals were taken exclusively for assay of the content of NA and the various peptides. Biochemical assay for NA content was performed using HPLC with electrochemical detection as described in Chapter 3.3.1. Peptide content of the tissues was measured by ELISA as in Chapter 3.3.2. Control and treated tissues were assayed at the same time. 10.3.7 Materials See Chapter 3.6. 10.3.8 Statistics See Chapter 3.5 Differences in tissue content of NA and the peptides between control and sympathectomised animals were assessed at 6 and 20 weeks of age. 10.4 RESULTS 10.4.1 Long-Term Sympathectomy with Guanethidine There were minor differences in the physical characteristics of the treated and control rats, similar to those previously described (Johnson et al. 1976; Nelson et al. 1988), in that the treated rats developed a transient ptosis bilaterally during the injection period and showed slightly retarded growth. The ptosis had, however, disappeared by the time the rats were studied at 6 weeks and although the treated rats were marginally lighter than the controls at 6 weeks there was no 206 difference in the weight of the rats at 20 weeks. These observations confirm that the animals thrive without their sympathetic nerves under laboratory conditions. The results for both histochemistry and biochemical assay within each group of animals studied were consistent. Sympathectomy has traditionally been assessed by viewing the condition of the SCG and analysing its NA content. The treated ganglia in both age groups in this study were atrophied (control ganglia mean weight 1.35 ± 0.12 mg; treated ganglia mean weight 0.60 + 0.06 mg) and had very few intact cells. A few NA-containing fibres were present in the treated ganglia, probably sprouting from the few cell bodies which remained (Figure 10.1 a, e). The appearances of the ganglia were similar at 6 and 20 weeks of age. On biochemical assay the NA content was depleted by 78.9% at six weeks and by 89.3% at 20 weeks (Table 10.1). The blood vessels and organs from guanethidine-treated animals all showed a large depletion in NA content (Table 10.1). The vas deferens and the right atrium of the heart, tissues which are normally heavily innervated with NA fibres, were almost devoid of fluorescent fibres after treatment in 6- and 20-week-old animals (Figure 10.2). Blood vessels were depleted so that noradrenergic fibres were rarely seen (Figure 10.2). This overall depletion of NA was maintained 16 weeks after the end of the treatment period, confirming that the regime employed had caused long-term sympathectomy. There most striking finding of this study was an increase in CGRP following guanethidine sympathectomy in most tissues studied (Figure 10.3, Table 10.2). In 6-week-old rats, CGRP levels in the SCG showed an 18-fold increase 207 over control values on immunochemical assay. The next highest rise was seen in the right atrium (10-fold), while in the other areas of the heart and in the vas deferens, the increase was approximately 3 times control levels (Figure 10.3). At 20 weeks, CGRP levels were still elevated, although in the right ventricle and vas deferens the increase was no longer stastically significant. There was, however, no significant increase on assay in the total CGRP content of the mesenteric vein or nodose ganglion after guanethidine treatment. Immunohistochemical examination confirmed increased innervation by CGRP-immunoreactive nerve fibres in the SCG (Figure 10.1 b,f), vas deferens, heart and bladder in guanethidine-treated animals (Figure 10.4, Table 10.2). Subjective immunohistochemical assassment of the blood vessels also suggested an increase in CGRP-innervation (Table 10.2). Fibres were more intensely fluorescent, with thick bundles being apparent in all the blood vessels viewed; increased immunolabelling was seen throughout the thick muscular coat of the vas deferens and within the muscle of the bladder and heart. On examining the slides "blind", it was generally possible to distinguish between the treated and the control tissues. Image analysis of stretch preparation of the mesenteric and femoral veins, however, did not show a significant difference in area of fluorescence (Table 10.3). In the treated animals, the nodose ganglion did not appear to be atrophied at the light microscopic level (Figure 10.1 d,h). However, cell bodies in the ganglion no longer labelled for CGRP but there was an increase in CGRP-containing fibres; the total CGRP content of the ganglion assessed by immunochemical assay did not change significantly. The levels of SP and VIP showed no change in any of the regions studied. Assay levels of SP in the treated nodose ganglion were not significantly different 208 from control values and immunolabelling of all tissues did not detect any change in SP fibre density. Where VIP concentrations were sufficient to be detected by immunoassay, no significant change was found between the control and treated animals (Figure 10.5). Histologically the immunolabelling was very similar in control and treated animals. 10.4.2 Short-Term Sympathectomy with 6-Hydroxydopamine Two days after animals had been treated with 6-OHDA to deplete sympathetic nerve terminals, CGRP-immunolabelled nerve fibres appeared to be identical to those in control animals, while NA-containing nerves were no longer seen around the blood vessels and were greatly depleted in the organs and ganglia. The appearance of the SCG is given as an example in Figure 10.1 c,g. Immunochemical assay of CGRP levels in tissues from treated animals showed no significantly difference from control levels (vas deferens: control 6.34 +1.69 pmol/g tissue, treated 6.78 ±1.48 pmol/g tissue; right atria: control 2.80 + 0.51 pmol/g tissue, treated 4.04 + 0.83 pmol/g tissue; all n=6). 10.5 DISCUSSION Guanethidine was first shown to produce long-term sympathectomy in 1971 (Burnstock et al. 1971); it destroys the cell bodies of sympathetic ganglia and the effect has been shown to persist for at least one year (Evans et al. 1979a). Using immunolabelling and assay techniques, the present study has shown an increase in the innervation of some tissues by non-sympathetic fibres in rats at 6 and 20 weeks of age after sympathectomy of neonates with guanethidine: a selective increase in CGRP expression with no change in SP or VIP has been demonstrated. 209 Since this effect is not seen after acute sympathectomy with 6-OHDA, the rise in CGRP seen 5 weeks after the beginning of guanethidine treatment appears to be a compensatory change. Although there is some decline, this change persists in 20-week-old rats. The most striking change in CGRP expression was seen in the SCG. This rise was due to a massive increase in CGRP fibres within the ganglion. In contrast, there was a loss of immunolabelling of nerve cell bodies for CGRP in the nodose ganglion, although CGRP fibres were still present. CGRP-immunoreactive fibres were also found hyperinnervating the cardiovascular system and the urogenital system; the origin of these fibres is not known, although they are likely to be the non-sympathetic innervating fibres that were reported previously (Evans et al. 1979a). Carvahlo and coworkers have also noted an increase in CGRP in the vas deferens after guanethidine sympathectomy of adult rats (Carvalho et al. 1986). In most tissues, immunohistochemistry supported the assay results and confirmed that the increase noted in CGRP content was due to changes in CGRP-containing nerves. But comparison of the assessment of changes in CGRP-immunoreactivity by histochemistry (Table 10.2) and in CGRP content by assay (Figure 10.3) revealed some differences. The apparent discrepancy in results for the nodose ganglion may be due to detection of CGRP in both nerve cell bodies and nerve fibres by assay: while CGRP immunoreactivity was undetectable histochemically in the cell bodies of the treated animals, there was an apparent increase in the content of CGRP in nerve fibres; immunoassay cannot distinguish between these sources and gives an average value for the content of the whole nodose ganglion. However, there is no obvious explanation for the discrepancy in results for the mesenteric vein. The differences between the two methods of 210 assessment do not alter the main finding of substantial increases in CGRP in most regions but suggest that subjective visual assessment of density of innervation in stretched tissue preparations is not completely reliable. Quantitative analysis of the density of innervation in immunohistochemically labelled whole-mount preparations appeared to give a closer correlation with assays (Table 10.3). Sympathectomy of the eye by removal of the superior cervical ganglion has been reported to cause a 2- to 3-fold rise in SP-like immunoreactivity in the iris and an increase in the number of CGRP-immunoreactive nerves (Cole et al. 1983; Terenghi et al. 1986). In the present study there was no rise in SP after guanethidine treatment. This may seem surprising, since CGRP is often colocalised with SP in sensory nerves (Gibbins et al. 1985; Lee et al. 1985b). However, it has been shown in the rat that although CGRP is found in most SP-containing nerve fibres, less than half of the CGRP-containing nerve fibres also contain SP (Terenghi et al. 1985). It is thus likely that there are two different populations of sensory nerves and that only one type - containing CGRP rather than SP - proliferates after long-term sympathectomy of the developing rat. Denervation has been shown to increase neurotrophic activity, particularly the production of sympathetic nerve growth factor (NGF) (Rush et al. 1986); a decrease in catecholamine concentration has also been shown to promote NGF production (Chun & Patterson, 1977). This factor stimulates the growth of sensory as well as sympathetic neurons (Levi-Montalcini & Angeletti, 1968; Thoenen & Barde, 1980) and it has been shown in certain tissues such as the iris that sensory and sympathetic neurons compete for NGF (Kessler et al. 1983; Korsching & Thoenen, 1985). NGF is also required for collateral branching of sensory nerves (Logan et al. 1988); hence increased availability of NGF in the 211 guanethidine-sympathectomised rats in this study may have promoted the growth of CGRP-containing sensory fibres. The hyperinnervation described here may be similar to that produced after daily injections of NGF into neonatal mice (Levi-Montalcini & Booker, 1960). The involvement of NGF may provide another explanation for the lack of change in SP levels that were found. Scott and Woolgar have shown that during the development of the nervous system, CGRP-immunoreactive nerve fibres are the first to develop a plexus in the mesenteric vascular bed of the rat (Scott & Woolgar, 1988). At the age when treatment was given in this study, SP-immunoreactive nerve fibres are only just beginning to appear. Hence it is possible that the CGRP fibres are able to take greater advantage of the increased availability of NGF than the comparatively undeveloped nerves in which SP and CGRP are co-localised. This may be related to a different sensitivity to NGF (Hill et al. 1985) or to the requirement of different trophic factors by each type of neuron (Rush et al. 1986). It is noteworthy that there was no increase in CGRP levels in the nodose ganglion, which is a sensory ganglion of placodal origin that does not seem to be dependent on NGF during development of the nervous system (Johnson et al. 1980). VIP was unchanged in the right atrium, vas deferens and bladder after guanethidine treatment in this study, in agreement with the findings of Gibbins and Morris in the cerebral vessels (Gibbins & Morris, 1988). VIP has been reported to increase in the lower gastrointestinal tract after 5 weeks treatment with guanethidine (Nelson et al. 1988), and there was a slight change in VIP levels in the vas deferens after 4 weeks of treatment of adult rats with guanethidine in the study by Carvalho et al., but this change was not significant (Carvalho et al. 1986). 212 The results presented here demonstrate a selective, compensatory increase in innervation by CGRP-immunoreactive nerves following long-term guanethidine sympathectomy and suggests that in CGRP-containing nerve fibres at a developmental stage, the expression of CGRP is susceptible to alteration by sympathetic denervation. This may be linked to the effects of NGF. The functional significance of the dramatic increase in innervation by CGRP-containing nerve fibres after removal of the sympathetic nerves with guanethidine remains to be assessed. 213 TABLE 10.1 The effects of long-term sympathectomy by guanethidine on noradrenaline levels Age 6 weeks Tissue Control Treated % Remaining SCG (ng/ganglion) 16.13 ± 1.60 (7) 3.41 ± 1.01 (6) 21.1 Heart (/xg/g tissue) Right atrium 1.71 + 1.20 (7) 0.16 ± 0.08 (6) 9.4 Left atrium 1.68 ±0.10 (7) 0.05 ± 0.03 (5) 2.9 Right ventricle 0.88 + 0.08 (7) 0.06 ± 0.02 (6) 6.8 Left ventricle 0.62 ± 0.06 (7) 0.06 ± 0.04 (6) 9.7 Vas deferens (ng/cm) 9.46 ± 0.39 (8) 1.34 ± 0.19 (8) 14.2 (epididymal) Mesenteric vein 3.86 ± 0.16 (7) 0.49 ± 0.06 (7) 12.7 (ng/cm) Bladder (ng/g tissue) 0.18 ±0.02 (7) 0.002± 0.002(6) 1.3 Age 20 weeks Tissue Control Treated % Remaining SCG (ng/ganglion) 22.21 ± 0.72 (5) 2.38 ± 0.20 (6) 10.7 Heart (jxg/g tissue) Right atrium 1.41 ± 0.10 (6) 0.013 ± 0.004 (6) 0.9 Left atrium 1.74 ±0.02 (6) 0.064 + 0.021 (6) 3.7 Right ventricle 0.64 + 0.04 (6) 0.018 ± 0.002 (6) 2.8 Left ventricle 0.47 ± 0.06 (6) 0.23 ± 0.001 (6) 4.9 Vas deferens (ng/cm) 94.65 ± 6.99 (6) 5.22 ± 1.72 (6) 5.5 (epididymal) Mesenteric vein 13.82 ± 1.25 (6) 0.33 ±0.15 (6) 2.4 (ng/cm) Bladder (ng/g tissue) 0.51 ±0.13 (4) 0.89 ± 0.009 (5) 17.3 Results are given as mean ± s.e.m. Numbers in brackets refer to the number of animals studied. All treated values are significant. 214 TABLE 10.2 Qualitative assessment of the effect of guanethidine sympathectomy on CGRP-like immunoreactivity in 6- and 20-week-old rats 6 weeks 20 weeks Tissue Control Treated Control Treated Mesenteric artery ++ +++ +++ ++++ Mesenteric vein ++ +++ +++ -H-H- Femoral artery ++ +++ ++ +++ Femoral vein ++ +++ ++ +++ I | i -i SCG + ++++ + TTTT Vas deferens (epididymal) + +++ + +++ Bladder ++ +++ ++ +++ Right atrium + ++ + +++ Scale: + = occasional fibre only, ++ = sparse plexus of fibres, +++ = moderate plexus, ++++ = dense fibre network. Blood vessels were examined as stretch preparations; other tissues were examined as cut sections. Ratings are an average of the assessment by two independent investigators of tissues from at least four animals in each case. 215 TABLE 10.3 Quantitative assessment of area of fluorescence in stretch preparations of blood vessels using computer image analysis CGRP immunoreactivity Saline Control Guanethidine Treated Mesenteric vein 6 weeks old 1.0660 ± 0.1339 (4) 1.6921 ± 0.2891 (5) NS 20 weeks old 2.4707 ± 0.0605 (4) 2.7630 ± 0.1840 (4) NS Femoral vein 6 weeks old 1.1130 + 0.0744 (4) 1.7440 ± 0.3039 (4) NS Substance P immunoreactivity Saline Control Guanethidine Treated Mesenteric vein 6 weeks old 0.6098 ± 0.0765 (3) 0.6833 ± 0.0450 (3) NS 20 weeks old 1.7730 ± 0.1392 (2) 1.6432 + 0.0347 (3) NS Values are mean area of fluorescence (xlO mm ) ± s.e.m. per standard frame area. Figures in brackets represent number of animals studied; NS = not significant. 216 FIGURE 10.1 Representative fluorescence micrographs showing the effect of sympathectomy on noradrenaline- and calcitonin gene-related peptide (CGRP)-containing nerve fibres in ganglia. Fluorescence micrographs were taken of tissues form animals 6 weeks old. Left-hand panels (a-d) show tissues from control animals and right-hand panels (e-g) are from animals after sympathectomy, a and e: Cell bodies in the SCG that stain for noradrenaline degenerate after guanethidine treatment; only a few damaged fibres and cells remain, b and f: CGRP-immunoreactive nerve fibres in the SCG increase dramatically after guanethidine treatment. c and g: CGRP-immunoreactive nerve fibres in the SCG are not increased after sympathectomy with 6-OHDA. d and h: Cell bodies in the nodose ganglion no longer show CGRP immunoreactivity after guanethidine treatment. Scale bar = lOO^im for all plates. FIGURE 10.2 Representative fluorescence micrographs showing depletion of noradrenaline after treatment with guanethidine. Tissues are from control (a-c) and treated (d-f) animals at 6 weeks of age. a and d: mesenteric vein (stretch preparation), b and e: right atrium. c and f: epididymal portion of the vas deferens. Scale bar = 100 /xm for all plates. FIGURE 10.3 The effects of long-term sympathectomy by guanethidine on CGRP levels. Two age groups were studied - 6 weeks and 20 weeks. Results were calculated as pmol CGRP /g tissue, pmol/cm, or pmol/ganglion, and have been given as mean + s.e.m. Clear bars represent control values, hatched bars represent treated values. The number of animals used for each group is indicated under each bar. RA = right atrium, LA = left atrium, RV = right ventricle, LV = left ventricle. * = p<0.05, ** = pcO.OOl. pmol CGRP per gm tissue pmol CGRP per ganglion FIGURE 10.4 Representative fluorescence micrographs showing CGRP-immunoreactive fibres in tissues from 6 week old control (a,b) and guanethidine-treated rats (c,d). a and c: mesenteric vein (stretch preparation), b and d: epididymal portion of the vas deferens. Scale bar =100 um for all plates. pmol SP per ganglion .1 5n IUE 10.5 FIGURE levels. Two age groups were studied - 6 weeks and 20 weeks. Results were were Results weeks. 20 and weeks 6 - studied were groups age Two levels. h nme o nml ue fr ah ru i idctd ne ec bar. each under indicated is group each for used animals of number The la br rpeet oto vle, ace br rpeet rae vle. No values. treated represent bars hatched values, control s.e.m. + mean represent as given bars been have Clear and pmol/ganglion or tissue pmol/g as calculated ifrne wr significant. were differences ganglion 6 nodose 6 7 WKS h efcs f ogtr smahcoy y untiie n P n VIP and SP on guanethidine by sympathectomy long-term of effects The ih ara a deferens vas atria right WKS6 6 6 6 6 7 bladder i X 0 WKS20 CHAPTER 11 NEUROPEPTIDE Y IN NON-SYMPATHETIC NERVES IN THE RAT: CHANGES DURING NORMAL MATURATION BUT NOT AFTER LONG-TERM SYMPATHECTOMY WITH GUANETHIDINE 11.1 SUMMARY Long-term chemical sympathectomy was seen in Chapter 10 to result in a compensatory increase in innervation of several tissues with sensory nerves containing calcitonin gene-related peptide (CGRP), presumably from extrinsic sources. In this Chapter, non-sympathetic neuropeptide Y (NPY)-containing nerves in several organs in the rat were demonstrated by their persistence after acute destruction of the sympathetic nerve terminals by treatment with 6-hydroxydopamine (6-OHDA) for 48 h. The potential for these non-sympathetic NPY-containing nerves to reinnervate the tissues after removal of NPY from sympathetic sources has been examined. The innervation of the superior cervical ganglion, femoral artery, mesenteric vein, right atrium, ileum, vas deferens and urinary bladder by noradrenaline (NA)- and NPY-containing nerves was assessed in 6- and 20-week-old Sprague-Dawley rats by biochemical and histochemical techniques after treatment of the rats as neonates with guanethidine (see Chapter 10). At six weeks of age, the reduction of NPY content in tissues from guanethidine-treated animals was similar to the reduction after acute sympathectomy with 6-OHDA, 222 indicating that there was no early reinnervation by non-sympathetic NPY-containing nerve fibres at a time when the sensory transmitters increase. Furthermore, there was no evidence for reinnervation by NPY-containing nerve fibres by the time the guanethidine treated animals had reached maturity at 20 weeks of age. With maturation, NPY levels increased in the SCG but decreased in the right atrium and prostatic end of the vas deferens. There was evidence for a non-sympathetic source of NPY in the immature rat vas deferens and right atrium which diminished with maturation. 11.2 INTRODUCTION Long-term guanethidine sympathectomy of neonatal rats leads to increased innervation of the cerebral arteries and iris by NPY-containing nerve fibres of non-sympathetic origin (Mione et al. 1990), probably through an increase in the expression of NPY in parasympathetic nerves that also contain vasoactive intestinal polypeptide (VIP) (Gibbins & Morris, 1988). While often co-localised with NA (Lundberg et al. 1982a; Ekblad et al. 1984), NPY has been found in non-sympathetic intrinsic nerve cell bodies in many tissues (Morris et al. 1985; Furness & Costa, 1987; Mione et al. 1990), including the heart (Hassall & Burnstock, 1984; Maccarrone & Jarrott, 1987), ileum (Furness & Costa, 1987), bladder (James & Burnstock, 1988) and pelvic ganglia (Mattiasson et al. 1985). Chapter 10 of this thesis describes how administration of guanethidine to neonatal rats resulted in a dramatic increase in CGRP-immunoreactivity in the the superior cervical ganglion, heart and vas deferens 3 weeks after treatment, a change which was maintained for 3 months. The focus of this Chapter is to 223 examine whether there is a similar increase in innervation of different tissues by non-sympathetic NPY-containing nerve fibres following the loss of sympathetic nerve fibres after long-term chemical sympathectomy. The animals were studied at 6 weeks (immature) and 20 weeks of age (mature). The tissues studied represent both those in which NPY is exclusively within sympathetic nerves and those with an intrinsic or extrinsic supply of non-sympathetic NPY-containing nerve fibres. 11.3 METHODS 11.3.1 Long-Term Guanethidine Sympathectomy Neonatal rats were injected with high doses of guanethidine sulphate as described in Chapter 3.4.1; age-matched controls were injected with the same volume of saline. The animals were sacrificed at 6 weeks or 20 weeks of age by an overdose of CO2 gas. In Chapter 10, plasticity of some peptidergic nerves was evident even by 6 weeks of age; allowing the rats to grow to maturity without their sympathetic nerves should thus provide sufficient time for any reinnervation of the tissues by intrinsic neurones to occur. 11.3.2 Acute 6-Hydroxydopamine Sympathectomy Rats of 6 or 20 weeks of age were treated with 6-OHDA to cause acute destruction of the sympathetic nerves as described in Chapter 3.4.2. This provided an indication of normal levels of non-sympathetic NPY which exist in immature 224 and mature animals as a control for the levels found after long-term sympathectomy. 11.3.3 Tissues Studied The following tissues were examined: superior cervical ganglion (SCG), superior mesenteric vein, femoral artery, vas deferens (epididymal and prostatic portions), right atrium of the heart, ileum and urinary bladder. These represent tissues with purely sympathetic NPY as well as those with intrinsic and extrinsic sources of NPY. 11.3.4 Fluorescence Histochemistry See Chapter 3.2.1 The mesenteric veins and femoral arteries were examined as whole mounts, the right atria and vas deferens were processed as cut sections. For visualisation of catecholamine-containing nerves in the SCG, ileum and urinary bladder, we found immunohistochemical staining with antibodies to dopamine beta-hydroxylase (DBH) to be more sensitive than the fluorescence histochemical technique (see below). 11.3.5 Immunohistochemistry See Chapter 3.2.2 Immunohistochemical demonstration of innervation by NPY-containing nerve fibres was performed for whole mounts using antibodies to NPY at a concentration of 1:200. The remaining tissues were sectioned and processed for immunolabelling of NPY (all tissues) and DBH (SCG, ileum and bladder only) using antibodies at a dilution of 1:400 (anti-NPY) and 1:200 (anti-DBH). 225 Histochemical preparations were coded to enable them to be assessed without prior knowledge of treatment. 11.3.6 Assay Biochemical assay for NA content by HPLC with electrochemical detection (see Chapter 3.3.1) and for NPY content by ELISA (see Chapter 3.3.2) was performed on all specimens apart from the ileum. Control and treated tissues were assayed at the same time. 11.3.7 Materials See Chapter 3.6 11.3.8 Statistics See Chapter 3.5 Differences in tissue content of NA and NPY following sympathectomy were assessed at 6 and 20 weeks of age 11.4 RESULTS 11.4.1 Control Levels of Neuropeptide Y: Effects of Normal Maturation The NPY content of several tissues from the control animals were found to change as the animals matured from 6 weeks (when they would be considered juvenile) to 20 weeks of age (i.e. sexually mature adults); these values are given in Table 11.1. In the SCG, NPY content per ganglion increased significantly with age (P<0.02); this parallels the increase found in the NA content (P<0.01, see Chapter 226 10). However, no similar increase in NPY content with maturity was seen in the mesenteric vein and urinary bladder, despite the fact that NA levels in these tissues also increased significantly over this period (PcO.OOl and P<0.05, respectively). The NPY content, but not the NA content, was significantly reduced in the prostatic portion of the vas deferens and in the right atrium of the heart in the mature rat compared with the younger animals (P<0.01). No fall in NPY was seen in the epididymal portion of the vas deferens. 11.4.2 Effects of Guanethidine Sympathectomy on 6-Week-Old Animals 2 weeks after cessation of treatment, assay of the NPY content showed that NPY levels were severely depleted in the SCG and mesenteric vein, but in the right atrium, vas deferens and urinary bladder considerable amounts of NPY were still present. To compare the relative degree of depletion of NPY and NA on assay following guanethidine sympathectomy, the percent reduction for both is given in Table 11.1. Fluorescence histochemical and immunohistochemical assessment of the tissues agreed with the assay data; the findings are summarised in Table 11.2. Furthermore, histochemical analysis demonstrated in that those tissues with residual NPY on assay, the peptide was present within nerve fibres. Both NPY- and DBH-immunoreactivity were lost from nerve cell bodies in the SCG (Fig 11.1 a,b,d,e) and, in parallel with the depletion of noradrenergic fibres, a drastic reduction in the number of NPY-immunoreactive (IR) nerve fibres was found in the mesenteric vein and femoral artery after sympathectomy (Fig 11.1 g,h). 227 The vas deferens, like the right atrium, had a dense adrenergic innervation in the controls which was drastically depleted in the sympathectomised animals (Figs 11.2 a,b). The right atrium and the vas deferens also received a dense innervation with NPY-IR nerve fibres in the control animals (Fig 11.2 c, Fig 11.5 c) but these were still present after sympathectomy in the muscle layers of both tissues, although with a reduced density of innervation (Fig 11.2 d, Fig 11.5 d). The adrenergic innervation of the urinary bladder was sparse even in the control rats but after guanethidine treatment no DBH-immunoreactive nerves were seen (Fig 11.3 a,b). As in the vas deferens and heart, some NPY-IR nerve fibres persisted in the bladder after sympathectomy, although again the density of innervation was reduced. No NPY-IR nerve cell bodies or small intensely fluorescent (SIF) cells were seen in these tissues. Histochemical sections of ileum in the control animals showed only sparse DBH-immunoreactive nerves in the muscle layers alone (Fig 11.4 b) but there was a dense innervation of both the mucosa and submucosa with NPY-containing nerves (Fig 11.4 c) and NPY-immunoreactive nerve cell bodies were seen in the submucosa (Fig 11.4 a). In animals following guanethidine treatment, there was no reduction in NPY-immunoreactive nerve fibres or cell bodies (Fig 11.4 d,f). 11.4.3 Effects of Guanethidine Sympathectomy on 20-Week-Old Animals In the mature rats that had undergone guanethidine sympathectomy as pups, the percent reduction in tissue NPY concentration compared to age-matched controls was similar to that found at 6 weeks of age in the SCG, mesenteric vein and urinary bladder. However, the level of non-sympathetic NPY in the right atrium of the heart was lower than in the younger animals. Interestingly, the 228 levels of non-sympathetic NPY in the vas deferens of the mature animals had also fallen and both the epididymal and prostatic portions were more severely depleted in the 20-week-old treated animals (91% and 81% reduction, respectively) than in the 6-week-old rats (65% and 63% reduction, respectively) (see Table 11.1). NA levels in all tissues were still drastically depleted (83-99% reduction in NA) compared with their age-matched litter-mate controls (Table 11.1 and see Fig 11.1 c). On histochemical and immunochemical examination of the tissues, little difference could be detected between the effects of sympathectomy at 6 weeks and 20 weeks of age; NPY-containing nerves in the sympathectomised animals were still present but reduced in density in the muscle layers of the bladder (Fig 11.3 e), vas deferens (Fig 11.2 c) and right atrium (not shown). The NPY-IR nerves in the ileum were still dense and showed no depletion (Fig 11.4 e), while NPY-immunoreactivity in the blood vessels and SCG was almost totally abolished (Fig 11.1 f,i). Qualitative assessment of the density of innervation of tissues by adrenergic and NPY-immunoreactive nerve fibres supported these assay findings (Table 11.2). Thus there was no evidence of reinnervation of tissues by the residual NPY-containing nerve fibres. 11.4.4 Effects of Acute Sympathectomy by 6-Hydroxydopamine Depletion of NA and NPY following 6-ODHA treatment was similar to that after long-term guanethidine sympathectomy in all tissues, except in the SCG (Table 11.3). At 6 weeks of age, levels of NPY were not significantly reduced in the SCG after 6-OHDA treatment and NA levels were reduced by only 24%. Even in the mature animals, there was no reduction in NA in the SCG, although NPY 229 levels were now significantly reduced. 6-OHDA treatment had a greater effect on NPY levels in the vas deferens of the the mature, 20-week-old rats than in the young animals. The density of innervation of the vas deferens and urinary bladder by NPY-immunoreactive fibres after acute sypathectomy appeared the same as that after long-term sypathectomy (Fig 11.2 f, 11.3 f). 11.5 DISCUSSION , It has been previously reported that NPY is found in sympathetic nerve cell bodies in the SCG (Jarvi et al. 1986; Mione et al. 1990) and in some perivascular sympathetic nerve fibres (Uddman et al. 1985; Morris et al. 1986). The experimental work reported here confirms this and suggests that there is no intrinsic or non-sympathetic source of NPY in these tissues, since there was an almost parallel reduction of NA and NPY in the SCG, mesenteric vein and femoral artery following guanethidine sympathectomy. However, it is known that in the rat heart about 50% of the NPY remains after sympathectomy (Allen et al. 1986; Maccarrone & Jarrott, 1987) and there is a dense population of intrinsic NPY-containing nerves in the gastrointestinal tract (Furness & Costa, 1987). In the present study, sympathectomy did not reduce NPY-containing nerve fibres to the same extent as NA in the right atrium and ileum, which agrees with these previous reports suggesting intrinsic NPY-innervation. NPY in the urinary bladder and vas deferens was also affected less than NA, with NPY-IR nerves persisting after sympathectomy, which indicates that there is also a non-sympathetic source of NPY in these tissues. 230 The reduction of NPY in the bladder, right atrium and vas deferens following long-term guanethidine sympathectomy was similar to that caused by acute 6-OHDA treatment. This suggests that destruction of sympathetic nerves with guanethidine did not stimulate reinnervation by non-sympathetic NPY-containing nerve fibres, either in tissues normally innervated by purely extrinsic, sypathetic nerve fibres or in tissues with an intrinsic, non-sympathetic source of NPY. This is in contrast to the effect of long-term guanethidine sympathectomy on cerebral blood vessels and the iris in which there was reinnervation by NPY-containing nerve fibres 2 weeks after cessation of treatment, possibly due to the new synthesis of NPY in parasympathetic nerves which also contain VIP (Mione et al. 1990). The striking increase in innervation of the SCG, heart and vas deferens by CGRP-containing sensory nerve fibres after long-term but not after acute sympathectomy occured within 2 weeks after the cessation of treatment with guanethidine (see Chapter 10). From these studies, it appears intrinsic or non-sympathetic NPY-containing nerves do not respond in the same way as CGRP-containing sensory nerves to increased availability of nerve growth factor (Levi-Montalcini & Angeletti, 1968; Cole et al. 1983; Terenghi et al. 1986). An unexpected finding of this study was the difference in the percentage reduction of NPY from both epididymal and prostatic ends of the vas deferens between the immature (6-week-old) and the mature (20-week-old) rats after both acute and long-term sympathectomy. It appears that in the mature rat all the NPY is contained within sympathetic nerve fibres, in agreement with previous reports (Lundberg et al. 1982a; Carvalho et al. 1986), but in the young rat there is another source of NPY resistant to 6-OHDA and guanethidine treatment. No nerve cell bodies immunoreactive to NPY were seen in the isolated vas deferens on 231 immunohistochemical examination. SIF cells, which are known to contain NA and to proliferate after neonatal sympathectomy (Eranko & Eranko, 1971; Mione et al. 1990), were unlikely to be the source of the non-sympathetic NPY in the immature animals since none were detected on immunohistochemical examination of either the young or the mature rat vas deferens. Ligation of the hypogastric nerve in young rats (Maccarrone & Jarrott, 1988) and ligation of the hypogastric and pelvic nerves in adult guinea-pigs (Carvalho et al. 1986) does not alter the NPY content or density of innervation of the vas deferens by NPY-IR nerve fibres, which indicates that most of the NPY-IR nerve fibres of the vas deferens originate from local pelvic ganglia. During maturation, hormonal changes may play a role in regulation of the synthesis of NPY in these nerve cell bodies, so that in the mature rat NPY is no longer synthesised in these local ganglia. In keeping with the finding of a predominance of intrinsic NPY-immunoreactivity in the immature vas deferens, there was a reduction in NPY content during maturation in the right atrium of the heart. In adult rats, it is known that approximately half the NPY is in non-sympathetic, intrinsic neurones (Hassall & Burnstock, 1984; Maccarrone & Jarrott, 1987). Unlike the vas deferens, there was still a significant number of non-sympathetic NPY-containing nerve fibres in the mature rat in the present study, but the level of NPY in the immature rat atrium was three times that of the 20-week-old rat. No such prevalence of intrinsic NPY-containing neurones was identified in the urinary bladder of the immature animals, despite the fact that in this tissue a major proportion of NPY-containing nerves are non-sympathetic (Mattiasson et al. 1985), with their nerve cell bodies located in the pelvic ganglia (Keast et al. 1989). There were no differences in NPY content of the bladder with maturation and the 232 proportion of residual NPY after sympathectomy was the same in the 6-week- and 20-week-old rat. The reasons for this are not clear, but it does seem that in certain tissues e.g. the right atrium and the vas deferens, but not the urinary bladder, intrinsic NPY-containing neurones are particularly prominent in the immature rat. Furthermore, this study shows that the expression of NPY within sympathetic nerves does not always parallel that of NA during development, for example, NPY levels in the mesenteric vein do not change with maturation but NA levels increase; a similar finding has been reported in cerebral blood vessels (Dhital et al. 1988). In conclusion, this work demonstrates that: 1) in immature rats the right atrium and vas deferens have high levels of non-sympathetic NPY-containing nerves in addition to the sympathetic supply; this diminishes in mature rats so that in the vas deferens the sympathetic nerves become the sole source of NPY 2) the degree of innervation by intrinsic rather than extrinsic NPY-containing nerves varies considerably in different organs, with no intrinsic source in the SCG and blood vessels but an almost exclusively intrinsic source supplying the mucosa in the ileum and 3) there is no reinnervation of the SCG, mesenteric vein, right atrium, urinary bladder, ileum or vas deferens by NPY-containing non-sympathetic nerves up to 4 months after long-term guanethidine sympathectomy, nor is there evidence for increased or de novo synthesis of NPY in non-sympathetic nerves despite the loss of sympathetic innervation. 233 TABLE 11.1 Levels of neuropeptide Y (NPY) and percent reduction in NPY compared with noradrenaline (NA) following guanethidine sympathectomy in 6-week- and 20-week-old rats. Age NPY NPY % reduction (weeks) Control (n) Treated (n) NPY Ni Superior Cervical 6 1.46 + 0.09 (7) 0.13 ±0.06 (6)*** 91 79 Ganglion a 20 2.42 + 0.29 (5) 0.20 ± 0.03 (5)*** 92 89 Mesenteric Vein ^ 6 0.78 + 0.26 (7) 0.13 ± 0.08 (6)* * 83 87 20 0.77 ± 0.11 (6) 0.13 ± 0.07 (6)*** 83 98 Urinary Bladder 6 115.5 + 15.8 (7) 52.7 ± 11.1 (6)** 54 99 20 51.8 :± 23.5 (6) 86.1 ± 0.13 (4)* 43 83 Vas Deferens 6 864.1 ± 43.4 (5) 299.0 ± 29.9 (6)*** 65 86 -epididymal 20 983.0 ± 185.5 (6) 89.2 ± 6.8 (6)*** 91 86 Vas Deferens 6 1732.6 ± 159.4 (9) 632.8 ± 203.9 (7)*** 63 94 -prostatic 20 996.1 ± 130.5 (6) 185.4 ± 53.6 (6)*** 81 93 Right Atrium 6 252.8 ± 28.7 (7) 136.4 ± 24.0 (6)** 46 91 20 75.4 ± 18.8 (6) 28.3 ± 5.8 (6)* 62 99 NPY levels are given as pmol/g tissue except for a, where they are pmol/ganglion and k, where they are pmol/cm. * = P<0.02, ** = PcO.Ol, *** = P<0.001 compared to control. 234 TABLE 11.2 Qualitative assessment of the effect of guanethidine sympathectomy on stretch preparations of blood vessels and sections of tissues 6- week- old rats 20- week-■old rats NPY NA NPY NA C T C T C T C T Superior Cervical ++ (+) +++ - ++ - +++ Ganglion Mesenteric Vein ++++ + ++++ + ++-H- (+) +++ Femoral Artery ++ - +++ - ++ - +++ Urinary Bladder +++ ++ + - +++ ++ ++ Vas Deferens- +++ ++ +++ (+) +++ + +++ epididymal Vas Deferens- ++++ ++ +++ (+) +++ + +++ (+) prostatic Heart- right atrium +++ ++ +++ - +++ ++ +++ Ileum- mucosa +++ ++-»- (+) +++ +++ + (+) and submucosa C = control; T = treated animals. Scale for density of innervation: - = absent, (+) = scant fibres in some sections only, + = scant, ++ = moderate, ++ = dense, +++ = very dense. 235 TABLE 11.3 Percentage reduction in noradrenaline (NA) and neuropeptide Y (NPY) content compared to age-matched control rats after acute 6-hydroxydopamine treatment. 6-week-old rats 20-week-old rats NPY NA NPY NA * ♦ Superior Cervical 37 24 46 0 Ganglion * Mesenteric Vein 78 * Urinary Bladder 66 *** *** *** *** Vas Deferens 57 99 82 96 -epididymal ** *** Right Atrium 33 89 = P<0.05, = P<0.01, = PcO.OOl compared to untreated rats. 236 FIGURE 11.1 The effect of long-term guanethidine sympathectomy on the mesenteric vein (stretch preparations, a,d,g) and the superior cervical ganglion, SCG (cut sections) of the rat. The NPY-immunoreactive fibres on the adventitial surface of the mesenteric vein of the control 6-week-old animal (a) were largely destroyed in the 6-week-old treated animal, although in some sections a few residual NPY-immunoreactive fibres could be seen (d). This was unchanged in the 20-week-old treated animal (g); there was no evidence of reinnervation with NPY-immunoreactive fibres. Intact DBH-immunoreactive cells in the SCG of control 6-week-old animals (c) were not seen in guanethidine-treated animals at 6 weeks (f) or at 20 weeks of age (i). (f) illustrates fluorescence from a few degenerating cells and/or SIF cells which were occasionally seen in the treated 6-week-old animals. NPY-immunoreactive cell bodies in the control 6-week-old SCG (b) were also lost in the guanethidine-treated 6-week-old (e) and 20-week-old animals (h). Calibration bar in (h) represents 50 /mi. Magnification is the same for all photomicrographs. FIGURE 11.2 The effect of acute and long-term sympathectomy on the catecholamine -containing (sections a,b) and NPY-containing nerve fibres (sections c-f) of the epididymal portion of the rat vas deferens. Abundant catecholamine-containing nerve fibres were seen in the muscle layers of the vas deferens of the control 6-week-old animal (a); these were almost completely lost in the 6-week-old guanethidine-treated animal (b). The vas deferens of the control 6-week-old animals also contained abundant NPY-immunoreactive nerve fibres (c). Following guanethidine sympathectomy, a moderate number of NPY-immunoreactive could still be seen in the 6-week-old animal (d). Acute sympathectomy with 6-OHDA at 6 weeks of age demonstrated a reduction in the NPY-immunoreactive nerve fibres (f) comparable to the reduction in the 6-week-old guanethidine-treated animal (d), indicating that the fibres remaining after guanethidine sympathectomy had not proliferated. In fact, there were fewer residual NPY-immunoreactive fibres in the guanethidine-treated animals at 20 weeks of age (e). Calibration bar in (e) represents 50 fnn. Magnification is the same for all photomicrographs. FIGURE 11.3 The effect of long-term sympathectomy on DBH- (a,b) and NPY-immunoreactive nerves (c-f) in the rat urinary bladder. In control 6-week-old animals, DBH- (a) and NPY-immunoreactive nerves (c) were seen running parallel to the muscle fibres of the bladder. No DBH-immunoreactive nerves were seen in the 6-week-old animal following guanethidine sympathectomy (b), but a moderate number of NPY-immunoreactive nerves were still present in the 6-week-old (d) and 20-week-old animals (e). Residual NPY-immunoreactive nerve fibres in the bladder of 6-week-old rats following acute sympathectomy with 6-OHDA (f) were similar to those following guanethidine sympathectomy. Calibration bar represents 50 /xm. Magnification is the same for all photomicrographs. FIGURE 11.4 The effect of long-term sympathectomy on NPY-immunoreactive nerves in the rat ileum. The specimens were cut in transverse section. In control 6-week-old animals, NPY-immunoreactive cell bodies could clearly be identified in the sub-mucosa (arrow in a) and there was a dense network of NPY-IR nerves in the sub-mucous plexus and villae (c). In these transverse sections, NPY-immunoreactive nerve fibres were seen only occasionally in the myenteric plexus (b). Following guanethidine treatment, cell bodies in the submucosa could still be clearly seen (d) and there was no reduction in the NPY-IR nerves in the villae (f). This was also unchanged at 20 weeks in the guanethidine treated animals (e). Magnification for (a-d,f) is the same while (e) is taken on a lower magnification. Calibration bar in (a) and (e) represents 50 fini. FIGURE 11.5 The effect of long-term sympathectomy on NPY-immunoreactive nerves in the right atrium of 6-week-old rats. Abundant NPY-immunoreactive nerve fibres were present in the control rats (b); following guanethidine sympathectomy, a moderate number of fine NPY-immunoreactive nerves could still be seen (d). Calibration bar represents 50 /xm. Magnification is the same for both photomicrographs. CHAPTER 12 FINAL DISCUSSION 12.1 USE OF THE RABBIT CORONARY MODEL 12.1 Smooth Muscle and Endothelial Responses The work in this thesis has established that the rabbit isolated coronary artery is a useful model to study coronary vasomotor responses. Several problems have, however, emerged. Firstly, considerable patience and practice are required to enable the artery to be dissected intact without damage to the endothelium. The responses of the vessel do appear to deteriorate if the time from removal of the heart to immersion of the preparations in the organ bath exceeds 45-60 min, in spite of continuous bathing in oxygenated physiological saline solutions, but several other workers are now using this model successfully. The ease with which the rabbit can be manipulated means that the model may be useful to study changes resulting from a variety of pathological stimuli such as chronic hypoxia or hormonal manipulation, and the Watanabe Hereditable Hyperlipidaemic rabbit seems to provide an interesting experimental model to study changes with the development of atherosclerosis. It is a disadvantage that only the epicardial coronary arteries may be studied; at present there is no way to reliably study the responses of the intramural rabbit coronary arteries without the complication of metabolic effects 242 from surrounding myocardium. Several groups are examining the use of myographs such as that designed by Mulvany to study responses from vessels less than 200 fin 1 in diameter but the coronary vessels are particularly thin walled and I have encountered problems with damage to the endothelium as these rather floppy vessels are threaded onto the wires. The principle of the technique is identical to that for the larger vessels. 12.1.2 Effects of Nerve Stimulation This thesis describes the postsynaptic responses to several of the autonomic neurotransmitters but not responses to nerve stimulation itself. This is because responses to sympathetic nerve stimulation in this vessel were disappointing. Transmural stimulation using a variety of parameters which produced excellent constrictor responses in other vessels, such as the saphenous, femoral and mesenteric arteries, gave no response in this coronary preparation. Destruction of the perivascular nerves was avoided by careful dissection rather than stripping of the preparations and the presence of these nerves was confirmed by fluorescence histochemistry of several stretch preparations similar to those in Chapters 10 and 11. Constrictor responses were readily obtained when the pulse duration exceeded 1 ms but these were not abolished by TTX which suggests that they were due to direct stimulation of the smooth muscle. Interestingly, when the coronary preparations were preconstricted (with either PGF2a or KC1) these same parameters produced profound and reproducible relaxation of the preparations which was also resistant to TTX and was not affected by removal of the endothelium. 243 A similar direct smooth muscle relaxation has been reported for the dog coronary artery and to a much lesser extent in other vessels such as the saphenous vein (Kalsner & Quillan, 1989b; Feletou & Vanhoutte, 1989). Some workers have shown TTX-sensitive responses to transmural field nerve stimulation in coronary arteries (usually of the dog) but these are always small compared to other vessels of similar size and with comparable noradrenergic innervation (Toda et al. 1986b). The reasons for this are unknown and may well be related to problems with in vitro experiments rather than a physiologically relevant phenomenon but this has made the study of neural control of the coronary arteries more difficult. A criticism of the work in Chapter 6 is that the evidence for inhibitory co-transmission by noradrenaline and ATP and could not be substantiated or refuted by nerve stimulation experiments. 12.2 CORONARY ARTERY RESPONSES TO NEUROTRANSMITTERS 12.2.1 Responses to the Classical Neurotransmitters As with most other species studied, the epicardial rabbit coronary arteries possesses -adrenoceptors which mediate vasodilatation to noradrenaline. However, the rabbit differs from the most frequently studied species, the dog, in that it possesses very few a-adrenoceptors. Furthermore, the response to acetylcholine is midway between the pig and the dog, as discussed in Chapter 4. This work emphasises that the differences between species and between blood vessels, extending even to the quality of the response to a given stimulus (i.e. vasoconstrictor or vasodilator), do not appear to reflect fundamental differences in 244 their anatomy or physiology but rather a variation in the density of smooth muscle and endothelial receptors, which are probably present to some degree in all species. 12.2.2 Purinergic Control of the Rabbit Coronary Artery The studies of the purinoceptors in Chapters 6 and 7 of this thesis have revealed an important variation between the coronary artery and most other vessels. Just as the coronary artery has jS-adrenoceptors mediating vasodilation while most other arteries possess vasoconstrictor a-adrenoceptors, so too does the coronary possess a dominant population of P 2 Y“PurinocePtors on the smooth muscle mediating direct smooth muscle relaxation rather than the *>2X~pur*noceptors mediating vasoconstriction which dominate in most other vessels. This is consistent with the concept that noradrenaline and ATP act as sympathetic inhibitory co-transmitters in the coronary arteries although, as noted above, this evidence is circumstantial. In the light of the limited responses obtained in coronary arteries to nerve stimulation, it is possible that nerve-mediated responses may play a relatively minor role in the control of epicardial coronary arteries. However, this does not mean that the particular distribution of adrenergic and purinergic receptors in the coronary arteries described in this thesis does not play an important role in the control of coronary vasomotor tone in response to physiological stimulation (such as exercise and stress) and pathophysiological stimulation (e.g. myocardial ischemia and heart failure). These conditions are known to be associated with sympathetic nerve stimulation and hence with markedly elevated levels of circulating catecholamines; because of the receptor population on the coronary arteries, 245 myocardial blood flow in response to these circulating agents will be preserved while other vascular beds (e.g. splanchnic) will vasoconstrict. ATP may play an important local role in the control of coronary tone: release of ATP has been demonstrated by Paddle and Burnstock in response to coronary vasodilation (Paddle & Burnstock, 1974). The distribution of the purinergic receptors in the coronary artery would allow this vessel to dilate in response to ATP, even in the presence of disease such as severe atheroma which disrupted the endothelium. It is intriguing to speculate that ATP might also stimulate primary sensory nerve endings in the heart, either directly via presynaptic P 2 -purinoceptors or via presynaptic Pj-purinoceptors after breakdown to adenosine, causing angina pectoris in the presence of hypoxic myocardial tissue (Crea et al. 1990). But the development of selective antagonists to ATP and adenosine which may be used in man are necessary to examine this hypothesis more fully. The degree to which these neurotransmitters mediate responses via the endothelium in vivo remains unknown but, as stated at the beginning of this thesis, it seems improbable that neurally-released transmitter is the physiological agonist of the endothelial receptors. The recent discovery that the endothelium is capable of the synthesis and release of several transmitter substances, which may then act locally on other endothelial cells, indicates that the endothelium itself is a more likely physiological source of these agents. Moreover, there are some early indications that chronic stimulation of the perivascular nerves may be able to influence the endothelial production of such agents. 246 12.2.3 Coronary Responses to the Neuropeptides The smooth muscle and endothelial responses of the rabbit epicardial coronary artery described in this thesis are similar to those which have been described for many other blood vessels in recent years. Thus substance P was found to be an endothelial-dependent vasodilator, while calcitonin gene-related peptide and vasoactive intestinal polypeptide acted only on the smooth muscle to cause relaxation. Neuropeptide Y is known to have a powerful direct effect on the coronary vascular bed in the isolated heart but the work in this thesis suggests that on the rabbit epicardial coronary arteries it is potent (i.e. it acts at low concentrations) but not powerful (i.e. it has a small maximal vasoconstrictor effect). Others have reported similar observations in the epicardial coronary arteries of dogs (Macho et al. 1989). This suggests that the main site of action of neuropeptide Y in the coronary bed is on the small coronary arteries or arterioles, a view supported by angiographic findings in man (Clarke et al. 1987). 12.3 PLASTICITY OF VASOMOTOR RESPONSES 12.3.1 Physiological Changes Many previous studies have described changes in coronary responses to the classical neurotransmitters noradrenaline and acetylcholine with development and aging, although these have not focussed on the period of sexual maturation examined in this thesis. Moreover, there are very few studies of changes in vasomotor responses to the peptide neurotransmitters; the work in this thesis is the first study to describe such changes in the coronary arteries. It shows that there 247 are profound and consistent changes in both vasoconstrictor and vasodilator responses to these peptidergic neurotransmitters with maturation. A limitation of this work is that it is phenomenological rather than analytical; I have described the changes which occurred but cannot comment on the mechanisms responsible. A number of studies would be useful in elucidating these mechanisms: for example, changes in receptor density could be examined using autoradiographic labelling, although the lack of specific antagonists for the peptides at present would complicate interpretation; measurement of adenylate and guanylate cyclase, cAMP and cGMP may indicate changes in the second messenger systems which mediate vasodilation; and alterations in intracellular calcium fluxes may occur in response to the vasoconstrictor agonists. From the wide variety of changes reported in other vessels and organs with aging, it is likely that several of these mechanisms may be operating. Recent preliminary work I carried out which has been followed up by a colleague, Dr. Jiang, indicated that oestrogen (as 17/?-oestradiol) has a powerful and longlasting vasodilator action on the smooth muscle of the rabbit coronary artery which is independent of the presence of the endothelium and may be related to a calcium channel-blocking action. Another colleague, Antonia Brizzolara, found differences in the nature and time course of the changes to several of the neurotransmitter peptides with aging in male New Zealand White rabbits compared to those in female rabbits. This suggests that hormonal influences may play an important role in controlling such age-related changes in responses; studies of oopharectomised female rabbits with and without hormone replacement therapy may reveal a role of the sex hormones in the control of vasomotor responses. Such studies could provide important clues to the 248 mechanisms protecting premenopausal women from coronary atheroma and myocardial infarction as well as their predisposition to suspected small vessel coronary disease in syndrome X. The widespread use of the oral contaceptive pill and hormone replacement therapy in postmenopausal women may also lead to long-term changes in coronary vasomotor responses in the same way as those described in maturing rabbits in this thesis; these merit further investigation. 12.3.2 Pathophysiological Changes The studies in Chapters 9, 10 and 11 of this thesis show that vasomotor peptidergic innervation and responses are affected not only by normal physiological development but also under pathophysiological conditions. The Watanabe Hereditable Hyperlipidemic rabbit is a good model for human familial hypercholesterolaemia and allowed me to study long-term changes in the responses to neurotransmitter agents during the early development of coronary atherosclerosis. This work showed the surprising result that endothelium-dependent responses in the coronary artery actually recovered early in the disease and suggests that, like perivascular nerves and vascular smooth muscle, the endothelium may also undergo compensatory changes in disease. The work is again descriptive rather than analytical and, in view of the unexpected findings, should be extended to examine whether an endothelium-derived relaxant factor mediates the response and, if so, whether this is due to increased synthesis or release of EDRF or to enhanced smooth muscle responses. Whatever the mechanism, support for the work reported in this thesis for coronary arteries came from other workers in the same laboratory who independently studied several other blood vessels (such as femoral, hepatic and mesenteric arteries) from the same 249 rabbits and also demonstrated increased endothelial-dependent relaxation in most vessels with the exception of the aorta, which showed extensive atheromatous damage at an early age. 12.4 PLASTICITY OF PEPTIDERGIC INNERVATION In addition to this ’plasticity’ of receptor-mediated responses in normal and disease states, it is well known that plasticity is a property of autonomic nerves. Again, there have been many studies on changes in noradrenergic innervation following section or destruction of sympathetic nerves. But with recent advances in immunohistochemical techniques, it has become clear that many other nerve types play an important role in autonomic neural control and indeed may dominate in some organ systems such as the gastrointestinal tract. Using guanethidine destruction of sympathetic nerves in rats, I and my co-workers have demonstrated that plasticity is also a property of certain peptidergic nerves which proliferate following release from the inhibitory presence of the normal sympathetic nerves. This appears to be a highly selective process - only one nerve subtype, containing CGRP, appeared to be able to undergo compensatory proliferation. Other nerve types, including nerves containing CGRP costored with SP, showed no such changes. This may be related simply to the maturity of the different nerve networks in the neonatal rats, since different subtypes have been shown to develop at different stages of maturation; continuing studies using guanethidine sympathectomy in adult rats and experiments with anti-nerve growth factor in neonates will help to establish the mechanisms involved. I have carried out selective sensory nerve destruction in neonatal rats with the neurotoxin capsaicin, with the aim of examining whether the CGRP-containing nerves or even noradrenergic nerves (which are both resistant to capsaicin) may also proliferate 250 when the primary SP-containing nerves are destroyed; this work has not yet been completed. It is interesting to note that non-sympathetic NPY-containing nerves do not appear to be capable of proliferating in the same way as the CGRP-containing nerves even though sympathectomy is associated with a profound reduction of the NPY contained in sympathetic nerves. This may reflect important differences in the factors controlling the growth and distribution of intrinsic neurones as distinct from extrinsic nerves. The preponderence of intrinsic NPY-containing nerves found in some organs in the younger animals suggests a trophic role for this peptide. 12.5 CONCLUDING REMARKS The work in this thesis has examined some of the factors controlling coronary vascular smooth muscle responses to autonomic transmitters and highlights the remarkable flexibility of the vascular control systems. It emphasises the similarities rather than differences between vessels and between species: responses which were considered to be fundamentally different in fact arise from variations in the quantity and distribution of the same underlying receptors. This may have important practical implications. 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Circ Res 16: 431-440. 334 PUBLICATIONS FROM WORK CARRIED OUT DURING THIS THESIS Laura Corr, J. Aberdeen, P. Milner, J. Lincoln, G. Burnstock. Sympathetic and nonsympathetic neuropeptide Y-containing nerves in the rat myocardium and coronary arteries. Circ. Res. 1990; 66: 1602-1609. J. Aberdeen, Laura Corr, P. Milner, J. Lincoln, G. Burnstock. Marked increases in calcitonin gene-related peptide-containing nerves following long-term sympathectomy with guanethidine. Neuroscience 1990; 35:175-184. Laura Corr, G. Burnstock. Magnesium inhibits the responses to Neuropeptide Y in the rabbit coronary artery. J. Mol. Cell. Cardiol. 1990; in press. Laura Corr, G. Burnstock, P. Poole-Wilson. Responses of the rabbit epicardial coronary artery to acetylcholine and adrenoceptor agonists. Cardiovasc. Res. 1990; in press. P. Milner, J. Lincoln, J. Aberdeen, L. Corr, G. Burnstock. 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