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The relationship between sinoaortic baroreceptors, atrial receptors and the release of vasopressin in… Courneya, Carol Ann Margaret 1987

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T H E R E L A T I O N S H I P B E T W E E N SINO AORTIC B A R O R E C E P T O R S , A T R I A L R E C E P T O R S A N D T H E R E L E A S E O F V A S O P R E S S I N IN T H E A N A E S T H E T I Z E D RABBIT.  by  Carol Ann Margaret Courneya B.Sc. University of Guelph M.Sc. University of Western Ontario  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F DOCTOR O F PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Physiology  We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y O F BRITISH C O L U M B I A March 1987 @  Carol Ann Margaret Courneya, 1987.  In presenting  this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  ft^  l( C I-TU  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6G/81)  OC^  ii ABSTRACT  Vasopressin, a hormone released from the neurohypophysis, contributes to the regulation of body fluid balance through its known actions on the kidney and the vasculature. Release of vasopressin is influenced by plasma osmolality and by afferent activity from sensory receptors in the high and low pressure vascular systems. Previous studies have not defined the relative importance of the carotid sinus baroreceptors, aortic baroreceptors and atrial receptors in the control of the plasma concentration of vasopressin in the rabbit. Experiments were carried out in anaesthetized rabbits to define the quantitative relationship between stimulation of the carotid sinus baroreceptors and the plasma concentration of vasopressin. This relationship was examined in the presence and absence of afferent input from the aortic and atrial receptors.  Changes in blood volume were  induced to produce a change in the stimulus to the aortic baroreceptors and atrial receptors at high or low, constant carotid sinus pressure. Section, of the aortic depressor nerves and the vagus nerves allowed examination of the individual contributions of atrial receptors or aortic baroreceptors on the plasma concentration of vasopressin. It was also possible to examine the interaction between the carotid sinus baroreceptors and the aortic and atrial receptors. The results showed that plasma concentration of vasopressin was reduced by minimal stimulation of carotid sinus baroreceptors and that maximal inhibition of the release of vasopressin was achieved with a relatively low total arterial baroreceptor input. No influence of carotid sinus baroreceptors on vasopressin release was seen in the presence of intact aortic baroreceptors demonstrating the important interaction between the effects of stimulation of these two sets of receptors. It was not possible to demonstrate, in the rabbits used in this study, a significant contribution of atrial receptors to the control of vasopressin release either in response to changes in carotid sinus pressure or in response to changes in blood volume. To minimize the inhibitory effect of arterial baroreceptors on  iii  the release of vasopressin the aortic depressor nerves were cut and carotid sinus pressure was set at a low level. It was still not possible to demonstrate an effect of a reduction in blood volume on vasopressin release, confirming the absence of a contribution from atrial receptors in the anaesthetized rabbit. There appears to be considerable variation between species in the contribution of the different receptor groups to the release of vasopressin. The results suggest that in the normal rabbit there is likely to be significant tonic inhibition of the release of vasopressin by stimuli arising from arterial baroreceptors. The absence of a demonstrable influence of atrial receptors in these rabbits is consistent with findings in primates but differs from those in dogs.  It is unlikely that changes in plasma vasopressin concentration induced by  small changes in blood volume contribute to the control of arterial pressure through direct effects on vascular resistance and capacitance.  T A B L E OF CONTENTS  ABSTRACT  ii  ACKNOWLEDGEMENTS  x  * i  TABLE OF CONTENTS  J-»  LIST O F FIGURES  vii  LIST O F T A B L E S  xii  HISTORICAL REVIEW  1  i  VASOPRESSIN G E N E R A L CHARACTERISTICS  1  ii  H I G H P R E S S U R E (SINOAORTIC) B A R O R E C E P T O R S  7  iii C A R D I A C (LOW PRESSURE) R E C E P T O R S  10  iv  O T H E R V O L U M E SENSITIVE RECEPTORS  15  v  CENTRAL CONNECTIONS  17  vi  I N T E R A C T I O N B E T W E E N H I G H A N D L O W P R E S S U R E R E C E P T O R S IN T H E  ".  R E L E A S E OF VASOPRESSIN vii E F F E C T S O F C H A N G E S IN V A S O P R E S S I N O N A R T E R I A L P R E S S U R E  HYPOTHESIS  24  ANIMALS, INDUCTION OF ANAESTHESIA AND SURGICAL PREPARATION  ii  21  23  GENERAL METHODOLOGY i  19  ANALYTICAL METHODS  iii R A D I O I M M U N O A S S A Y F O R A V P  24 28 30  V  CHAPTER 1: CAROTID SINUS PRESSURE AND VASOPRESSIN RELEASE  36  i  INTRODUCTION  36  ii  PROTOCOL  36  iii RESULTS CHAPTER 2: BLOOD VOLUME CHANGES AND VASOPRESSIN RELEASE  39 64  i  INTRODUCTION  64  ii  PROTOCOL  64  iii RESULTS  68  CHAPTER 3: INTERACTION BETWEEN CAROTID SINUS BARORECEPTOR AND ATRIAL VOLUME RECEPTORS IN VASOPRESSIN RELEASE  97  i  INTRODUCTION  97  ii  PROTOCOL  97  iii RESULTS  100  CHAPTER 4:HAEMORRHAGE AND VASOPRESSIN RELEASE (AORTIC BARORECEPTORS VS. ATRIAL VOLUME RECEPTORS) i  INTRODUCTION  115 115  ii PROTOCOL  115  iii RESULTS  118  DISCUSSION  126  i  CAROTID BARORECEPTORS AND PLASMA VASOPRESSIN  127  ii  BLOOD VOLUME CHANGES AND VASOPRESSIN  132  a) VASOPRESSIN RESPONSES TO HAEMORRHAGE  132  b) VASOPRESSIN RESPONSES TO VOLUME EXPANSION  134  c) CARDIOVASCULAR RESPONSES  135  vii  d) INTERACTION BETWEEN HIGH AND LOW PRESSURE RECEPTORS  136  e) ATRIAL VOLUME RECEPTORS AND AORTIC BARORECEPTORS AND VASOPRESSIN RELEASE iii ROLE OF VASOPRESSIN IN CARDIOVASCULAR CONTROL REFERENCES  140 143 145  LIST OF FIGURES  Figure l a  Amino acid sequence of arginine vasopressin  2  Figure lb  Illustration of carotid sinus perfusion system  26  Figure lc  Iodination profile and standard curve for arginine vasopressin  32  Figure 2  Protocol for experiments in Chapter 1  37  Figure 3  Changes in mean arterial pressure in response to alterations in carotid sinus pressure. Aortic depressor nerves were sectioned before vagus nerves  Figure 4  41  Changes in mean arterial pressure in response to alterations in carotid sinus pressure. Vagus nerves were sectioned before aortic depressor nerves  Figure 5  43  Records of responses of mean arterial pressure and right atrial pressure to step changes in carotid sinus pressure. Rabbits had had both aortic depressor nerves and vagus nerves intact  Figure 6  45  Records of responses of mean arterial pressure and right atrial pressure to step changes in carotid sinus pressure. Rabbits had intact aortic depressor nerves and sectioned vagus nerves  Figure 7  47  Records of responses of mean arterial pressure and right atrial pressure to step changes in carotid sinus pressure. Rabbits had sectioned aortic depressor nerves and sectioned vagus nerves  49  viii--.  Figure 8  Changes in immunoreactive vasopressin in response to alterations in carotid sinus pressure. Aortic depressor nerves were sectioned before vagus nerves  Figure 9  51  Changes in immunoreactive vasopressin in response to alterations in carotid sinus pressure. Vagus nerves were sectioned before aortic depressor nerves  Figure 10  53  Changes in immunoreactive vasopressin in response to alterations in carotid sinus pressure Comparison was made between rabbits before and after sectioning both the aortic depressor nerves and vagus nerves (n= 19)  Figure 11  55  Changes in mean arterial pressure in response to alterations in carotid sinus pressure. Comparison was made between rabbits before and after sectioning both the aortic depressor nerves and vagus nerves (n= 19)  Figure 12  58  Changes in heart rate in response to alterations in carotid sinus pressure. Comparison was made between rabbits before and after sectioning both the aortic depressor nerves and vagus nerves (n=19)  Figure 13  60  Effects of serial nerve section on baseline levels of immunoreactive vasopressin, mean arterial pressure, right atrial pressure and heart rate  62  Figure 14  Protocol for experiments in Chapter 2  65  Figure 15  Record of response of mean arterial pressure and right atrial pressure to bilateral vagotomy  69  ix  Figure 16  Changes in immunoreactive vasopressin in response to haemorrhage before and after vagal section in rabbits with intact aortic depressor nerves  Figure 17  73  Changes in immunoreactive vasopressin in response to volume expansion carried out before and after vagal section in rabbits with intact aortic depressor nerves  Figure 18  76  Record of immediate and 10 minute response of mean arterial pressure and right atrial pressure to haemorrhage of 10% of the blood volume in aortic barodenervated rabbits  Figure 19  79  Changes in immunoreactive vasopressin in response to haemorrhage carried out before and after vagal section in rabbits with sectioned aortic depressor nerves  Figure 20  82  Changes in immunoreactive vasopressin in response to volume expansion carried out before and after vagal section in rabbits with sectioned aortic depressor nerves  Figure 21  Relationship between immunoreactive vasopressin and mean arterial pressure  Figure 22  85  87  Effects of vagal section on baseline levels of immunoreactive vasopressin  90  Figure 23  Effects of vagal section on baseline levels of right atrial pressure  92  Figure 24  Effects of vagal section on baseline levels of mean arterial pressure  94  Figure 25  Protocol for experiments in Chapter 3  98  X  Figure 26  Changes in mean arterial pressure in response to haemorrhage carried out in rabbits at two levels of carotid sinus pressure  Figure 27  Changes in right atrial pressure in response to haemorrhage carried out in rabbits at two levels of carotid sinus pressure  Figure 28  102  104  Changes in immunoreactive vasopressin in response to haemorrhage carried out in rabbits at two different levels of carotid sinus pressure  Figure 29  Changes in mean arterial pressure in response to volume expansion at two levels of carotid sinus pressure  Figure 30  Figure 32  Protocol for experiments in Chapter 4  Figure 33  Changes in mean arterial pressure in response to haemorrhage (CSP = 60mmHg)  112  116  120  Changes in right atrial pressure in response to haemorrhage (CSP=60 mmHg)  Figure 35  110  Changes in immunoreactive vasopressin in response to volume expansion at two levels of carotid sinus pressure  Figure 34  108  Changes in right atrial pressure in response to volume expansion at two levels of carotid sinus pressure  Figure 31  106  122  Changes in immunoreactive vasopressin in response to haemorrhage (CSP = 60 mmHg)  124  xi  LIST OF T A B L E S  Table I  Changes in mean arterial pressure and right atrial pressure measured 10 minutes after haemorrhage in rabbits with intact aortic depressor nerves  Table II  71  Changes in mean arterial pressure and right atrial pressure measured 10 minutes after volume expansion in rabbits with intact aortic depressor nerves  Table III  75  Changes in mean arterial pressure and right atrial pressure measured 10 minutes after haemorrhage in rabbits with sectioned aortic depressor nerves  Table IV  78  Changes in mean arterial pressure and right atrial pressure measured 10 minutes after volume expansion in rabbits with sectioned aortic depressor nerves  Table V  84  Changes in mean arterial pressure and right atrial pressure measured within 1 minute of haemorrhage or volume expansion  96  xii  ACKNOWLEDGEMENTS I sincerely thank Dr.John Ledsome for his unending assistance, enthusiasm and patience despite a heavy administrative load. There was never any laboratory problem too minor or question too small which he would not take time to discuss and for that I am very grateful. I thank Andrew Rankin for assistance in our collaborative studies and for making Zedsome Labs a fun place to work. I thank Marie Greene for her excellent technical assistance and her Scottish sense of humor. I am grateful to Anna Maria Azzarola for her skilled handling of the vasopressin assay. I thank Dr. John Brown, Dr. S. Katz and the members of my advisory committee (Dr.B. Milsom, Dr. F.Lioy, Dr. P.Vaughan, D r . N . Wilson) whose constructive criticisms were essential in the completion of this thesis. I acknowledge the financial support of the Canadian Heart Foundation. I extend a sincere thank you to Mary Forsythe for her good humor and excellent administrative assistance and to John Sanker, Jeff Russel and Joe Tay for their assistance in the completion of the figures for this thesis. Thank you to Pat Leung for his timely help in the final drafting of this thesis and to Dr. Pearson for providing me with a space where I could write. I am especially grateful to Ray Pederson for. his unwavering support and friendship throughout the course of my time at U B C . I thank the Brew Crew (+Martin) for all the good times we had and Lori Mudrick for sharing with me her enthusiasm for life. Finally I thank Rob Thies for always understanding exactly what I meant.  1  HISTORICAL REVIEW J.P.  Peters (1935) was the first to suggest that there was a link between blood  volume and a regulatory system involving the kidney. Peters stated that "the fullness of the blood stream may provoke a diuretic response on the part of the kidney." This provided the basis for the hypothesis of a negative feedback system of blood volume control (Henry and Pearce, 1956).  The components of this system are now known to include volume  sensitive receptors, afferent nerves which communicate information to central neurons and effector mechanisms (both humoral and neural) which act via the kidney to establish normal blood volume.  The efferent mechanisms controlling kidney function and therefore  blood volume include both neural (sympathetic nervous system) and humoral components (vasopressin).  The major focus of this review is on the receptors and the central neurons  which are involved in the hormonal control of blood volume.  Particular emphasis will be  placed on factors promoting or inhibiting the release of vasopressin, a hormone known to be involved in body fluid regulation.  Vasopressin General Characteristics Vasopressin is an octapeptide in which an amino acid ring structure is formed by the closure of an S-S bond between two cysteine molecules to form cystine (figure la). The form of vasopressin most commonly found in mammals is arginine vasopressin.  Lysine  vasopressin (lysine is substituted for the arginine in position 8) is the form of vasopressin found in pigs.  Unless otherwise stated, the generalized term vasopressin will be used in  this review to signify arginine vasopressin. The  synthesis  and  release  of  vasopressin  takes  place  in  the  hypothalamoneurohypophysial tract. The two principal groups of neurons in this tract are located within the supraoptic and paraventricular nuclei of the hypothalamus. Axons from the supraoptic nuclei and the paraventricular nuclei project ventrally and caudally to the median eminance and posterior pituitary (neurohypophysis).  Vasopressin is synthesized  2  Figure la: The amino acid structure of the octapeptide arginine vasopressin.  3  0 V 2 || 0 = C - C H - N H - C - CC HH - -NH, C HH  o-  2 C=NH N H  NH  CH -CH  NH  2  C—O  CH,  NH i -C •CH2 CH2 — CH  O  —  II  PH  2  0  \ II  CH, 0 O II 9 2 II C-NH-CH-C-NH- C H - C - N H H  C-NH- CH-C •NH-CH-C-N-CH  O  + 0  2  CH,  c=o NH  O CHo  0  B Jyr Phe  ' I  Gin S \ 1 Asn-Cys-Pro-Arg-Gly-NH2  2  4  along with oxytocin and neurophysin. common precurser protein (Sachs et al.  These three peptides are thought to arise from a 1969, Pickering et al.  1975, Gainer and Sarne  1977) and are transported in secretory granules down the axons to the terminal buds. The granules are released into the blood stream by exocytosis (Douglas 1973) in conjunction with axonal depolarization and the subsequent inward movements of calcium and other ions (Hays 1976).  It is believed that once in the blood stream the distribution of  vasopressin is confined to the plasma in humans, rats and dogs (Ginsburg and Heller, 1953, Lauson and Bocanegra, 1961, Czaczkes and Kleeman 1964,  Smith and Thorn  1965). The half life of vasopressin in blood has been reported to be between 0.9 and 7.5 minutes depending on the animal species under examination (Ginsburg and Heller 1953, Ginsburg 1957, Lauson and Bocanegra 1961, Silver et al. 1961, Chaudhury 1961, Share, 1962, Czaczkes and Kleeman, 1964).  More recent studies suggest that in dogs the  disappearance of vasopressin has two components one having a half time of 1.4 +/1.4 minutes and the other a half time of 4.1 +/-0.2 minutes (Weitzman and Fisher, 1978) . Plasma concentration of vasopressin may reflect either release or degradation. There are two proposed sites for the metabolic clearance of vasopressin, one being the kidney and the other the splanchnic viscera. Until recently it was believed that the bulk of the vasopressin clearance which occurred in the splanchnic viscera took place in the liver. Matsui et al.  (1983) quantified the clearance of vasopressin which occured in the pre-  hepatic viscera (intestines) and the total clearance of vasopressin by the splanchnic circulation.  The pre-hepatic vasopressin clearance accounted for almost half of the total  splanchnic clearance of vasopressin and the liver accounted for the rest. In the kidney the one determinant of clearance is how much vasopressin is freely filtered.  Share and Crofton (1980) have demonstrated that the higher the plasma  concentration, the greater the amount of vasopressin bound to plasma protein and the less  5  which is freely filtered. A t normal plasma concentrations, however, glomerular filtration is only minimally inhibited by protein binding.  Once vasopressin is filtered at the  glomerulus it is either degraded or reabsorbed in the proximal nephron and then secreted into the  distal nephron (Kimura and Share, 1981).  Post glomerular clearance of  vasopressin has also been shown to occur in isolated rat kidneys perfused with an artificial colloid solution (Rabkin et al.  1979).  Since vasopressin has vasoactive properties and  renal clearance of vasopressin involves tubular processes Matsui et al. (1983) investigated the direct effects of different concentrations of vasopressin on the renal clearance of vasopressin.  In spite of alterations in the plasma vasopressin up to 14 times the normal  levels there was no significant change in the renal clearance of vasopressin. Finally there is evidence that in dogs both kidney and splanchnic clearance of vasopressin account for only half of the total metabolic clearance of vasopressin (Weitzman and Fischer 1978, Montani et al. 1980) therefore one cannot rule out the possibility that there are other sites of metabolic clearance. The secretion of vasopressin is controlled by receptors which are sensitive to changes in the osmolality of the plasma. This osmoreceptor hypothesis was formulated by Verney in 1947 based on experiments in conscious dogs.  He showed that infusion of a  hyperosmolar solution into the common carotid arteries inhibited a water diuresis in hydrated dogs. Therfore it was suggested that receptors sensitive to alterations in osmolar concentration of extracellular fluid were located in the area of the brain supplied by the common carotid arteries. Later Jewel (1953) suggested that these receptors were located in the anterior hypothalamus. vitro  from  tissue  which  Sladek and Joynt (1978) stimulated vasopressin release in  contained  supraoptic  neurons  which  were  linked to  the  neurohypophysis by altering the osmolality of the medium in which the tissue was suspended.  The specificity of this response was demonstrated by Sladek and Knigge  (1977) when they showed that vasopressin was  not changed when osmolality  was  increased by the addition of glucose or urea as opposed to sodium chloride. Robertson et  6  al. 1977 showed that increases in blood osmolality of 1% or less stimulated vasopressin release in vivo.  Therefore these central osmoreceptors were exquisitely sensitve to the  tonicity of the hypothalamic extracellular fluid.  Bie (1980) has given a comprehensive  description of the work in the area of osmoreception since Verney's work. Receptors sensitive to changes in osmolality have been reported to exist in the hepatic or portal circulations (Haberich 1968, Chwalbinska-Moneta 1979, Baertschi and Valet, 1981), in the renal circulation (Recordati et al. 1980) and in the area of distribution of the common carotid artery (Montani et al. 1980, Wade et al. 1982). Recently Liard et al. (1984) have cast doubt on the existance of hepatic osmoreceptors. Quillen and Cowley (1983) have examined the release of vasopressin over a range of plasma osmolalities in conscious dogs and found a linear relationship between plasma vasopressin and plasma osmolality. correlating  plasma  osmolality  They found that during normovolaemia the line and  plasma  vasopressin  0.21 pg/ml / mosmol/kg and an intercept of 277 mosmol/kg.  had  a  slope  of  To date the relationship  between plasma osmolality and vasopressin release has not been examined in rabbits. In addition to osmoreceptor control of vasopressin secretion it has been shown that alterations in blood volume influence the plasma concentration of vasopressin (Cowley 1982).  In the event that the body is faced with a situation where the homeostasis of  osmotic pressure and that of blood volume mutually interfere, protection against loss of blood volume predominates over restoration of osmotic pressure (McCance 1936, Epstein 1956,  Welt 1960).  This concept was  nicely phrased by Leaf and Frazier (1961):  "teleologically, dilutional hyponatraemia is a lesser evil than circulatory collapse." Quillen and Cowley (1983) demonstrated that in the dog changes in blood volume exerted a significant  modulation over  the  relationship between  the  plasma  concentration  of  vasopressin and plasma osmolality.  Hypovolaemia resulted in a greater amount of  vasopressin  level  released  at  a  similar  of  plasma  osmolality  as  compared  to  normovolaemia. The opposite was true for hypervolaemia. Because of this modulation the  7  plasma concentration of vasopressin was regulated so that contraction or expansion of blood volume was more expeditiously corrected than would occur if osmoreceptors alone regulated the plasma concentration of vasopressin.  A n interaction between the volume  and osmoreceptor control of vasopressin release has also been demonstrated in conscious sheep and rats (Dunn et al. 1973, Johnson et al. 1970). The  major thrust of this review is the control of vasopressin release caused by  alterations in volume rather than alterations in plasma osmolality.  The volume sensitive  receptors are divided into three classes, each of which will be dealt with individually: high pressure (sinoaortic) baroreceptors, low pressure (cardiac) receptors and "other" volume sensitive receptors.  High Pressure (sinoaortic) Baroreceptors Baroreceptors are specialized nerve endings which are found in the vessel walls of the. aortic arch, right subclavian-carotid angle and at the carotid sinuses close to the bifurcation of the internal and external carotid arteries. These receptors respond to stretch by increasing the number of impulses generated per unit time (Kirchiem 1976). The wall of the carotid sinus contains two types of sensory nerve endings which have been described as distortion receptors since they respond to deformation of the vessel wall in any direction (Heymans and Neil 1958, Paintal 1972).  In general in the carotid  sinus the receptor endings are located only in the adventitia (Abraham, 1967, Rees, 1967a, Rees, 1967b) whereas in the aortic arch they have been shown to penetrate into the outer media (Boss, 1956). receptors.  The receptors have been classified as type 1 and type 2  Type 1 receptors consist of myelinated fibres that form diffuse arborizations,  type 2 receptors consist of a single thick myelinated fibre with a rich arborization and terminal neurofibrillar end plates  (Kirchiem  1976).  Abraham  (1967) described the  majority of receptors in the aortic arch as being similar to type 2 receptors. Paintal (1972) described the process of activation of baroreceptors as involving deformation of the  8  receptor-generator region by the surrounding fibroelastic tissues.  The most conclusive  evidence that baroreceptors were activated by stretch and not by an increase in pressure was given by Hauss et al. (reported in Kircheim, 1976). The reflex hypotension, caused by balloon inflation inside the carotid sinus, was prevented by first surrounding the sinus with a plaster of Paris cast.  Therefore although balloon inflation increased endosinus  pressure there was no stretch of the sinus wall.  Similarily the increased baroreceptor  activity which occurred with increased intra-aortic pressure was abolished by concomittant increases in extra-mural pressure (Angel-James, 1971) The baroreceptors are connected to medullated type A nerve fibres with a diameter ranging between 2 and 12 um in the dog and rabbit (Gerard and Billingsley  1923,  Eyzaguirre and Uchizono 1961) or non-medullated C fibres in most species including the aortic nerve of rabbits (Sarkar 1922, Aars 1971) and the sinus nerve in cats (Eyzaguirre and Uchizono 1961, Fidone and Sato 1969). The afferent fibres from the carotid sinus form the carotid sinus nerve which joins the glossopharyngeal nerve before entering the brain.  Afferents from the aortic arch and  the right subclavian artery form the left and right aortic depressor nerves (ADN) respectively.  In dog and man the aortic depressor nerve is contained within the vagal  sheath but may be identified at its junction with the superior laryngeal nerve.  In the  rabbit however, the aortic depressor nerve is separate from the vagus nerve until it joins with the superior laryngeal nerve. The superior laryngeal nerve then joins with the vagus nerve to enter the brain. In the early 1900's Koster and Tschermak (reported in Heymans and Neil, 1958) demonstrated that action potentials were recorded in the aortic depressor nerve during distension of the isolated aorta with saline.  Einthoven et al.  (1908) later showed that  electrical activity of the aortic depressor nerve could be recorded with every heart beat. Bronk and Stella (1931, 1932) correlated the impulse activity in the carotid sinus nerve with the arterial pulse wave in the rabbits. They noted that single unit activity increased  9  during systole, with the frequency greatest during the systolic upstroke of the pressure wave.  The firing frequency decreased or ceased as pressure fell from the systolic level. A  second important contribution  made by Bronk  and Stella (1931,  1932)  was  the  observation that if the mean arterial pressure was lowered (by haemorrhage) to below the normal diastolic level (at which point sinus nerve firing had stopped) there was still an increase in firing correlated with the  upstroke of the  arterial pulse wave.  This  demonstrated that it was the rate of rise of pressure and not just the mean level of arterial pressure which contributed to the firing of the carotid sinus nerve.  The third important  observation made by Bronk and Stella (1931, 1932) was that different receptors within the sinus had different thresholds of firing and as mean arterial pressure was increased more of the receptors began firing.  This was a clear demonstration of recruitment of receptors  contributing to the reflex fall in arterial pressure. As early as  1866  Cyon and Ludwig (reported in Heymans and Neil,  1958)  demonstrated that afferent stimulation of the cut end of the aortic depressor nerve caused marked bradycardia and systemic hypotension.  Shortly after the turn of the century  Hering showed that stimulation of the central end of the carotid sinus nerve depressed systemic  arterial  pressure  and  heart  rate.  Furthermore Hering  showed  that  administration of atropine abolished the decrease in heart rate but not the hypotension, demonstrating that there were two separate cardiovascular reflexes involved (Heymans and Neil  1958).  Since then many scientists have confirmed that increased arterial  pressure caused a reflex inhibition of sympathetic nervous activity (Iggo and Vogt 1962, Downing and Siegel 1963, Okada 1964, Kezdi and Geller 1968, Ninomya and Irisawa, 1969) and an increase in vagal efferent activity (Katona et al. 1970, Kunze 1972). Both of these efferent mechanisms can be abolished by section of the afferent fibres from the carotid sinus and the aortic arch (Iggo and Vogt 1962, Downing and Siegel 1963). In addition to reflex hypotension and bradycardia, baroreceptor activity has been shown to influence the plasma concentration of vasopressin.  Although the plasma  10  concentration of vasopressin was not measured in their studies Yamashita and Koizumi (1977, 1979), showed that selective stimulation of carotid sinus baroreceptors (isolated sinus preparation, distension of balloons in the sinus) and aortic arch baroreceptors, inhibited the activity of nuclei in the supraoptic region of the hypothalamus in cats. Investigation  of the relationship between plasma vasopressin and the high pressure  baroreceptors is most often carried out by decreasing arterial pressure in an effort to "unload the baroreceptors." Two mechanisms baroreceptors are hypotensive  which have been used to unload the  haemorrhage and carotid occlusion.  Haemorrhage in  relation to the release of vasopressin will be discussed in more detail in the section on the low pressure receptors.  Carotid occlusion in dogs, cats and sheep specifically unloads the  carotid sinus baroreceptors and this manoeuvre causes the  plasma concentration of  vasopressin to rise only if the vagus nerves have previously been sectioned (Share and Levy 1962, Clark and Silva 1967, Wood et al.  1984).  It is thought that the reflex  hypertension caused by carotid occlusion stimulated the aortic arch baroreceptors to inhibit vasopressin release (Wood et al.  1984).  This inhibitory influence was removed following  section of the vagus nerves since afferents from the aortic baroreceptors travel in the vagus nerves in these animals.  It is important to note that although carotid occlusion  stimulated a rise in vasopressin concentration in the above studies (Share and Levy 1962, Clark and Silva 1967, Wood et al. 1984), arterial pressure was not measured distal to the site of the carotid occlusion therefore it was impossible to quantitate the relationship between carotid sinus pressure and vasopressin release (Cowley 1982).  Cardiac (Low Pressure) Receptors Gauer and Henry (1963) described the low pressure side of the circulation as encompassing the pulmonary circulation, the systemic capacitance vessels, the right atria and ventricles and the left ventricle (during diastole).  Gauer and Henry (1963) postulated  the existance of receptors which would monitor the state of filling of the intrathoracic blood  11  vessels and suggested that a likely location for these receptors was in the atria of the heart. Histological evidence for sensory nerve endings in the atria had been provided by a number of workers (reviewed by Linden and Kappagoda, 1982). Nonidez (1937) described sub-endothelial receptors which were distributed close to the entry of the vena cavae and encircling the circumference of the pulmonary veins in newborn kittens.  These complex  unencapsulated endings were later described in the lamb, monkey and dog (Miller and Kasahara 1964).  That these receptors discharged into nerve fibres in the vagus was  demonstrated by Coleridge et al.(1957) who recorded afferent impulses in the cervical vagus produced by probing the atrial endocardium of the dog. These sensitive atrial areas were then examined histologically and found to contain complex unencapsulated endings which were stimulated by deformation of surrounding tissue and could sense changes in tension in any direction (Miller and Kasahara 1964).  This made them well suited as  stretch receptors in the heart. Degeneration studies have demonstrated that the complex unencapsulated endings were connected to sensory rather than motor fibres in the vagus nerves (Nettleship, 1936, Holmes 1957). Using  electron  microscopy  Tranum-Jensen  (1975)  showed  that  complex  unencapsulated endings appeared as a disk like aggregation of cells into which a thick nerve fibre entered. Once inside the end-organ the nerve fibre split into branches forming a dense arborization of very irregular fibres with bulky varicosities containing many mitochondria.  Tranum-Jessen (1975) suggested that since the varicosities were in close  proximity to collegen fibrils they could form part of the generator membrane of the receptor. The functional classification of atrial receptors by Paintal (1963) was based on early electrophysiological studies in the cat (1953a,  1953b).  Paintal classified the three  types of atrial receptors as either type A receptors which discharged mainly during atrial systole, type B receptors which discharged mainly during atrial filling or intermediate receptors which discharged during both atrial systole and atrial filling.  Kappagoda et al.  12  (1976, 1977) examined the relative occurance of these three types of receptors in dogs, cats and rabbits.  They found type A receptors were rare, intermediate receptors were  common and conversion among receptor types occured frequently (1976, 1977). Miller and Kasahara (1964) observed a second type of receptor (other than the complex unencapsulated endings) in the atrial endocardium and referred to it as an endnet, which they suggested was formed by the junctions of the branched dendrites of several different myelinated fibres and was located throughout the endocardium and thought to be sensory in nature (Miller and Kasahara 1964).  The functional relationship between the  end-net and the complex unencapsulated endings remains undefined (Wahab et al. 1975). Unless otherwise specified the general term atrial receptors will be used in this thesis to describe the complex unencapsulated endings in the atria connected to myelinated fibres in the vagus nerves. There are also receptors located in the ventricles which have afferents in the vagus nerves.  Thames et al.  (1980) and Zucker et al.  (1983) have demonstrated that intra-  coronary injection of cryptenamine or veratrine (veratrum alkaloids) into dogs prevented the rise in vasopressin which accompanied hypotension.  They suggested that veratrine  and cryptenamine stimulated left ventricular receptors causing inhibition of vasopressin release.  The role played by veratrine sensitive ventricular receptors in the normal control  of the vasopressin release remains unclear. It was Henry and Pearce (1956) who first provided evidence that volume sensitive receptors on the low pressure side of the circulation were responsible for a diuretic response associated with distension of the atria.  The time course of the diuretic response  (5 to 10 minute delay) suggested a hormonal rather than a neural response. several studies reported that  Although  vasopressin (measured b3' bioassay) decreased during atrial  distension (Shu'ayb et al. 1965, Baisset and Montastruc, 1957, Share 1965, Brennan et al.  1971), the values for plasma vasopressin in these animals (even after atrial distension)  exceeded that which was necessary for maximal antidiuresis.  Therefore no conclusions  13  could be made as to the contribution made by vasopressin to the diuresis associated with left atrial distension.  It was not until the application of sensitive radioimmunoassays for  vasopressin that the role of vasopressin release during atrial distension was clarified in normally hydrated conscious, (Schultz et al.  1982, Fater et al.  dogs (DeTorrente et al.  1975). Although atrial distension decreased  plasma  concentration  1975, Zucker et al. of vasopressin,  the  values  1982) and anaesthetized  of vasopressin  measured  in  the  anaesthetized dogs were higher than normal plasma vasopressin levels in conscious dogs (Cowley 1982). In 1983 Ledsome et al. showed that in anaesthetized dogs with a resting plasma  concentration of vasopressin  within the  normal  range  (2-8  pg/mL),  mitral  obstruction significantly decreased vasopressin concentration. Mitral obstruction involved inflating a balloon, placed in such a manner as to partially obstruct the mitral orifice and increase left atrial pressure.  The time course of this response involved a rapid fall in  plasma concentration of vasopressin within 2 minutes followed by a slow decrease to a steady state within 4 minutes.  In the same study they demonstrated that cooling of the  vagus nerves to 1 0 ° C (removal of afferent input from atrial receptors with myelinated afferent fibres) abolished the decrease in vasopressin associated with mitral obstruction. Mitral obstruction has been shown to suppress plasma concentration of vasopressin in conscious dogs (Schultz et al.  1982, Fater et al.  1982) and this response could be  abolished by cardiac denervation. A technique was developed by Ledsome and Linden (1964) for stimulating the left atrial receptors by inflating balloons in the pulmonary vein/atrial junction. In this way a discrete stretch of the left atrial receptors could be applied without a significant change in left  atrial pressure or mean arterial pressure.  Using this technique it has been  demonstrated that stimulation of the left atrial receptors caused an increased heart rate (Ledsome and Linden, 1964)  decreased renal nerve activity (Karim et al.  1972) a  decreased renal vascular resistance (Mason and Ledsome, 1974) and an increased urine output (Ledsome and Linden 1968). Pulmonary balloon distension is also associated with a  14  fall in plasma concentration of vasopressin which is abolished by cooling the vagus nerves to 8 - 1 0 ° C (Wilson and Ledsome 1983). The above studies clearly demonstrated that stimulation of the atrial receptors significantly decreased plasma concentration of vasopressin and caused a dilute diuresis. This does not necessarily mean that the fall in vasopressin was the cause of the diuresis. In fact several studies have reported a lack of correlation during the changes in plasma vasopressin and the urinary diuresis associated with left atrial distension (Bennett et al. 1983, Kaczmarczyk et al. 1983). In a recent review Ledsome (1985) addressed this issue. He showed that when the urine osmolality was compared to the plasma vasopressin measured at the start of each collection period rather than the end, then the relationship between urine osmolality and plasma vasopressin during atrial distension was similar to the known relationship between urine osmolality and plasma osmolality in conscious dogs (Ledsome and Wilson 1984). Haemorrhage is a potent stimulus for the release of vasopressin in a variety of species.  It is thought that the fall in central venous pressure and arterial pressure  "unloads" the atrial receptors and the sinoaortic baroreceptors resulting in a decreased inhibitory input on vasopressin release. In  an  attempt  to  specifically  unload  the  low  pressure  receptors  several  investigators have shown that non-hypotensive haemorrhage (10% blood volume) caused increased release of vasopressin in dogs (Share 1968, Henry et al. 1983, Goetz et al.  1984).  1968, Wang et al.  Non-hypotensive haemorrhage and the application of lower  body negative pressure have both been shown to be relatively ineffective as stimuli for the release of vasopressin in human and non-human primates (Goetz et al. al.  1977, Gilmore and Zucker 1980, Gilmore et al.  1974, Arnauld et  1982, Goldsmith et al.  1984). This  suggests that in contrast to dogs, primates have a lowered sensitivity of the low pressure receptors to alterations in volume.  The relative importance of high pressure versus low  pressure receptors in the control of the release of vasopressin will be discussed in the  15  section entitled "Interaction between high and low pressure receptors".  Other Volume Sensitive Receptors There have been reports of other volume sensitive receptors in the vasculature. Notable among these are the hepatic, mesenteric and renal volume receptors. Although there has been no anatomical description of the hepatic volume receptors investigators  have recorded afferent discharge in the hepatic nerve in response  to  increased hepatic or portal volume or pressure (Andrews and Palmer, 1967, Niijima, 1977, Kostreva et al. nerve activity.  1980).  Associated with these afferent nerve impulses is increased renal  The increased renal activity is likely to be part of the efferent limb of a  reflex response since it can be abolished by section of the hepatic nerve (Kostreva et al. 1980).  In addition Ashton et al.  (1982) have demonstrated  that  administration of capsaicin reflexly decreases arterial pressure and this response could be abolished by section of the anterior hepatic plexus.  intra portal hypotensive  The physiological  relevance of these hepatic receptors is as yet unclear, however, Lautt (1977) proposed the presence of a liver-gut neural axis.  Decreased intra-hepatic pressure would reflexly  stimulate a vasodilation in the splanchnic vasculature therefore restoring hepatic pressure by shunting blood from the gut into the portal circulation.  Whether hepatic volume  receptors operate in the normal control of the cardiovascular system has yet to be investigated. The pioneers in the investigation of mesenteric volume receptors were Gammon and Bronk (1935). They showed that cats possessed Pacinian corpuscles located at branch points along the mesenteric and arcuate arteries.  Recordings made from the afferent  nerves arising from the Pacinian corpuscles showed pulsatile discharge which could be increased by increasing perfusion pressure in a section of isolated mesentery.  Intravenous  injection of Ringers fluid in an intact cat resulted in an increase in impulse activity while haemorrhage significantly decreased or abolished afferent impulse activity.  Gammon and  16  Bronk (1935) concluded that these receptors signal the degree of distension of the mesenteric vessels. In addition Gammon and Bronk (1935) increased perfusion pressure in steps over a wide range in a section of mesentery and were able to record no changes in arterial pressure.  This suggested that the increased activity in these receptors does not  affect arterial pressure. There has been a great deal of interest recently in the possible role of the renal afferents in the control of the cardiovascular system. demonstrate  the presence  of renal afferents.  Tower (1933) was the first to  Since then many investigators  have  examined renal afferents which respond to increased renal venous pressure with an increase in activity (Ueda et al.  1967, Pines 1968, Astrom and Crawford 1968).  The  reflex effects of stimulation of renal afferents include decreased cervical sympathetic efferent nerve activity, decreased systemic blood pressure and decreased renal sympathetic efferent nerve activity (Ueda et al. 1967, Aars and Akre 1970). Recently Kostreva et al. (1981) confirmed the above observations  and also showed that stimulation of renal  afferents decreased contractile force of the right ventricle probably due to a decrease in sympathetic efferent nerve activity.  One must be cautious when interpreting the studies  involving electrical stimulation of renal afferents  since these nerves  carry  sensory  information from mechanoreceptors (Niijima 1971) and chemoreceptors (Recordati et al. 1978).  Therefore one can only attribute reflex changes  to renal mechanoreceptor  activation if the stimulus applied was limited to changes in renal venous pressure or intrarenal pressure. Electrophysiological studies have shown that stimulation of afferent renal nerves altered activity in medullary and hypothalamic nuclei (Calaresu and Ciriello 1981), including putative vasopressin magnocellular neurons in the supraoptic nuclei (Day and Ciriello, 1985) and neurons in the paraventricular nuclei which project to the spinal cord and the neurohypophysis (Caverson et al. 1986). These electrophysiological data suggest that information from renal receptors (mechano- or chemoreceptors) could play a role in  17  the release of vasopressin.  Finally it has been shown that the paraventricular nuclei  neurons which respond to stimulation of the afferent renal nerves  also respond to  stimulation of the carotid sinus and aortic depressor nerves (Caverson et al.  1986)  suggesting some integration of renal afferent and arterial baroreceptor activity in the control of vasopressin. Further conclusions on the relevance of neural connections between renal afferents and vasopressinergic neurons await studies utilizing discrete physiological stimulation of the renal mechanoreceptors. In summary there appear to be areas in the vasculature which respond to alterations in the systemic blood volume or to alterations in blood volume of a certain section of the vascular bed. The major volume sensitive areas are in the atria of the heart, the aortic arch and the carotid sinus.  Although previous studies have described the  qualitative relationship between carotid sinus baroreceptor activity and the release of vasopressin there have been no studies which have attempted to quantitate levels of carotid sinus pressure with vasopressin release (Cowley 1982). It has been shown that a part of the increased plasma vasopressin response seen during haemorrhage in cats, dogs and rabbits cannot be attributed to input from sinoaortic baroreceptors and atrial receptors (Ginsburg and Brown, 1956, Clark and Silva 1967, Chien and Usami 1974, Rankin et al. 1986) suggesting the presence of other volume sensitive receptors. hepatic, mesenteric  It is possible that  or renal receptors may be capable of modifying cardiovascular  variables and plasma vasopressin in response to changes in blood volume.  Further  detailed work in this area is needed before any conclusions can be made as to the individual contributions made by these receptors in the vasopressin response to changes in blood volume.  Central Connections Afferents originating in the sinoaortic baroreceptors and atrial receptors travel to the brain within the vagus nerves and glossopharyngeal nerves.  The first synapse  18  encountered by these afferent impulses occurs in the medulla at the nucleus of the tractus solitarius (NTS) (Crill and Reis 1968, Kumada and Nakajima 1972, Palkovitis 1977, Jordan and Spyer 1977, Sumal et al. 1981).  1977, Wallach and Lowey 1980, Ciriello et al.  Aortic baroreceptor afferent fibres enter the rostral medulla into the ipsilateral  solitary tract and give off fibres terminating throughout the ipsilateral nucleus.  A few  fibres cross the midline to terminate in the contralateral nucleus (Ciriello and D'Ippolito 1981). Afferents from the sinoaortic baroreceptors and the atrial receptors all synapse at the N T S . Since these receptors have all been shown to affect the release of vasopressin it is not surprising to find a monosynaptic pathway between the N T S and the hypothalamic vasopressinergic neurons (Ciriello and Calaresu, 1980).  In addition to the monosynaptic  connections, norepinephrine containing neurons from the A l and A2 areas of the ventral lateral medulla as well as some neurons from the pons have been shown to project to the hypothalamus. These neurons travel within the reticular formation of the brain stem and enter  the  medial  forebrain  bundle  and  then  supply  the  supraoptic  nuclei  and  paraventricular nuclei (Sladek and Sladek, 1985). Sved et al. (1985) have demonstrated that lesions of the fastigial nucleus attenuated the release of vasopressin seen with haemorrhage. Thus there are neural connections between the N T S and the hypothalamic neurosecretory neurons which may contribute to normal secretion of vasopressin. Yamashita and Koizumi (1977, 1979)showed that stimulation of carotid sinus and aortic baroreceptors decreased the activity of neurons in the supraoptic nuclei which was indirect evidence that barorececeptor activity inhibited vasopressin release. They did not however, measure plasma vasopressin concentrations during baroreceptor stimuluation. Ciriello et al.  (1983) showed that discrete stimulation of aortic baroreceptors increased  metabolic activity in both paraventricular nuclei and supraoptic nuclei in the rat as evidenced by an increased uptake of tritiated deoxyglucose.  In the same study they found  similar results when aortic baroreceptors were bilaterally stimulated by increasing arterial pressure in carotid sinus denervated rats.  Since stimulation of aortic baroreceptors is  19  associated with inhibition of vasopressin containing neurons (Koizumi and Yamishita 1978, Yamishita 1977) Ciriello et al.  (1983) suggested that the increase in metabolic activity  associated with stimulation of aortic baroreceptors seen in their study was due to increased activity of inhibitory interneurons that synapse on vasopressinergic neurons.  There are  also neurons in the hypothalamus which are activated by stimulation of atrial (low pressure) receptors. Menninger and Frazier (1972) showed that there were neurons in the paraventricular nuclei which responded only to increases in osmolality (increased activiy), there were paraventricular neurons which responded only to atrial stretch (decreased activity) and there were neurons which responded to both stimuli. This may represent the first step in some integration of osmotic and volume control of vasopressin secretion. Recently Swanson and Sawchenko (1980) showed that parvocellular neurons in the paraventricular  nuclei  of the  hypothalamus  (which  are  activated  by baroreceptor  stimulation) have axonal connections with autonomic nuclei in the brain stem and spinal cord thus implicating the paraventricular nuclei in a possible reflex control of autonomic function.  It is also possible that vasopressin released into the systemic circulation may  feed back across the blood brain barrier and effect autonomic preganglionic neurons (Schmid et al.  1984).  Although the cerebro-spinal fluid uptake of vasopressin from the  peripheral circulation is limited in dogs (Wang et al. rabbits (Heller et al.  1981) it is extremely effective in  1968) suggesting that some species specificity exists. Therefore the  possibility exists in some animals that there is a neuro-humoral feedback system in operation involving baroreceptors and the central actions of vasopressin.  Interaction Between High Pressure Sinoaortic Baroreceptors and Low Pressure Atrial receptors in the Release Of Vasopressin. It has been well documented in dogs that unloading of the low pressure atrial receptors is a potent stimulus for the release of vasopressin (Share 1968, Henry et al. 1968, Wang et al.  1983, Goetz et al.  1984).  It has also been shown that the rise in  20  vasopressin during haemorrhage is not affected by sinoaortic denervation but is severely attenuated by cardiac denervation (Goetz et al.  1984).  This control over vasopressin  release by cardiac receptors has not been found in human and non-human primates. Unloading of the low pressure atrial receptors does not increase plasma concentration of vasopressin in man or monkey (Goetz et al.  1974, Arnauld et al.  Zucker 1980, Gilmore 1982, Goldsmith et al.  1984).  1977, Gilmore and  In fact the only time one sees an  increase in vasopressin in response to haemorrhage in the monkey is if the haemorrhage is severe enough to decrease the arterial pressure (Arnauld et al. 1982).  1977, Gilmore et al.  Zucker and Gilmore (1975) found a reduced sensitivity of the atrial receptors to  atrial stretch in primates as compared to dogs. teleological  argument for the  Goldsmith et al.  (1984) have used a  apparent insensitivity of the primate atrial  receptors.  Goldsmith stated that " This discrepancy between canines and primates has an appealing teleological rationale in that if vasopressin were as responsive to mild and moderate changes in reflex tone in primates as it is in dogs, interference in body water homeostasis could result since primates by virtue of frequent alterations in posture are more subject to frequent changes in intracardiac and mean arterial pressure than are animals that spend the majority of their time in one horizontal position." Larsson showed that in goats it was the high pressure receptors rather than the low pressure receptors which caused the vasopressin response to haemorrhage (Larsson et al. 1978). Therefore not all quadrupeds rely on their low pressure receptors rather than their high pressure receptors. Generally in situations such as haemorrhage or volume expansion the sinoaortic baroreceptors and atrial receptors are both acting to buffer the fall in pressure/volume. If these two sets of receptors are artificially manipulated so that they receive opposing stimuli there appears to be an interaction between the two inputs.  Thames and Schmid  (1981) unloaded the atrial receptors by vagal cold block (VCB) at three different levels of carotid sinus pressure.  When carotid sinus pressure was held constant at 50 mmHg  (minimal inhibition) V C B caused a significant increase in the plasma concentration of  21  vasopressin.  If the carotid sinus baroreceptors were concomitantly stimulated (carotid  sinus pressure from 50 to 135 mmHg), V C B did not change the plasma concentration of vasopressin.  Finally if the carotid sinus baroreceptors were further stimulated (carotid  sinus pressure from 50 to 200 mmHg) there was a decrease in vasopressin despite V C B . Therefore there is a complex interaction between these two sets of receptors.  As  previously mentioned this shows qualitatively that there is a relationship between carotid sinus pressure and vasopressin release and that input from carotid sinus baroreceptors can interact with input from cardiac receptors.  It does not, however, quantitatively describe  the relationship between carotid sinus pressure and vasopressin release.  In addition the  plasma levels of vasopressin in these dogs were extremly high making interpretation of the results difficult. In other experiments it has been shown that unloading of the carotid sinus baroreceptors (carotid occlusion) caused a rise in vasopressin only if the animal was previously vagotomized (Share and Levy 1962; Clark and Silva, 1967; Thames and Schmid, 1979).  Given the results from the experiment by Thames and Schmid (1981) it  would seem plausible that the aortic baroreceptors or the atrial receptors were responding to the reflex rise in arterial pressure which accompanied carotid occlusion by inhibiting vasopressin release.  It was not possible to ascertain if the buffering of the rise in  vasopressin was caused by aortic baroreceptors or atrial receptors since both sets of afferents are carried in the vagus nerve in the dog and cat.  Effects of Changes in Plasma Vasopressin The changes in plasma vasopressin concentration induced by haemorrhage or expansion of blood volume may regulate blood volume b}' causing changes in the renal excretion of water. To have done this the changes in plasma vasopressin must have occur within the range of vasopressin concentration which was associated with changes in renal water excretion (1-6 pg/mL in man; Bie 1980). Many authors have pointed out that this  22  would provide a poor regulation of volume since it would lead to changes in plasma osmolality which would limit the response (e.g. Ledsome, 1985). For efficient regulation, a mechanism which also influences sodium excretion would be required. The concept that plasma vasopressin concentration may contribute to the normal control of blood pressure by direct effects on vascular resistance or capacitance has been reviewed by Cowley (1982). Increased plasma vasopressin concentration has been shown to oppose falls in blood pressure induced by haemorrhage, in dogs in which other buffering systems had been eliminated.  However, in intact animals, it is unlikely that changes in  plasma vasopressin contribute to the maintenance of blood pressure unless the changes in plasma vasopressin concentrations are large (see review Rossi and Schrier, 1986).  23  HYPOTHESIS  The hypothesis to be tested was that the release of arginine vasopressin may be inhibited by stimulation of three groups of cardiovascular receptors; carotid sinus baroreceptors, aortic baroreceptors and atrial receptors.  To test this hypothesis  experiments were designed to allow stimulation of each of the three receptor regions independently of the other two regions in anaesthetized rabbits.  The design of the  experiments made possible quantification of the relationship between stimulation of the carotid sinus baroreceptors and plasma vasopressin concentration.  24  G E N E R A L METHODOLOGY  Animals, induction of anaesthesia and surgical preparation. All experiments  were performed on male New Zealand White rabbits.  The  anaesthetic used was a mixture of urethane (lgm/kg, Sigma Chemical Co., St. Louis) and alpha-chloralose  (lOOmg/kg,  Calbiochem, Anaheim).  Urethane  and chloralose were  dissolved in saline and kept at 6 0 ° C in a water bath. Following induction of anaesthesia b3' injection into an ear vein, the rabbits were ventilated with room air supplemented with 100% oxygen through a cannula placed in the trachea and connected to a respirator (Harvard Apparatus, Newport Beach, C A , model 665). blood samples  A t intervals during the procedures  (< lmL) were taken and p H , PCO2 and PO2 were measured  appropriate electrodes (Corning, Medfield, M A , model 165/2).  using  Prior to the equilibration  period adjustments to the respiratory rate or infusion of N a H C O g (IM) were made to bring P a C 0 2 to 25-28 mmHg and arterial p H within the range of 7.35 - 7.55.  These values of  PaC02 and p H are similar to those measured in the laboratory in conscious rabbits (26.3 mmHg, 7.436 units unpublished observation). Cannulae (PE190, 15 cm long) placed in the right femoral artery and the right atrium were connected to strain gauges (Statham Instruments Co. Puerto Rico: P23DB) and after direct current amplification, systemic arterial pressure (MAP) and right atrial pressure (RAP) were recorded on an ultra violet light recorder (Honeywell, Visicorder, Denver,  C O , model  manometers.  1508).  Calibration  was  performed using mercury and  saline  Zero right atrial pressure was referred to the cannula tip at heart level, free  in air at the end of the experiment.  Mean pressures were obtained using an R-C circuit  with a time constant of 2 seconds. A cannula (PE190) placed in the right femoral vein and connected to a constant flow infusion pump (Cole-Parmer, Masterflex, Chicago IL, model 7520-25) allowed infusion of 0.45% saline at 0.5 mL/min throughout the course of the experiment. Infusion of saline at this rate maintained plasma osmolality between 290 and  25  310  mOsmol/kg.  Two E C G electrodes were attached to the neck and leg for the  measurement of heart rate and the output was recorded on an ultraviolet light recorder (described above).  The number of beats were counted over 10 sec. and the value obtained  was expressed as beats/minute. In the rabbits in Chapters 2, 3 and 4  the bladder was exposed ventrally and a  flared cannula (PE240) was inserted to ensure constant drainage of bladder contents. Oesophageal temperature was maintained between 3 7 ° and 3 9 ° C by heating bars beneath the surgical table. Blood from a donor rabbit provided a reservoir from which replacements were obtained for withdrawn samples,  haemorrhaged volumes and for volume expansion.  The  blood removed from the donor rabbit was mixed with heparin, filtered and exchanged with the experimental rabbit.  This allowed replacement of blood in the experimental rabbit  without alteration of the composition of the circulating blood. The blood reservoir was kept at 3 7 ° C in a water bath over the course of the experiment. estimated as a percent of the rabbits blood volume.  Blood volume changes were  Previous measurements  in the  laboratory found the blood volume of the New Zealand White rabbit to be 59.3 +/- 1.2 ml/kg (unpublished observation).  The protocol for haemorrhage and volume expansion  involved withdrawal (or infusion) of the appropriate volume of blood through a femoral artery cannula into a plastic syringe.  The time span for the haemorrhage (or volume  expansion) was 1-3 min.. Restoration of normal blood volume following the haemorrhage and volume expansion was carried out in the same manner. Isolation of the carotid sinuses was performed through an incision in the ventral surface of the neck.  The carotid sinuses were perfused at controlled pressure with an  extracorporeal circuit (figure lb).  Blood was withdrawn from the left common carotid  artery and passed through a roller pump (Watson Marlow, H.R. Flow Inducer), a heated damping chamber, a filter and was finally fed back into the distal ends of the cut right and left common carotid arteries.  Ties were placed around the external carotid artery, the  26  Figure lb: A representation of the carotid sinus perfusion system. Left common carotid artery (LCC), right common carotid artery (RCC), external carotid artery (EC), occipital artery (OC), internal carotid artery (IC).  27  N  Pressure transducer  /,  /EC  LCC  Perfusion System Heated  Carotid Sinus  damping Roller  chamber  pump  RCC  Tl  LCC  28 occipital artery, the internal carotid artery and one branch of the internal maxillary artery to create sinus sacs.  Another branch of the internal maxillary artery was left open on  each side so as to allow flow through the sinus at all times. A cannula (PE50) placed in one branch of the left internal maxillary artery was connected to a pressure transducer (Statham Instruments Co. Puerto Rico P23DB) . The signal from this pressure transducer was fed into a servo-control unit which automatically adjusted the pump speed.  In this  way carotid sinus pressure could be maintained constant at 100 mmHg or changed in steps throughout the course of the experiment. A t the end of each experiment the carotid sinus pressure was increased and decreased (to 160 mmHg and 40 mmHg respectively) to test the efficacy of the carotid sinus baroreflex, as demonstrated by the reflex change in arterial pressure. In all animals arterial pressure decreased and increased as carotid sinus pressure was increased and decreased. In appropriate groups both aortic depressor nerves were sectioned at their junction with the superior laryngeal nerves.  Following all surgery a bolus dose of heparin (2000  International Units) was administered intra-arterially followed by 300 I U every 30 min.. At this time a supplemental dose of anaesthetic (10% of the initial dose) was given. A l l rabbits were allowed a 60 minute equilibration period prior to the start of the experimental period during which time blood volume remained constant and carotid sinus pressure was held at 100 mmHg.  Analytical Methods Samples of arterial blood (4mL) were taken into cold dry syringes.  Three  millilitres of blood were transferred into E D T A tubes (Vacutainer) for measurement of vasopressin and l m L was transferred into a plastic test tube for measurement of osmolality.  The E D T A tubes and plastic tubes were centrifuged in a refrigerated  centrifuge (4°C) at 2500g (Silencer, H-103NA Series) for 5 min.. Plasma osmolality was measured by freezing point depression (Advanced DigiMatic Osmometer Model 3D 2,  29  Advanced Instruments Inc.).  Plasma osmolality was measured on 6 aliquots of rabbit  plasma and was found to be accurate to within a range of +/-0.67 mosmol/kg.  Plasma  from the E D T A tubes was stored at - 2 0 ° C and later used for determination of vasopressin levels by radioimmunoassay (RIA).  The plasma was not extracted prior to RIA, because  non-specific interfering factors in rabbit plasma did not interfere with the antibody antigen binding in this RIA (Leighton et al., 1982).  30  Radioimmunoassay For Arginine Vasopressin (AVP) The R I A for arginine vasopressin employed i n these studies reported i n this thesis w a s s i m i l a r to that reported earlier for m e a s u r e m e n t of this hormone i n tissue extracts and p l a s m a (Keeler and W i l s o n , 1976; W i l s o n and Ngsee, 1980; Ledsome et a l . 1982)  Iodination. Phosphate monobasic  sodium  buffer  was  prepared  phosphate  ( N a H P 0 ) to a p H of 7.2 units. 2  4  from  (Na2HPU2)  a  stock  titrated  solution  with  (24.8g/1.2L  dibasic  sodium  H 0) 2  of  phosphate  The phosphate buffer (0.15M) w a s stored at 4 ° C for use  in the a s s a y . The reactants were m i x e d i n the  iodination vessel according to the  following  protocol: 1  10 ug lysine vasopressin (Sigma grade I X )  2  10 u L acetic acid ( 5 0 m M )  3  15 u L phosphate buffer (0.15M)  4  10 u L chloramine T ( l m g / m L i n H ^ O , E a s t m a n Organic C h e m i c a l s Rochester N . Y . ) ; vortex for 10 sec.  5  15 u L (1.5 m C i ) of ^ I o d i n e ( I M S - 3 0 , A m e r s h a m , Toronto, Ontario); vortex 12  briefly and w a i t for 50 sec. 6  100 u L bovine serum a l b u m i n (0.25 m g / m L dissolved i n saline, M i l e s Scientific, N a p e r v i l l e 111.); vortex for 10 sec.  7  2 0 0 u L A 6 1 X 1 0 (0.25 m g / m L dissolved i n H 0 , B i o r a d 9 9 9 5 , R i c h m o n d Ca.); 2  vortex for 10 s e c .  31  The reaction mixture was centrifuged for 3 min. (2500g) and the  supernatant  applied to a C M Sephadex G25 Column (Pharmacia Fine Chem.), and eluted at a flow rate of 0.6 mL/min with 0.6 M acetate buffer (pH=4.85).  A representative column profile is  shown in figure lc. The range of specific activities of ^ ^ I - L V P was 650 to 1830 uCi/ug.  Antibody. The vasopressin antibodies were raised in guinea pigs using a procedure previously reported by Goodfriend et al. (1964).  The coupling reagent was l-ethyl-3(3-  dimethylaminopropyl) carbodiimide hydrochloride (Calbiochem, L a Jolla, Calif. U S A ) and the carrier molecule was human serum albumin (Sylvana F r . V).  The anti-vasopressin  serum obtained (GP-15) showed no cross-reactivity with oxytocin, 4-ser-9 ileu-oxytocin, arginine  vasotocin  or angiotensin  I.  The final dilution of the  antiserum in these  experiments was 1: 7.2 x IO"*.  Standards. (U.S.  Vasopressin standards were prepared from posterior pituitary extract  Pharmacopia, Reference  Standard), first diluted to 1.0 mg/mL ( in acetic acid).  Further dilutions were made and stored in 2.0uL (10 ng AVP) aliquots at - 2 0 ° C .  For each  RIA the standard was thawed and diluted to 1.0 ng/mL (in 2 M acetic acid). To construct a standard curve the prepared standard (1.0 ng/mL) was serially diluted in RIA buffer to 0.1, 0.25,  0.5,  1.0, 2.5, 5.0,  10.0, 25.0 pg/tube).  A representative  standard curve is  shown in figure lc.  Quality Controls.  Blood that was obtained from anaesthetized rabbits by heart  puncture was centrifuged for 5 min. at 3000 g in a refrigerated centrifuge. plasma was divided into aliquots of l m L and stored at - 2 0 ° C .  The pooled  Measurements of quality  control plasma were included in each assay and the mean +/- standard error of the mean of the quality control values in all the assays gave an indication of the variablility.  inter-assay  The calculated value of the A V P concentration in the quality control samples  32  Figure lc:  (A) Chromatography of iodinated vasopressin on a column of Sephadex G25 eluted  with  (fractions  0.6M acetate buffer 60-80)  (B) A representative  represents  (pH = 4.85). the  iodinated  The 2nd peak vasopressin.  standard curve relating bound iodinated  vasopressin / free iodinated vasopressin with concentrations of noniodinated vasopressin.  Maximum binding of iodinated vasopressin  with the GP-15 antibody at 1:7.2 x 1 0 dilution was 0.61. 4  33  34  between assays was  10.1 +/- 0.5 pg/mL (n = 48).  Six aliquots of a single sample of  plasma were included in one assay and the mean +/- standard error of the mean of these 6 aliquots gave an indication of the intra-assay variability. The calculated mean value of the vasopressin in the 6 aliquots was 16.4 +/- 0.6 pg/mL, (n = 6).  Incubation.  A l l measurements were conducted in triplicate.  Unknowns were  assayed both in the presence and the absence of antiserum (3 specific binding, specific binding) to account for nonspecific binding of varied between  lOuL  I-LV P .  2 non-  The volumes of plasma,  and 300uL per tube, depending on hormone concentration.  Incubation was continued for 5 days: 2 days preincubation and 3 days following the addition of - ^ ° I = L V P (2000 cpm added to each tube). L  The total incubation volume was  1.0 m L , and all assay components were diluted in 0.15 M phosphate buffer, p H 7.2 .  Separation.  The non-specific adsorption of free  ^ ^ I - L V P to dextran coated 1 or  charcoal was used as a method for the separation of bound and free  I-AVP.  A mixture  of charcoal (Norit, Fisher Scientific Co., Fair Lawn N.J.) and dextran (Pharmacia Fine Chem. Upsala, Sweden) was prepared as follows : 30 ml phosphate buffer (0.15M) 0.167g dextran 20 ml gamma globulin (0.28g/ml RIA buffer) 0.835 g charcoal  The total volume was 50 ml; 250ul of this mixture was added to each assay tube. The tubes were centrifuged at 2500 g for 45 min.. The supernatants were decanted into separate tubes and both the supernatants (containing bound iodinated antigen) and the charcoal pellet (containing free iodinated antigen) were counted for 4 min. in an automatic gamma counter (LKB Wallace 1272 Clinigamma).  35 Peptide Quantification: The results were calculated using the following equations:  Damage = B / B + F B / F = B- (B + F) x Damage/ F (B: C P M bound; F: C P M free)  The standard curve was plotted as a semi-logarithmic plot of bound free - ^ ^ I - L V P  against the concentration of vasopressin (figure ii).  I-LVP /  The value of the B / F  distribution for any unknown sample was located on the curve and the corresponding standard value read from the horizontal axis.  Assay Sensitivity.  Displacement of 20% and 50% of the tracer from the antibody  was at 0.52 +/- 0.02 pg and  1.68 +/- 0.06 pg respectively (n = 74).  36  CHAPTER 1  CAROTID SINUS PRESSURE AND VASOPRESSIN R E L E A S E Introduction There were three aims for the experiments reported in Chapter 1. determine the  Firstly to  quantitative relationship between carotid sinus pressure and plasma  vasopressin concentration.  Secondly to compare and contrast this relationship with that  seen between carotid sinus pressure and mean arterial pressure.  Thirdly to assess the  buffering effect on the vasopressin response to alterations in carotid sinus pressure by the aortic baroreceptors and the atrial receptors with vagal afferents.  Protocol The experiments were carried out in 27 rabbits divided into three separate groups. Prior to the start of the experiment the rabbits had intact vagal nerves (VN), intact aortic depressor nerves (ADN) and the carotid sinus pressure was set at lOOmmHg (figure 2). In the first group of 10 rabbits carotid sinus pressure was altered in steps as follows: lOOmmHg, 160mmHg, 120mmHg, 80mmHg, 40mmHg, lOOmmHg. This series of carotid sinus pressure changes was repeated in each rabbit first while all buffer nerves were intact and second after bilateral aortic depressor nerve section and finally after bilateral vagotomy. The second group of rabbits (n = 8) were used as controls to examine the effect of serial nerve section (aortic depressor nerves sectioned before vagus nerves) on the plasma concentration of vasopressin at a constant carotid sinus pressure of lOOmmHg. In the third group of rabbits (n=9) similar step changes in carotid sinus pressure were carried out, but the order of nerve section was reversed. First carotid sinus pressure changes were performed while all buffer nerves were intact, secondly after bilateral vagotomy and finally after bilateral aortic depressor nerve section.  A  15 minute  37  Figure 2: An illustration of the experimental protocol for the experiments in Chapter 1. For a description of the protocol see the text of the thesis.  VN intact  (  ADN Intact  VN intact ADN sectioned (  »••••• ****** V* • * * •  0  5  10  15  20  25  40  45  50  VN sectloned^ADN  sectioned  •#•••*  55  60  65  80  85  90  95 100  105  TIME (MINUTES)  CO 00  39  stabilization period was allowed after each nerve section before the next set of changes in carotid sinus pressure. All recording and sampling of blood was done 5 minutes after each change in carotid sinus pressure.  A l l blood removed for analysis was replaced with  exchanged whole blood from the reservoir.  Statistical Analysis To determine whether decreases in carotid sinus pressure were associated with changes in the cardiovascular variables the values corresponding to a carotid sinus pressure of 160 mmHg were compared to the values at carotid sinus pressure of 120, 100, 80 and 40 mmHg using a one way analysis of variance and a Duncans Multiple Range test (Wallenstein 1980). Since the vasopressin values did not conform to a normal distribution a Wilcoxon's Signed Ranks Test for paired data was used for statistical evaluation.  A  value of P<0.05 was considered statistically significant.  Results The perfusion experiments were carried out on 27 New Zealand White rabbits (weight:3.34 +/- 0.13kg). 0.02 units and P C 0  2  At the start of the experimental period the p H was 7.49  was 24.6 +/- 1.2 mmHg.  +/-  The respiratory gas was supplemented  with oxygen so that in all the rabbits PO2 was greater than 200mmHg.  Plasma  osmolality was 299.6 +/- 2.4 mOsmol/kg at the start and 306 +/- 2.7 mOsmol/kg at the finish of the experiment respectively.  A blood sample was taken and cardiovascular  measurements made at the end of the 60 minute equilibration period which followed the surgery. The baseline levels of mean arterial pressure (MAP), right atrial pressure (RAP), heart rate (HR) and immunoreactive vasopressin (iAVP) were as follows: M A P = 9 9 . 8 +/4.7 mmHg; R A P = 4.1 +/- 0.8 cmHoO; HR=279.2 +/- 8.2 bts/min and i A V P = 9.24 +/1.2 pg/mL. Decreasing carotid sinus pressure, resulted in a reflex  rise in M A P in both groups  40  of rabbits regardless of the order of nerve section (figure 3, 4).  The actual recordings of  M A P and R A P during the alterations in carotid sinus pressure in a representative rabbit can be seen in figures 5, 6 and 7.  These three figures represent alterations in carotid  sinus pressure in the same rabbit, firstly with both aortic depressor and vagus nerves intact (NI, figure 5), secondly after section of the vagus nerves (VNX, figure 6) and finally after section of the aortic depressor nerves ( A D N X , figure 7). There were no significant changes in R A P in response to decreases in carotid sinus pressure either with nerves intact or after section of aortic depressor nerves or vagus nerves. The average R A P (CSP=100 mmHg), calculated from both groups of rabbits, was 3.35 +/- 0.9 cimH^O with both the aortic depressor nerves and vagus nerves intact and 3.68 +/- 0.7 cmT^O after both aortic depressor nerves and vagus nerves sectioned. In rabbits before the aortic depressor nerves and vagus nerves were sectioned (figure 8, panel A and figure 9, panel A), decreasing carotid sinus pressure did not change the plasma concentration of vasopressin.  There was also no change in the plasma  concentration of vasopressin in rabbits with intact aortic depressor nerves and sectioned vagus nerves (figure 9, panel B).  It was clear, however, that once the aortic depressor  nerves had been sectioned (figure 9, panel C , figure 8, panel B and C) decreased carotid sinus pressure caused a significant rise in plasma concentration of vasopressin.  The  inverse relationship between the plasma concentration of vasopressin and carotid sinus pressure appeared to be slightly augmented in the aortic barodenervated rabbits following section of the vagus nerves (figure 8, panel C). However the individual values of i A V P in panel B at each carotid sinus pressure were not significantly different from those in panel C. The  vasopressin  and M A P data from  the  two  groups of rabbits in these  experiments were combined (n=19) in order to illustrate two points.  Firstly the total  baroreceptor input necessary to maximally inhibit the release of vasopressin was relatively low (figure 10).  With zero afferent input from the aortic baroreceptors (ADN sectioned),  41  Figure 3: Changes in mean arterial pressure (MAP, mmHg) in response to alterations in carotid sinus pressure (CSP, mmHg). The rabbits underwent three sets of C S P changes, first (A) with both aortic depressor nerves (ADN) and vagus nerves (VN) intact, (NI), secondly (B) after A D N section  (ADNX)  and  * P<0.05 all values pressure of 160 mmHg.  thirdly  (C)  after  compared to the  VN  value  section  (VNX).  at carotid sinus  42  A  B ADN s e c t i o n e d  40 80 100120 160  C .  _  40 80 100120 160  C a r o t i d Sinus P r e s s u r e (mmHg)  VN s e c t i o n e d  40 80 100120 160  43  Figure 4: Changes in mean arterial pressure (MAP, mmHg) in response to alterations in carotid sinus pressure (CSP, mmHg). The rabbits underwent three sets of C S P changes, first (A) with both aortic depressor nerves (ADN) and vagus nerves (VN) intact, (NI), secondly (B) after V N section  (VNX) and  thirdly  (C)  after  A D N section  (ADNX).  * P<0.05, ** P<0.01 all values compared to value at carotid sinus pressure of 160 mmHg.  44  B 200  VN s e c t i o n e d  ADN s e c t i o n e d  1G0  ~  O)  X  120  I E  j= o_ <  *  X  80  X 40  40 80 100120 160  40 80 100 120 160  C a r o t i d S i n u s P r e s s u r e (mmHg)  40 80 100120 160  45  Figure 5:  The effect of changing carotid sinus pressure (CSP) on the mean arterial pressure (MAP) and right atrial pressure (RAP). Consecutive parts of the femoral arterial pressure record and right atrial pressure record in one experiment. Each section of the trace corresponds to 5 seconds. Records taken 5 minutes after change in CSP. Rabbits had intact aortic depressor nerves and vagus nerves (NI).  46  47  Figure 6:  The effect of changing carotid sinus pressure (CSP) on the mean arterial pressure (MAP) and right atrial pressure (RAP). Consecutive parts of the femoral arterial pressure record and right atrial pressure record in one experiment. Each section of the trace corresponds to 5 seconds. Records taken 5 minutes after change in CSP. Rabbits had intact aortic depressor nerves and sectioned vagus nerves (VNX).  48  100  160  120  80  CAROTID SINUS PRESSURE (mmHg)  40  100  49  Figure 7:  The effect of changing carotid sinus pressure (CSP) on the mean arterial pressure (MAP) and right atrial pressure (RAP). Consecutive parts of the femoral arterial pressure record and right atrial pressure record in one experiment. Each section of the trace corresponds to 5 seconds. Records taken 5 minutes after change in CSP. Rabbits had sectioned aortic depressor nerves and vagus nerves (ADNX).  50  51  Figure 8:  Changes in immunoreactive vasopressin  (iAVP, pg/mL) in response  alterations in carotid sinus pressure (CSP, mmHg). underwent three sets of C S P changes, firstly (A)  to  The rabbits  with both aortic  depressor nerves (ADN) and vagus nerves (VN) intact, secondly (B) after A D N section (ADNX) and thirdly (C) after V N section (VNX). * P<0.05, ** P<0.01 all values compared to value at carotid sinus pressure of 160 mmHg.  52  C  B BO  ADN s e c t i o n e d  VN s e c t i o n e d **  40 cn  I I  CL  > <  20  40  80 100 120 160  40  80 100120160  C a r o t i d Sinus P r e s s u r e (mmHg)  40  80 100 120  160  53  Figure  9:  Changes in immunoreactive vasopressin  (iAVP,  pg/mL) in response  to  alterations in carotid sinus pressure (CSP, mmHg). The rabbits underwent three sets of C S P changes, firstly (A)  with both aortic  depressor nerves (ADN) and vagus nerves (VN) intact, secondly (B) after V N section (VNX) and thirdly (C) after A D N section (ADNX). * P<0.05, ** P<0.01  all values compared to value at carotid  sinus pressure of 160 mmHg.  54  B 200  VN  R  ADN  sectioned  sectioned  160  120  80  40  40 80 100120 160  40 80 100 120160  C a r o t i d Sinus Pressure  (mmHg)  40 80 100 120 160  55  Figure 10: Changes in immunoreactive vasopressin (iAVP, pg/mL) in response to changes in carotid sinus pressure (CSP).  The results were from all 19  rabbits before and after section of both the aortic depressor nerves (ADN) and vagus nerves (VN). * P<0.05,  * * P<0.01 all values  compared to value at carotid sinus pressure of 160 mmHg.  56  125 r  ADN intact  ADN sectioned  VN intact  VN sectioned  100 •  **  75 50 .25 I  40  80  100  120  1G0  40  80  Carotid Sinus Pressure (mmHg)  100  120  1 GO  57  the carotid sinus pressure had to be decreased to concentration of vasopressin rose appreciably.  100 mmHg before the plasma  Secondly, although the presence of intact  vagus and aortic depressor nerves does not prevent reflex changes in systemic arterial (mean) pressure (figure 11), it clearly prevents a rise in vasopressin. When changes in HR for both groups of rabbits were combined (n= 19) the H R was significantly increased during decreases in carotid sinus pressure only in the rabbits with aortic depressor nerves and vagus nerves sectioned (figure 12).  In the rabbits with both  aortic depressor nerves and vagus nerves sectioned carotid sinus pressure had to be decreased to 100 mmHg before a significant (P<0.05) increase in H R was observed. This was in contrast with the M A P results where decreasing carotid sinus pressure from 160 to 120 mmHg elicited a significant (P<0.05) rise in M A P even in the presence of the aortic depressor nerves. In a separate group of eight rabbits, while the carotid sinus pressure was held constant at lOOmmHg, cutting the aortic depressor nerves (ADNX) did not significantly change the levels of R A P , M A P , H R or the plasma concentration of vasopressin from control levels (figure 13).  Cutting the vagi in these aortic barodenervated rabbits did not  significantly change R A P , H R or the plasma concentration of vasopressin, however, following vagotomy M A P was significantly (P<0.05) increased as compared to the rabbits with both nerves intact (figure 13).  58  Figure 11:  Changes in mean arterial pressure (MAP, mmHg) in response to changes in carotid sinus pressure (CSP).  The results were from all 19 rabbits  before and after section of both the aortic depressor nerves (ADN) and vagus nerves (VN).  * P<0.05,  * * P<0.01 all values  compared to value at carotid sinus pressure of 160 mmHg.  59  60  Figure 12:  Changes in heart rate (HR, mmHg) in response to changes in carotid sinus pressure (CSP).  The results were from all 19 rabbits before and  after section of both the aortic depressor nerves (ADN) and vagus nerves (VN).  * P<0.05,  ** P<0.01 all values compared to  value at carotid sinus pressure of 160 mmHg.  350  ADN sectioned VN sectioned  ADN intact VN intact  40  80  100  120  1G0  40  80  Carotid Sinus Pressure (mmHg)  100  120 1G0  62  Figure 13:  The effects of serial nerve section on  right atrial pressure (RAP, panel A),  heart rate (HR, panel B), mean arterial pressure (MAP, panel C) and  plasma  immunoreactive  vasopressin  (iAVP, panel  D).  Following a control period when both nerves were intact (NI) the aortic depressor nerves were sectioned (ADNX) followed by the vagus nerves  (VNX).  Carotid sinus pressure (CSP) was held  constant at 100 mmHg. Each time period, either with nerves intact (NI) or after A D N X and V N X was 60 minutes. The bars represent the mean of the measurements minutes.  taken at  10 minutes and 60  63  "  400  HR tbts/nin)  300  40  AVP (pg/«U  30  64  CHAPTER 2  BLOOD VOLUME CHANGES AND VASOPRESSIN RELEASE  Introduction The aims of the experiments relationship  between  increases  concentration of vasopressin.  and  reported in Chapter decreases  in  blood  2 were volume  to establish and  the  the  plasma  In particular to establish the contribution to this response  made by the aortic baroreceptors and atrial receptors with vagal afferents.  To eliminate  any influence from carotid sinus baroreceptors, the carotid sinus pressure was maintained at 100 mmHg.  Protocol The experiments were carried out on 4 groups of 10 rabbits in which carotid sinus pressure (CSP) was maintained constant (CSP = lOOmmHg) throughout the experimental period (figure 14). Half of the rabbits had intact aortic depressor nerves (ADNI) while the other half underwent bilateral section of their aortic depressor nerves experimental period (ADNX).  prior to the  In both the A D N I rabbits and the A D N X rabbits blood  volume (BV) was either increased (n = 10) or decreased (n = 10) by 10% and 20% of the estimated blood volume (60 mL/kg), before and after bilateral vagotomy.  Blood samples  for the measurement of immunoreactive vasopressin were taken immediately before blood volume change (pre-BVC), 10 minutes after B V C and 10 minutes after blood volume was returned to normal (post BVC). The pre- and post-BVC measurements were averaged and compared to the values measured 10 minutes after B V C . The step changes in blood volume were as follows: control B V ; +/- 10% B V ; control B V ; +/- 20% B V ; control B V . These steps were then repeated in the same rabbit after bilateral vagotomy. A 15 minute stabilization period was allowed after bilateral vagotomy before the next set of blood  65  Figure 14: A n illustration of the experimental protocol for the experiments in Chapter 2. For a description of the protocol see the text of the thesis.  ADNI  VN intact  VN sectioned 1  +/-  r  20 • (n = 20) CSP=100  +/ -  10  C UJ  Z£  ZD  ADNX  _l o > Q O O  VN sectioned  VN intact +/ -  20 -  (n =  _j CD  20)  CSP=100 +/-  10•  C •  1 0  10  20  30  40  TIME  55  65  75  85  95  (MINUTES) ON ON  67 volume changes.  The order of blood volume changes, either 10% or 20% was alternated  with each experiment. Sampling of blood was done 10 minutes after each change in blood volume. All blood removed was replaced with exchanged blood from the reservoir. The four experimental groups are shown below. I  Haemorrhage, ADN intact  II  Haemorrhage, ADN sectioned  III  Volume Expansion, ADN intact  IV  Volume Expansion, ADN sectioned In addition to these four groups of experimental rabbits, six rabbits were used in a  time control study to examine the changes in baseline MAP, RAP, HR and plasma concentration of vasopressin. The carotid sinus pressure of these rabbits was maintained at 100 mmHg. The protocol for these six rabbits was similar to the experimental groups of rabbits however blood volume was not changed throughout the experiment.  Three  rabbits had intact aortic depressor nerves (ADNI) and in the other three rabbits aortic depressor nerves were sectioned (ADNX). As the data was not significantly different for the ADNI and the ADNX rabbits the 2 groups were combined (n = 6). All six rabbits had their vagus nerves sectioned half way through the protocol.  Statistical Analysis For all measured variables there were two controls; one prior to and one 10 minutes after each volume change.  The pre-control and post-control values were pooled  and the resulting mean control values were compared to the experimental values using a two tailed Student's T-Test for paired data. Since the vasopressin values did not conform to a normal distribution a Wilcoxon's Signed Ranks Test for paired data was used for statistical evaluation. A value of P< 0.05 was considered statistically significant.  68  Results The experiments were carried out on 46 male New Zealand White rabbits (3.2 +/0.1 kg).  At the start of the experimental period the p H was 7.45 +/- 0.01 units, and  PCC"2 was 24.9 +/- 1.2 mmHg.  The respiratory gas was supplemented with oxygen so  that in all the rabbits PO2 was greater than 200 mmHg.  Plasma osmolality was  significantly higher (P<0.05) at the start of the experiment than at the end (307.2 +/1.4 mOsmol/kg and 301.2 +/- 1.6 mOsmol/kg respectively).  Haematocrit was 31.2 +/-  0.8 % at the start and 32.2 +/- 0.7 % at the finish of the experiment.  The values for  arterial pressure, right atrial pressure, heart rate and immunoreactive plasma vasopressin at  CSP=  lOOmmHg  were  M A P = 101.1 +/-3.7 mmHg,  R A P = 2 . 7 +/- 0.3 c m H 0 , 2  HR=270 +/- 6.0 bts/min and iAVP=14.8 +/- 1.4 pg/mL respectively. The results from these experiments were divided into two parts. The first section deals with the results from those rabbits with intact aortic depressor nerves and the second with those rabbits that underwent bilateral section of the aortic depressor nerves. Both groups of rabbits had constant carotid sinus pressure (lOOmmHg) throughout the experiment and the blood volume was either increased or decreased before and after sectioning the vagus nerves.  The pattern of the change in M A P which accompanied  section of the vagus nerves was similar in rabbits whether the aortic depressor nerves were intact (ADNI) or sectioned (ADNX) (figure 15).  Immediately after vagotomy M A P  decreased followed by a return to pre-vagotomy levels of M A P . The fall in M A P was likely due to stimulation of vagal afferents during nerve section.  Aortic Depressor Nerves Intact: Haemorrhage of 10% and 20% of the blood volume significantly decreased M A P and R A P in rabbits with intact or sectioned vagus nerves (table I). The magnitude of the fall in M A P and R A P was greater after haemorrhage of 20% of the blood volume than after haemorrhage of 10% of the blood volume.  Baseline H R was 258 +/- 10 bts/min at  69  Figure 15: The effect of cutting the vagus nerves (VNX) on mean arterial pressure (MAP) and right atrial pressure (RAP) in rabbits with intact (ADNI) or sectioned aortic depressor nerves (ADNX). Consecutive parts of the femoral arterial pressure and right atrial pressure record in one experiment. Carotid sinus pressure (CSP) was maintained constant at 100 mmHg.  i  10  seconds  100  i  seconds  71  TABLE  I:  (RAP)  RECORDEO  WITH  MEAN  INTACT  PERFORMED STANDARD  ARTERIAL 10  AORTIC  BEFORE ERROR,  PRESSURE  MINUTES  OEPRESSOR  (VNI)  AND  (MAP)  AFTER  AND  HAEMORRHAGE  NERVES  AFTER  (ADNI).  BILATERAL  RIGHT  ATRIAL  CARRIED  OUT  PRESSURE IN  HAEMORRHAGES VAGOTOMY  RABBITS WERE  (VNX).  MEAN  •  N-10.  ADNI  C  c  -10  -20  VNI MAP  i 8  102  *  9  93  • 8»  2.2  i  0.5  1.5  1  122  •  6  108 •  5"  119 • 8  2.1  1  0.6  1.3  0.6«"  2.M  11 5  75  1  7  ..  (MMHG)  RAP  0.4«*  0.6  2.4  l.H 1  0.5"  (CMH20)  VNX MAP  88 i  6"  (MMH6)  RAP  •  •.  0.6  0.8  i  0.5"  (CMH20)  (MEAN  • P  0.05 ( A S  COMPARED TO CONTROL)  +/-  SE)  72 the start and 250 +/- 7 bts/min at the finish of the experiment and was not altered significantly by haemorrhage. In the rabbits with intact aortic depressor nerves and intact vagus nerves (figure 16) haemorrhage of 20% of the blood volume but not 10% of the blood volume, significantly increased the plasma concentration of vasopressin.  In these rabbits following bilateral  section of the vagus nerves (no input from atrial receptors with vagal afferents, figure 16), the magnitude of the increase in the concentration of vasopressin as observed with haemorrhage of 20% of the blood volume was not significantly different from that prior to section of the vagus nerves. Volume expansion of 10% and 20% of the blood volume significantly increased MAP and RAP in rabbits with intact or sectioned vagus nerves (Table II). The magnitude of the increases in MAP  and RAP in response to volume expansion was greater after  expansion of 20% of the blood volume than after expansion of 10% of the blood volume. Baseline heart rate was 276 +/- 7 bts/min and 283 +/- 6 bts/min at the start and finish of the experiment respectively. Heart rate was not altered by volume expansion. In rabbits with intact aortic depressor nerves and vagus nerves volume expansion of 10% and 20% of the blood volume did not significantly change the plasma concentration of vasopressin (figure 17). In these same rabbits following section of the vagus nerves, however, volume expansion was  accompanied by a significant fall in the plasma  concentration of vasopressin (figure 17).  Aortic Depressor Nerves Sectioned: In the rabbits with no input from aortic baroreceptors, haemorrhage significantly decreased MAP  and RAP both before and after section of the vagus nerves (Table III).  The actual alterations in MAP and RAP were recorded during haemorrhage of 10% of the blood volume (figure 18) and these responses were compared before and after vagus nerve section in aortic barodenervated rabbits with carotid sinus pressure at lOOmmHg. Ten  73  Figure  16:  Changes in immunoreactive vasopressin (iAVP, pg/mL) in response  to  haemorrhage of 10% and 20% of the estimated blood volume. The haemorrhages were carried out in the same rabbits before ( V N intact) and after bilateral vagotomy (VN sectioned). average  of  measurements.  the  pre-haemorrhage  and  C is the  post-haemorrhage  All rabbits (n=10) had intact aortic depressor  nerves (ADNI). * P<0.05, ** P<0.01 haemorrhage compared to control.  74  50 ADNI  VN  VN s e c t i o n e d  intact  40  (n=10)  1I  30 iAVP (pg/mL) 20  I  I  10  0  C  -10  C  C -10  -20  Blood Volume  (%l  C -20  75  TABLE I I : (RAP)  MEAN ARTERIAL  PRESSURE (MAP) AND RIGHT  RECORDED 10 MINUTES  RABBITS  WITH INTACT  AORTIC  WERE PERFORMED BEFORE MEAN *  ATRIAL  PRESSURE  AFTER VOLUME EXPANSION CARRIED OUT IN DEPRESSOR NERVES ( A D N I ) .  ( V N I ) AND AFTER B I L A T E R A L  EXPANSIONS  VAGOTOMY  (VNX).  ERROR. N - 1 0 .  - STANDARD  ADNI + 10  c  c  + 20  VNI i  93 1 6  103  2.3 • 0.5  2.8 • 0.5*  MAP  7 "  89 1 7  107 *  8»»  (MMHG)  RAP  1.8 • 0 . 5  3.8 • O.M««  (CHH20)  VNX MAP  100 •  7  UM i 8««  99 • 6  122 1 7"  (MMHG)  RAP  1.6 1 0.5  2.9 l 0.6»«  1.3  •. 0.6  3.9  0.8*»  (CMH20)  • P  0.05  •• P  0.01  (MEAN • / - SE) (AS  COMPARED  TO  CONTROL)  76  Figure 17: Changes in immunoreactive vasopressin (iAVP, pg/mL) in response to volume expansion of 10% and 20% of the estimated blood volume. The volume expansions were carried out in the same rabbits before (VN intact) and after bilateral vagotomy (VN sectioned).  C is the  average of the pre-volume expansion and post-volume expansion measurements. nerves  (ADNI).  All rabbits (n=10) had intact aortic depressor * P<0.05,  compared to control.  * * P<0.01  Volume  expansion  77  Blood Volume (X)  78  TABLE I I I : (RAP)  MEAN ARTERIAL PRESSURE  (MAP) AND RIGHT ATRIAL PRESSURE  RECORDED 10 MINUTES AFTER HAEMORRHAGE CARRIED OUT IN RABBITS  WITH SECTIONED AORTIC DEPRESSOR NERVES PERFORMED BEFORE STANDARD ERROR,  (ADNI).  HAEMORRHAGES WERE  ( V N I ) AND AFTER BILATERAL VAGOTOMY (VNX).  MEAN *  N-10.  ADNX  c  —  10  C  •  -20  VNI 107 • 8«  118 • 6  92 l 8"  3.5 • 0.5  2.5 • 0.4"  3.6 • 0.6  2.2 i 0.5"  130 • 5  116 • 7 "  132 ± 4  104 ± 11"  3.8 i 0.9  2.3 • 1.0"  3.9 • 0.9  2.3 • 1.0"  MAP  118  1  7  (MMHG)  RAP (CHH20)  VNX MAP (MMHG)  RAP (CMH20)  '  "  P  P  ° '  0  5  0.01  (AS COMPARED TO CONTROL)  <  M  E  A  N  +  /  "  S  E  >  79  Figure 18:  The effect of haemorrhage of 10% of the blood volume on mean arterial pressure  (MAP) and  right  atrial  pressure  barodenervated rabbits with carotid sinus  (RAP) in  aortic  pressure maintained  constant at 100 mmHg. Haemorrhages were carried out before (VNI) and after bilateral section of the vagus  nerves  (VNX).  Records were taken in order to demonstrate the immediate (left),and the recovery from haemorrhage (right).  ADNX  VNI  140 120  CSP  100  eo MAP 60 40 20 0  RAP  i_  100 seconds  10 seconds  ADNX  VNX  140  CSP  120 100 80 60 40 20 0  I  10 seconds  100  I  seconds  81  minutes  after  haemorrhage M A P had recovered to within  10 mmHg of the pre-  haemorrhage value in the rabbits with no input from sinoaortic baroreceptors and no atrial volume receptors (bottom panel).  There were no qualitative differences in either the  pattern of these responses or the magnitude of the falls in the cardiovascular variables (measured 10 minutes after the blood volume change) between the aortic barodenervated rabbits and the rabbits with intact aortic depressor nerves. There was a difference in the response of vasopressin to haemorrhage between rabbits with and without input from aortic baroreceptors (figure 19).  In the rabbits with  sectioned aortic depressor nerves haemorrhage of 10% and 20% of the blood volume did not increase the plasma concentration of vasopressin either before or after section of the vagus nerves (figure 19).  Therefore without input from aortic baroreceptors haemorrhage was  ineffective as a stimulus for the release of vasopressin in rabbits. Volume  expansion  significantly  increased  M A P and  R A P in  the  aortic  barodenervated rabbits in a manner both qualitatively and quantitatively similar to that seen in the rabbits with intact aortic depressor nerves (table IV). Consistent with the haemorrhage data presented above, volume expansion (figure 20) did not significantly alter the plasma concentration of vasopressin either before or after the vagus nerves were sectioned. Further analysis of the data showed there was a high correlation between M A P and immunoreactive vasopressin in the rabbits with intact aortic depressor nerves and sectioned vagus nerves (r = 0.973).  The slope of the relationship was negative with the  immunoreactive vasopressin decreasing as M A P increased (figure 21). There was however no correlation between M A P and immunoreactive vasopressin in the rabbits with sectioned aortic depressor nerves (whether the vagus nerves were intact or sectioned).  82  Figure  19:  Changes in immunoreactive vasopressin (iAVP, pg/mL) in response  to  haemorrhage of 10% and 20% of the estimated blood volume. The haemorrhages were carried out in the same rabbits before (VN intact) and after bilateral vagotomy ( V N sectioned). average  of the pre- and post-haemorrhage  C is the  measurements. All  rabbits underwent aortic depressor nerve section (ADNX) prior to the experiment.  83  50 ADNX  VN sectioned  VN i n t a c t  40  (n=10) 30 i AVP (pg/mL) 20 X  10 0  C -10  1  C -20 Blood Volume ('/.)  1 C -10  C  -20  84  TABLE I V : (RAP)  ME AN ARTERIAL  RECORDED  RABBITS  10  WITH SECTIONED  WERE PERFORMED BEFORE MEAN ± STANDARD  (MAP)  PRESSURE  MINUTES AFTER VOLUME  ERROR,  AORTIC  (VNI)  AND RIGHT EXPANSION  DEPRESSOR NERVES  AND AFTER  BILATERAL  ATRIAL PRESSURE CARRIED  (ADNI).  OUT IN  EXPANSIONS  VAGOTOMY  (VNX).  N-10.  ADNX  C  + 10  C  -•-  + 20  VNI HAP  105 i 6  117  2-2 1 0.5  5«  103 • 6  125  3.1 • 0 . 7 "  2.1 1 0.6  3.6 1 0 . 6 "  116 • 5.7  129 • 5.7«  113 1 4  130 • 5 "  2.2 • 0.6  3.2 • 0.7*  1.9 • 0.7  4.H 1 0 . 7 "  1  M'  (MMHG)  RAP (CMH20)  VNX MAP (MMHG)  RAP 'cnH20>  (MEAN • / -  "  • P  0.05  P  0.01  (AS  COMPARED  TO  CONTROL)  SE)  85  Figure 20: Changes in immunoreactive vasopressin (iAVP, pg/mL) in response to volume expansion  of 10% and 20% of the estimated blood volume. The  volume expansions were carried out in the same rabbits before (VN intact) and after bilateral vagotomy (VN sectioned).  C is the  average of the pre-volume expansion and post-volume expansion measurements. All rabbits ,(n=10) underwent bilateral aortic depressor nerve section (ADNX) prior to the experimental period.  86  50 ADNX  VN sectioned  VN i n t a c t  40  (n=10) 30  I  iAVP (pg/ml) 20 10 0  X  I  C +10  C +20 Blood Volume (%)  C  +10  C  +20  87  Figure 21: The linear relationship between the piasma concentration of immunoreactive vasopressin (iAVP) and mean arterial pressure (MAP). Rabbits with intact aortic depressor nerves before (circles, dashed line, r = 0.604) and after vagal section (diamonds, solid line, r = 0.973)  88  89 Effect of Vagotomy In a separate group of rabbits we examined the effect of bilateral vagotomy on the baseline levels of M A P , R A P and vasopressin (figures 22,23,24). pressure, time course and the sampling times were experiments described above.  The carotid sinus  similar to those found in the  Since there was no significant difference in the baseline  levels of the variables between the rabbits with intact aortic depressor nerves and the rabbits with sectioned aortic depressor nerves, the data were combined (n = 6).  Bilateral  section of the vagus nerves did not significantly alter M A P , R A P or vasopressin concentration (figures 22,23,24). Because the plasma concentrations of vasopressin may have been influenced more by the vascular pressures immediately after the volume change than by those present at the time of blood sampling, 10 minutes later, the data showing the vascular pressure 1 minute after the volume changes are given in Table V . The magnitude of the changes in R A P were significantly greater in the rabbits after their aortic depressor nerve were sectioned than in the rabbits with intact aortic depressor nerves. This was not the case for the magnitude of the change in M A P .  90  Figure 22: Time control study of plasma vasopressin (iAVP). (Vn), vagus nerve. Three of six rabbits had intact aortic depressor nerves (ADNI) while three of the rabbits underwent bilateral aortic depressor nerve section (ADNX) prior to the experiment.  91  50 ADNI/ADNX (n=6)  VN sectioned  VN i n t a c t  40 30  iAVP Ipg/mL) 20  X  x  10 0  0  10  3D  40  "  Time (minutes)  65  75  95 105  92  Figure 23:  Time control study of right atrial pressure (RAP).  Three of six rabbits had  intact aortic depressor nerves (ADNI) while three of the rabbits underwent bilateral aortic depressor nerve section (ADNX) prior to the experiment.  93  VN intact  VN sectioned  ADNI/ADNX (n=6)  RAP (cmH20)  4  ix 0  10  30  40  AV  Time (minutes)  65  75  95 105  94  Figure 24:  Time control study of mean arterial pressure. Three of six rabbits had intact aortic  depressor  nerves  (ADNI)  while  three  of  the  rabbits  underwent bilateral aortic depressor nerve section (ADNX) prior to the experiment.  95  140 ADNI/ADNX (n=B)  1 2 0  100 • 80 •  VN s e c t i o n e d  VN i n t a c t  r  X  X  XI  MAP (mmHg) BO 40 20 0  0  10  30  40  "  Time (minutes)  65  75  95 105  96  TABLE V : RIGHT  THE ABSOLUTE CHANGE I N MEAN ARTERIAL  CHANGE OF 1 0 1 OR 2 0 1 OF THE BLOOD VOLUME. UNDERWENT THE VOLUME CHANGES EITHER NERVES  PRESSURE (MAP) A N D  ATRIAL PRESSURE ( R A P ) MEASURED ONE MINUTE AFTER A BLOOD VOLUME  (ADNI)  OR THE AORTIC  PRIOR TO THE E X P E R I M E N T . AND AFTER B I L A T E R A L  THE RABBITS  WHICH  HAD INTACT AORTIC DEPRESSOR  DEPRESSOR NERVES WERE SECTIONED  (ADNX)  VOLUME CHANGES WERE PERFORMED BEFORE ( V N I )  VAGOTOMY  (VNX).  MEAN • STANDARD  RIGHT A T R I A L PRESSURE  ERROR. N - 4 0 .  MEAN ARTERIAL PRESSURE  (CMH 0)  (MMHG)  2  ADNX  ADNI  ADNI  ADNX  VNI +10  •l.M • / - 0.4  •1.6  •/-  •20  *3.7 • / - 0.3  •3.8  -10  -1.3 • / - 0.3  -20  -1.2 • /- 0.5  •/- 2  •32.1 • / - 4  NS  •/- 0 . 5 "  •29.3 • / - 5  •35.4 • / - 5  NS  -1.5  •/-  0.3»»  -30.8  •/- 6  -30.6  •/- 4  NS  -2.1  •/-  0.4«»  -60.2  •/- 8  -57.6  •/- 5  NS  0.4»»  •19.2  1  VNX •10  *1.8 • / - 0.3  •2.1  •/-  0.4««  •20.5  •/- 5  •19.3  •/- 5  NS  •20  *3.7 • / - 0.6  •3.8  • / - 0.5"»  •25.5  •/- 4  •21.7  •/- 8  NS  -10  -1.3 • / - 0.3  -1.9  •/-  0.4»»  -29.4  •/- 7  -19.4  •/- 8  NS  -20  -1.9 • / - 0.3  -2.0  •/-  0.6»«  -66.4  • / - 11  -66.4  • / - 1 1 NS  (MEAN +/-  P P  0 . 0 5 (ADNI V S . ADNX) 0 . 0 1 (ADNI V S . ADNX)  SE)  97  CHAPTER 3  I N T E R A C T I O N B E T W E E N C A R O T I D SINUS B A R O R E C E P T O R S A N D A T R I A L V O L U M E R E C E P T O R S IN V A S O P R E S S I N R E L E A S E  Introduction The aim of the experiments reported in Chapter 3 was to assess specifically the role of the atrial receptors with vagal afferents in the vasopressin response to blood volume changes in a rabbit with minimal input from either carotid sinus baroreceptors or aortic baroreceptors. These rabbits had their aortic depressor nerves sectioned and carotid sinus pressure was maintained at either high or low pressure.  It was anticipated that  with little inhibition of vasopressin release from either the carotid sinus baroreceptors and the aortic baroreceptors then the effect of the atrial receptors with vagal afferents would be more apparent.  Protocol The experiments were divided into two groups of rabbits which underwent either haemorrhage (n=10) or volume expansion (n=10).  In all rabbits carotid sinus pressure  (CSP) was maintained constant (CSP= lOOmmHg) throughout the 60 minute equilibration period (figure 25). Following the equilibration period carotid sinus pressure was decreased to 60 mmHg.  After 15 minutes blood volume was either increased or decreased by 10%  and 20% of the estimated blood volume (60 mL/kg).  Blood samples for the measurement  of MAP, RAP, HR and immunoreactive vasopressin were taken immediately before blood volume change (pre-BVC), 10 minutes after BVC and 10 minutes after blood volume was returned to normal (post BVC).  The pre- and post-BVC measurements were averaged and  compared to the values measured 10 minutes after BVC.  In the second half of the  98  Figure 25: A n illustration of the experimental protocol in Chapter 3.  All rabbits (n = 20)  had intact vagus nerves and sectioned aortic depressor nerves. Between each volume change the blood volume was restored to the control level (C).  CSP=60  CSP=120  VN intact  0*  ~  +/-  ADN sectioned  20 -•  (n = 20)  LU 3  O >  + / - 10 -  Q O O  _j CD  c•  1 0  10  20  30  40  55 65 75 85 95  TIME (MINUTES)  100  experimental protocol carotid sinus pressure was increased from 60 to 120 mmHg and after 15 minutes the blood volume changes were repeated. Sampling of blood was done 10 minutes after each change in blood volume.  Statistical Analysis For all measured variables there were two controls; one prior to and one 10 minutes after each volume change.  The pre-control and post-control values were pooled  and the resulting mean control values were compared to the experimental values using a two tailed Student's T-Test for paired data. Since vasopressin values did not conform to a normal distribution a Wilcoxon's Signed Ranks Test for paired data was used for statistical evaluation. A value of P<0.05 was considered statistically significant.  Results The experiments (weight: 3.25 +/- 0.08 kg).  were carried out on 20 male New Zealand White rabbits A t the start of the experimental period the p H was 7.49 +/-  0.01 units and PCO2 was 27.6 +/- 0.6 mmHg.  The respiratory gas was supplemented  with oxygen so that in all the rabbits PO2 was greater than 200 mmHg.  Plasma  osmolality was 310 +/- 2.0 osmol/kg at the start, and 300 +/- 2.0 mosmol/kg at the finish of the experimental period. The haematocrit was 33 +/1.3 % at the start and 30.9 +/1.0 % at the finish of the experiment. The values of arterial pressure (MAP), right atrial pressure (RAP) and heart rate (HR) at carotid sinus pressure of lOOmmHg were 99.2 +/5.5 mmHg,  3.1 +/0.6 c m H 2 0 and 274 +/- 4.5 bts/min respectively.  immunoreactive vasopressin  at carotid sinus  pressure  of  100  The value of  mmHg was  9.2 +/-  2.2 pg/mL. The alterations in blood volume (both haemorrhage and volume expansion) were carried out in aortic barodenervated rabbits with carotid sinus pressure maintained at either 60 mmHg or 120 mmHg.  Haemorrhage of 20% of the blood volume significantly  101  (P<0.01) reduced M A P at both levels of carotid sinus pressure (figure 26). Haemorrhage of 10 % of the blood volume significantly (P<0.01) decreased M A P when carotid sinus pressure was 60 mmHg but not when carotid sinus pressure was 120 mmHg.  The  baseline level of M A P as well as the magnitude of the reductions in M A P were attenuated when carotid sinus pressure was 120 mmHg as compared to carotid sinus pressure of 60 mmHg.  This was not true for R A P since baseline R A P was similar at high and low CSP.  Haemorrhage of 10 and 20% of the blood volume significantly decreased R A P in rabbits with C S P of 60 mmHg  (figure  27).  However, when carotid sinus  pressure  was  120 mmHg, only haemorrhage of 20% significantly (P<0.01) decreased RAP. Haemorrhage of 10% or 20% of the blood volume was not an effective stimulus for the release of vasopressin in these aortic barodenervated rabbits (figure 28).  A t both  levels of carotid sinus pressure haemorrhage of 20% of the blood volume resulted in a rise in vasopressin in some of the rabbits and not in others. Therefore due to the variability of the response the rise in vasopressin did not achieve statistical significance. Volume expansion of 10% and 20% of the blood volume significantly (P<0.01) increased M A P at high and low carotid sinus pressures (figure 29).  The baseline levels of  M A P were significantly lower in the rabbits with high carotid sinus pressure than in rabbits with lower carotid sinus pressure, however, the magnitudes of the increases in M A P were not affected by alterations in background carotid sinus pressure.  Volume  expansion of 20% of the blood volume was accompanied by a significant rise in R A P at both levels of carotid sinus pressure (figure 30).  Right atrial pressure was significantly  (P<0.05) increased in response to volume expansion of 10% of the blood volume only when carotid sinus pressure was 120 mmHg.  Volume expansion did not alter the plasma  concentration of vasopressin at either high or low carotid sinus pressure (figure 31). Neither haemorrhage nor volume expansion significantly changed heart rate from the baseline level (pre-volume change) at either high (257 +/- 7 bts/min) or low carotid sinus pressure (281 +/- 5 bts/min). The groups of rabbits which underwent haemorrhage  102  Figure 26:  Changes in arterial pressure (MAP, mmHg) in response to haemorrhage of 10% and 20% of the estimated blood volume.  C is the average of  the pre-haemorrhage and post-haemorrhage  measurements. All  rabbits (n=10) underwent bilateral aortic depressor nerve section (ADNX)  prior  to  the  experiment.  haemorrhage compared to control.  * P<0.05,  * * P<0.01  103  B l o o d Volume (%)  104  Figure 27: Changes in right atrial pressure (RAP, cmH^O) in response to haemorrhage of 10% and 20% of the estimated blood volume.  C is the average of  the pre-haemorrhage and post-haemorrhage measurements.  All  rabbits (n=10) underwent bilateral aortic depressor nerve section (ADNX)  prior  to  the  experiment.  haemorrhage compared to control.  * P<0.05  ** P<0.01  105  CSP 120  CSP BO ADNX  (n=10)  RAP (cmH20)  4  C -10  C -20 Blood Volume (%)  C -10  C  -20  106  Figure 28:  Changes in immunoreactive vasopressin  (iAVP, pg/mL) in response  haemorrhage of 10% and 20% of the estimated blood volume. the  average  measurements.  of  the  pre-haemorrhage  and  to  C is  post-haemorrhage  All rabbits (n=10) underwent bilateral aortic  depressor nerve section (ADNX) prior to the experiment.  107  Blood Volume (%)  108  Figure 29:  Changes in arterial pressure (MAP, mmHg) in response to volume expansion of 10% and 20% of the estimated blood volume. C is the average of the  pre-volume  measurements. depressor  nerve  expansion  and  post-volume  expansion  All rabbits (n=10) underwent bilateral aortic section  (ADNX)  prior  to  the  experiment.  * P<0.05, ** P<0.01 volume expansion compared to control.  109  Blood Volume (%)  110  Figure 30:  Changes in right atrial pressure (RAP, cmH^O) in response to volume expansion of 10% and 20% of the estimated blood volume. C is the average of the pre-volume expansion and post-volume expansion measurements. depressor  All rabbits  nerve  section  (n=10) (ADNX)  underwent prior  to  bilateral aortic the  experiment.  * P<0.05, * * P<0.01 volume expansion compared to control.  Ill  Blood Volume (X)  112  Figure 31: Changes in immunoreactive vasopressin (iAVP, pg/mL) in response to volume expansion of 10% and 20% of the estimated blood volume. C is the average of the pre-volume expansion and post-volume expansion measurements.  All rabbits  (n=10)  underwent  bilateral aortic  depressor nerve section (ADNX) prior to the experiment.  113  50  ADNX  CSP 60  CSP 120  40  (n=10) 30 AVP (pg/mL)  20  10  0  I C +10  x  I C +20  C +10  Blood Volume C.)  X C +20  114  or volume expansion were combined (n = 20) and the baseline levels of vasopressin were calculated for both levels of carotid sinus pressure in order to confirm that carotid baroreceptors could influence the plasma concentration of vasopressin.  The plasma  concentration of vasopressin at carotid sinus pressure of 60 mmHg (12.0 +/- 2.6 pg/mL) was significantly  higher than the plasma concentration of vasopressin at carotid sinus  pressure of 120 mmHg (8.75 +/- 2.4 pg/mL, {P<0.05}).  115  CHAPTER 4  HAEMORRHAGE AND VASOPRESSIN RELEASE  Introduction The  aim of the  experiments  reported in Chapter  4 was  to challenge  the  cardiovascular system of the rabbit with a severe haemorrhage (30% of the blood volume) and examine the role of the aortic baroreceptors in the release of vasopressin in response to this haemorrhage. Carotid sinus pressure was maintained at 60 mmHg to minimize the inhibitory effect of the carotid sinus baroreceptors since it was clear from Chapter 1 that vasopressin  release  was  almost  maximally inhibited at  carotid sinus  pressure  of  lOOmmHg.  The haemorrhages were performed in rabbits before and after section of the  vagus nerves to eliminate the influence of the atrial receptors with vagal afferents.  Protocol Following the equilibration period carotid sinus pressure was decreased to 60 mmHg and the rabbit was allowed a further 15 minute stabilization period (figure 32). After that the blood volume was decreased by 10%.  Ten minutes later blood volume was  once again decreased by 10% and finally 10 minutes later blood volume was decreased by a third 10% making the total decrease in blood volume 30%.  After 10 minutes, at this  lowest blood volume, the withdrawn blood (30% of the blood volume) was returned to the rabbit through a cannula in the femoral artery. In the second half of the experiment the vagus nerves were sectioned bilaterally and the changes in blood volume were repeated. Measurements of M A P , R A P HR and immunoreactive vasopressin were made immediately before haemorrhage, 10 minutes after each step decrease in blood volume (-10, -20, -30%) and 10 minutes after the withdrawn blood was replaced.  All recording of pressures and  sampling of blood was done 10 minutes after each change in blood volume.  116  Figure 32: A n illustration of the experimental protocol for the experiments in Chapter 4. For details on the protocol see the text of the thesis.  TIME  (n = 10) 0  I  UJ  _l O > Q O O  10  20  30  (MINUTES)  40  55  65  75  85  J  [  10 •  20 ••  _j  CD  -30  •  J  L  VN intact  VN CSP=60  sectioned  95  118  Statistical Analysis In order to assess whether haemorrhage or volume replacement was associated with a change in the measured variables control values were taken to be the value measured immediately prior to the haemorrhage.  Experimental values were taken to be  the values measured 10 minutes after each step change in blood volume.  A one way  analysis of variance and a Duncans Multiple Range Test were used for statistical evaluation of the cardiovascular data.  Since the vasopressin values did not conform to a  normal distribution a Wilcoxon's Signed Rank Test for paired data was used to assess statistical significance. A value of P<0.05 was considered statistically significant.  Results The experiments were carried out on 10 New Zealand White rabbits (weight: 3.0 +/- 0.2 kg).  At the start of the experimental period the p H was 7.48 +/- 0.01 units  and PCO"2 was 30.7 +/- 1.2 mmHg.  The respiratory gas was supplemented with oxygen  so that in all the rabbits PO2 was greater than 200mmHg.  Plasma osmolality was  315 +/- 2.4 mOsmol/kg at the start and 307 +/- 2.4 mOsmol/kg at the finish of the experiment respectively.  A blood sample was taken and cardiovascular measurements  made at the end of the 60 minute equilibration period which followed the surgery.  These  baseline levels of mean arterial pressure (MAP), right atrial pressure (RAP), heart rate (HR) and immunoreactive vasopressin (iAVP) were as follows: M A P = 89.0 +/- 7.0 mmHg; R A P = 1.9 +/- 0.7 c m H 0 ; HR=261 +/- 5.0 bts/min and i A V P = 6.6 +/- 2.2 pg/mL. 2  These haemorrhage experiments were done in rabbits with intact aortic depressor nerves and carotid sinus pressure maintained at 60 mmHg. The initial decrease in carotid sinus pressure from 100 mmHg (during equilibration period) to 60 mmHg resulted in a significant increase in baseline M A P . In the rabbits with intact aortic depressor nerves and vagus nerves haemorrhage of 20% and 30% 'of the blood volume significantly (P<0.05) reduced M A P and volume  119  replacement restored M A P to a level not significantly different from the pre-haemorrhage control value (figure 33).  After vagal section the three successive step-like haemorrhages  resulted in significant. (P< 0.05) falls in M A P and volume replacement restored M A P to control levels. Section of the vagus nerves did not significantly alter the magnitude of the fall in M A P in response to any of the haemorrhages. Although there was a trend towards decreasing R A P with haemorrhage and increasing R A P after volume replacement these changes in R A P were not statistically significant (figure 34). In the rabbits with intact aortic baroreceptors and atrial receptors with vagal afferents haemorrhage of 10% and 30% of the blood volume significantly (P<0.05) increased  the  plasma concentration of vasopressin  (figure  35).  Although plasma  concentration of vasopressin decreased following volume replacement the vasopressin concentration was still significantly higher than the pre-haemorrhage control value 10 minutes after volume replacement.  Following section of the vagus nerves the pattern of  vasopressin changes was similar to that seen with intact vagus nerves but the magnitude of the changes in vasopressin was attenuated.  After vagal section vasopressin was  significantly (P<0.05) increased only after 30% haemorrhage and volume replacement restored vasopressin to levels not significantly different than control levels.  120  Figure 53: Changes in arterial pressure (MAP, mmHg) in response to serial step changes in blood volume. Blood volume was decreased in increments of 10% until the rabbit had been haemorrhaged 30% of the estimated blood volume.  All rabbits (n=10) had intact aortic depressor nerves  (ADNI).  (C), Control blood volume. (VR), volume replacement.  * P<0.05 experimental control value.  value  compared  to  pre-haemorrhage  121  VN i n t a c t  Blood Volume ('/.)  VN sectioned  122  Figure 34:  Changes in right atrial pressure (RAP, cmH^O) in response to serial step changes in blood volume. Blood volume was decreased in increments of  10% until the rabbit had been haemorrhaged 30% of the  estimated blood volume. (C), control blood volume, (VR), volume replacement. (ADNI).  All rabbits (n=10) had intact aortic depressor nerves  8  VN i n t a c t  VN sectioned  ADNI  (n=10)  G•  RAP (cm H20)  4-  Blood Volume (%)  124  Figure 35:  Changes in immunoreactive vasopressin (iAVP, pg/mL) in response to serial step changes in blood volume. Blood volume was decreased in increments of 10% until the rabbit had been haemorrhaged 30% of the estimated blood volume.  All rabbits (n=10) had intact aortic  depressor nerves (ADNI). (C), control, (VR), volume replacement. * P<0.05 experimental control value.  value  compared  to  pre-haemorrhage  125  50  ADNI  VN  intact  VN s e c t i o n e d  40  (n=10) 30 iAVP (pg/mL)  20  T !  T  10  C  -10  -20  -30  VR  Blood Volume  C (%)  -10  -20  I  -30  VR  126  DISCUSSION  S e v e r a l physiological and anatomical characteristics of the N e w Zealand W h i t e rabbit made  this species  a n excellent a n i m a l for examination of the  cardiovascular responses to alterations i n blood volume. cross reactivity to blood from other rabbits.  h o r m o n a l and  The rabbit does not show a n y  Therefore, whole blood from donor rabbits  could be used for replacement of all blood samples and for volume expansion.  A l s o the  afferent fibres from the aortic baroreceptors t r a v e l i n the aortic depressor nerves w h i c h are anatomically distinct from the vagus nerves (carrying afferent fibres from the a t r i a l receptors).  U s e was made of this anatomical feature i n order to separate the individual  contributions of aortic baroreceptors and a t r i a l receptors towards the vasopressin response to alterations i n blood volume. Previous studies investigating vasopressin release  have reported high baseline  levels of vasopressin i n anaesthetized a n i m a l s (DeTorrente et a l . 1975, Zucker et a l . 1975, T h a m e s and S c h m i d , 1981,).  It has been suggested t h a t some anaesthetics (Lehtinen et  al. 1984) and surgical stress (Bonjour and M a l v i n 1970, L e h t i n e n et a l . 1984) can increase baseline vasopressin concentrations and t h a t these levels of vasopressin can contribute to elevations i n a r t e r i a l pressure (Pang 1983, M c N e i l l and P a n g 1982). have demonstrated  t h a t it was possible i n anaesthetized  In contrast others  a n i m a l s to m a i n t a i n p l a s m a  vasopressin at levels w h i c h were comparable to those found i n conscious animals (Leighton et a l . 1982, Ledsome et a l 1985). T h e rabbits i n the present study had baseline levels of vasopressin which ranged from 5 p g / m L to 15 p g / m L . p l a s m a vasopressin  These values were only slightly higher than the range of  (0.8 p g / m L - 12.5 p g / m L ) w h i c h  has  been  previously reported  in  conscious rabbits (Leighton et a l . 1982, A r n o l d a et al. 1985). Quillen and C o w l e y (1983) have demonstrated  a linear relationship between the  p l a s m a concentration of vasopressin and osmolality i n dogs, the slope of which can be  127  influenced by changes plasma  i n blood volume.  osmolality i n the  range  from  T h i s relationship suggests that m a i n t a i n i n g 300 m O s m o l / k g to  associated w i t h a p l a s m a vasopressin of 7-10 p g / m L .  310 m O s m o l / k g would  be  I n this range of osmolality clear  distinctions would be expected between the p l a s m a vasopressin measured between hypoand h y p e r v o l a e m i a . I n the present studies it was possible to show significant increases i n the p l a s m a concentration of vasopressin d u r i n g haemorrhage and significant suppression of vasopressin d u r i n g volume expansion. In only one of the groups (Chapter 2, group 2) was there a significant change i n osmolality over the course of the experimental period.  E x a m i n a t i o n of the relationship  between p l a s m a osmolality a n d p l a s m a vasopressin i n each of the 4 groups of experiments showed  no  significant correlation i n the  individual  measurements,  between  plasma  osmolality and p l a s m a vasopressin at the s t a r t of the experiment.  CAROTID BARORECEPTORS AND PLASMA VASOPRESSIN The results from the data presented i n C h a p t e r 1 show t h a t i n the  anaesthetized  rabbit, w i t h a l l nerves intact, changes i n carotid sinus pressure have no effect on p l a s m a immunoreactive vasopressin.  T h i s agrees w i t h previous work i n cats and dogs i n w h i c h  carotid occlusion did not increase p l a s m a vasopressin i f the vagus nerves were intact (Share and L e v y 1962, C l a r k and S i l v a 1967).  Share and L e v y (1962) clearly showed  t h a t carotid occlusion w a s ineffective as a stimulus for vasopressin release i n dogs w i t h intact vagus nerves.  H o w e v e r , following section of the vagus nerves i n these same dogs,  carotid occlusion resulted i n significant elevations i n p l a s m a concentration of vasopressin. C l a r k and S i l v a (1967) repeated  these observations i n cats b y showing that carotid  occlusion increased p l a s m a concentration of vasopressin following cervical section of both the vagus and sympathetic nerves.  In these e a r l y studies it w a s not possible to determine  i f the receptors w h i c h were inhibiting the rise i n vasopressin were the aortic baroreceptors or the cardiac volume receptors w i t h v a g a l afferents, since i n the dog and cat both sets of  128  receptors have afferents w h i c h r u n i n the vagus nerves. T h e present data suggest that receptors w i t h afferents  i n the aortic depressor  nerves are m a i n l y responsible for buffering the rise i n immunoreactive vasopressin induced b y a fall i n carotid sinus pressure i n the anaesthetized rabbit.  A n inverse relationship  between carotid sinus pressure and immunoreactive vasopressin became apparent aortic depressor nerve section i n the rabbits w h i c h had intact vagus nerves.  after  I f however,  the vagus nerves were sectioned before the aortic depressor nerves there w a s no change i n i m m u n o r e a c t i v e vasopressin w i t h carotid sinus pressure changes.  These  observations  suggest t h a t i n this preparation it is p r i m a r i l y the aortic baroreceptors w h i c h are acting to suppress the changes i n immunoreactive vasopressin seen d u r i n g alterations i n carotid sinus pressure. To  explain  why  plasma  concentrations  of immunoreactive  unchanged d u r i n g decreased carotid sinus pressure  in the  vasopressin  rabbits  were  w i t h intact aortic  baroreceptors, the different inputs to the carotid sinus and aortic baroreceptors m u s t be considered.  A l t h o u g h the  protocol of this experiment involved both decreasing  and  increasing carotid sinus pressure, the discussion of the immunoreactive vasopressin results w i l l relate to decreasing carotid sinus pressure. nerves  decreased  vasopressin. pressure  carotid  sinus  pressure  In rabbits w i t h intact aortic depressor  reduced  the  inhibition  of the  release  of  Decreased carotid sinus pressure also caused a reflex increase i n a r t e r i a l  and therefore  vasopressin release.  stimulated the aortic baroreceptors increasing the inhibition of  Since these two inputs had opposing effects, there was no change i n  i m m u n o r e a c t i v e vasopressin.  After section of the aortic depressor nerves the stimulus to  release immunoreactive vasopressin r e s u l t i n g from decreased carotid sinus baroreceptor stimulation  was  unopposed and, therefore,  p l a s m a immunoreactive vasopressin  rose  sharply. It is interesting to note the a m o u n t of total afferent input from necessary  to  inhibit  the  release  of  immunoreactive  vasopressin.  baroreceptors In  the  aortic  129  barodenervated rabbits (zero i n p u t from aortic baroreceptors) i t w a s only after carotid sinus  pressure  fell  to  lOOmmHg  that  i m m u n o r e a c t i v e vasopressin  started  to  rise  appreciably (figure 9). I n the anaesthetized rabbit, carotid sinus baroreceptor activity is at a m a x i m u m at a carotid sinus pressure of 140-160 m m H g and at about 70% of m a x i m a l at carotid sinus pressure of 100 m m H g (Holmes and Ledsome 1984) T h i s indicated the total baroreceptor afferent activity should be decreased to about 70% of m a x i m a l carotid sinus  input  and  minimal  aortic baroreceptor  input before  a  significant release  of  immunoreactive vasopressin i n the rabbit w a s observed. Afferent fibres from aortic chemoreceptors also r u n i n the aortic depressor nerves of  some  species  and  the  possibility  existed  that  some  of  the  buffering  of  the  immunoreactive vasopressin response w a s due to changes i n chemoreceptor s t i m u l a t i o n . T h i s is u n l i k e l y i n this preparation since the i n s p i r a t o r y gas was supplemented w i t h 100% oxygen.  T h i s exposure to o x y g e n m a i n t a i n e d P a 0 2 at levels i n excess of 200 m m H g  throughout the experiment. A t this level of PaC-2 significant chemoreceptor s t i m u l a t i o n w a s u n l i k e l y to occur (Hornbein 1968).  I n addition, few (if any), chemoreceptor fibres  originate from the aortic a r c h of the rabbit ( C h a l m e r s et a l . 1967). It w a s surprising, that while the vasopressin s y s t e m w a s completely buffered, there seemed to be m u c h less buffering effect on the reflex responses of M A P to changes i n carotid sinus pressure.  F o r the sake of c l a r i t y the discussion of the a r t e r i a l pressure  responses w i l l deal w i t h reflex response of M A P to increases i n carotid sinus pressure.  In  the rabbits w i t h intact aortic depressor nerves increasing the carotid sinus pressure from 40 m m H g to 80 m m H g caused a decrease i n the M A P (figure 11).  I n the rabbits w i t h  sectioned aortic depressor nerves the carotid sinus pressure h a d to be increased from 80 m m H g to 100 m m H g before any appreciable fall i n M A P w a s seen. barodenervated rabbits h a d no input from the aortic baroreceptors afferent i n p u t from carotid sinus baroreceptors  (CSP = 40mmHg).  These aortic  and only m i n i m a l Consequently there  would be little baroreceptor restraint of efferent sympathetic vasoconstrictor tone and  130  there  would  be  maximal  sympathetic  vasoconstriction.  The  relationship  between  sympathetic activity and vasoconstriction has been s h o w n to be non-linear (Mellander and J o h a n s s o n 1968).  Therefore, i n the present study, a significant increase i n baroreceptor  restraint of sympathetic activity would have been needed before there w a s a decrease i n vasoconstriction.  T h u s carotid sinus pressure  h a d to be increased to greater  8 0 m m H g before there is a decrease i n a r t e r i a l pressure.  than  I n the rabbit w i t h intact aortic  baroreceptors the reflex increase i n a r t e r i a l pressure i n response to a decrease of carotid sinus pressure to 40 m m H g would cause a n increase i n activity from aortic baroreceptors. Increased aortic baroreceptor activity would prevent m a x i m u m sympathetic activity and m a x i m u m vasoconstriction. Therefore a n increase i n carotid sinus pressure from 40 to 80 m m H g would have been likely to cause a w i t h d r a w a l of sympathetic a c t i v i t y sufficient to decrease a r t e r i a l pressure. pressure,  These differences were observed w h e n the changes i n a r t e r i a l  i n a l l 19 rabbits, were plotted before a n d after  depressor nerves and the vagus nerves (figure 11). pressure of 4 0 m m H g w a s less intact,  compared  with  section of both the aortic  A r t e r i a l pressure at a carotid sinus  w h e n the vagus nerves and aortic depressor nerves were  when  they  were  cut,  providing  evidence  that  maximal  vasoconstriction w a s not present. It has been reported i n other species that the threshold carotid sinus  pressure  required to induce changes i n heart rate w a s higher t h a n t h a t carotid sinus pressure needed to induce changes i n M A P (Bolter and Ledsome 1976).  T h i s w a s apparent i n our  experiments (figure 12) i f the pattern of the H R response at increasing levels of carotid sinus pressure w a s examined.  T h e largest consistent change i n H R occurred between  carotid sinus pressure of 100 m m H g a n d 120 m m H g .  I n contrast the first consistent  change i n M A P (in the range from 40 m m H g to 160 m m H g ) occurred between carotid sinus pressure of 40 m m H g and 80 m m H g i n the rabbits w i t h intact aortic depressor nerves  and  between  barodenervated.  80 m m H g  and  100 m m H g  i n the  rabbits  which  were  aortic  A change i n the threshold for changes i n heart rate after section of either  131  the vagus nerves or the aortic depressor nerves could not be demonstrated.  N e i t h e r was  there a n y change i n the pattern of the changes i n H R w i t h alterations i n carotid sinus pressure after section of the vagus nerves or aortic depressor nerves.  T h i s indicated t h a t  the reflex decrease i n heart rate was not likely to be mediated b y a n increase i n v a g a l efferent a c t i v i t y , but more likely to be due to a w i t h d r a w a l of cardiac sympathetic activity. There appeared to be v e r y little tonic v a g a l control of heart rate i n the rabbit.  anaesthetized  T h i s was i n contrast to the findings i n the dog i n w h i c h there was a significant  tonic v a g a l control over the heart rate (Thames and S c h m i d 1979). R i g h t a t r i a l pressure w a s unchanged b y either changes i n carotid sinus pressure or b y the presence or absence of the aortic depressor or vagus nerves. There was a s t r i k i n g difference between the changes i n p l a s m a vasopressin w i t h changes i n carotid sinus pressure and those of M A P (figures 10, 11).  W i t h intact aortic  baroreceptors changes i n carotid sinus pressure had no effect on p l a s m a vasopressin but there were significant changes i n a r t e r i a l pressure. there were significant  In the absence of aortic baroreceptors  changes i n the p l a s m a concentration of vasopressin w h e n carotid  sinus pressure was raised from 40 to 80 m m H g but no changes i n m e a n a r t e r i a l pressure. Maximum  inhibition of p l a s m a vasopressin release  was achieved at a carotid sinus  pressure of 120 m m H g whereas r a i s i n g the carotid sinus pressure to 160 m m H g had further effects on a r t e r i a l pressure.  These findings emphasize the quantitative differences  i n the m e c h a n i s m s w h i c h control the release of vasopressin and the sympathetic outflow. Maximal baroreceptor decreased  inhibition  input.  of vasopressin release  T h i s was  likely  i n m e a n a r t e r i a l pressure  vasopressin.  to m e a n  was  achieved w i t h  relatively little  that d u r i n g haemorrhage  were likely to be necessary  significant  to increase  plasma  A l s o i f vasopressin was already m a x i m a l l y inhibited at n o r m a l a r t e r i a l  pressure there would be little further inhibition w i t h volume expansion. It appears that i n the  rabbit the m a x i m a l inhibition of vasopressin release  that can be achieved w i t h  baroreceptor s t i m u l a t i o n did not decrease p l a s m a vasopressin below measurable values.  132  Since there were some rabbits i n the present study i n w h i c h p l a s m a vasopressin did decrease to such low values this m a y have been a function of the p l a s m a osmolality together w i t h baroreceptor s t i m u l a t i o n i n this series of experiments.  T h e influence of  p l a s m a osmolality on p l a s m a vasopressin w a s not examined i n these experiments. There w a s no evidence i n the rabbit of a tonic inhibition of immunoreactive vasopressin release b y afferents i n the vagus nerves since v a g a l section did not cause a significant increase i n immunoreactive vasopressin even after aortic nerve section and at low carotid sinus pressure.  These findings were i n contrast to those i n the dog. Section of  the vagus nerves failed to alter p l a s m a immunoreactive vasopressin i n these experiments, whereas section of the vagus nerves i n barodenervated dogs caused a significant rise i n p l a s m a concentration of vasopressin (Thames and Schmid, 1979).  T h e technique of E d i s  and Shepherd (1971) used b y T h a m e s a n d S c h m i d (1979) to identify and section the aortic depressor nerve i n the dog, cannot guarantee that a l l of the aortic baroreceptor fibres i n the vagus nerves were sectioned. It is possible that there were r e s i d u a l aortic baroreceptor fibres  in  the  vagus  nerves  which  could  have  contributed  to  the  suppression  of  immunoreactive vasopressin release, w h i c h w a s removed w h e n the vagus nerves were cut i n these dogs. H o w e v e r , since i t w a s clear t h a t i n the dog afferent inpulses from left a t r i a l receptors inhibited the release of immunoreactive vasopressin (Ledsome et a l . 1983) it w a s reasonable to suppose that i n the dog there w a s some tonic inhibition of immunoreactive vasopressin release attributable to v a g a l afferent fibres.  Nevertheless even i n the dog,  cooling the vagus nerves to block m y e l i n a t e d afferent, fibres from a t r i a l receptors has only m i n i m a l effects on p l a s m a vasopressin (Ledsome et a l . 1983, Bennett et a l . 1983).  BLOOD V O L U M E CHANGES A N D VASOPRESSIN  Vasopressin Response to Haemorrhage: In  previous  studies  haemorrhage  has  been  shown  to  increase  the  plasma  133  concentration of vasopressin i n a v a r i e t y of species ( H e n r y et a l . 1968, Hock et a l . 1984, Sved et a l . 1985, L e d s o m e et a l . 1985, R a n k i n et a l . 1986). T h e contributions made b y volume sensitive receptors to this response were usually thought to include the high pressure sino-aortic baroreceptors and the low pressure a t r i a l receptors.  In the present  study i n p u t from the carotid sinus baroreceptors w a s eliminated b y perfusing the sinuses at constant pressure.  I n this w a y a distinction could be made between the aortic a r c h  baroreceptors and the a t r i a l receptors i n the vasopressin response to haemorrhage. ' Haemorrhage  i n the  vasopressin to be increased.  rabbits  with  intact aortic baroreceptors  T h e increase i n vasopressin i n response to haemorrhage of  20% of the blood volume before v a g a l section after v a g a l section.  caused p l a s m a  w a s not different from the increase seen  I n addition, i n rabbits without input from aortic baroreceptors and  constant carotid sinus pressure, decreases i n blood volume did not cause any significant increases i n p l a s m a vasopressin either before or after bilateral v a g o t o m y . T h i s suggested t h a t the increase i n vasopressin secondary to haemorrhage w a s not due to w i t h d r a w a l of afferent input from a t r i a l receptors but w a s more likely to be due to w i t h d r a w a l of afferent input from aortic baroreceptors.  These results were inconsistent w i t h w h a t has been  shown i n dogs and cats ( H e n r y et a l . 1968, H o c k et a l . 1984, S v e d et a l . 1985, Ledsome et al. 1985) where the l o w pressure a t r i a l receptors appeared to be p a r a m o u n t i n the release of vasopressin d u r i n g haemorrhage. In primates (in contrast to dogs a n d cats), haemorrhage did not cause a release of vasopressin unless M A P fell, thus i m p l i c a t i n g sino-aortic baroreceptors rather t h a n low pressure receptors (Goetz et a l . 1974, A r n a u l d et a l . 1977, G i l m o r e et a l . 1982).  These  observations were not limited to primates since since similar results have been shown i n conscious goats ( L a r s s o n et a l . 1977). In a recent study, R a n k i n et a l . (1986) reported that i n the rabbit, haemorrhage of 10% of the blood volume could cause a n increase in p l a s m a vasopressin after section of the carotid sinus, aortic depressor a n d vagus nerves.  This release of vasopressin following  134  buffer nerve section has also been shown i n cats (Clark and S i l v a 1967), r a t s (Ginsburg and  Brown  1956) and dogs (Chien and U s a m i  1974).  These studies suggested  that  w i t h d r a w a l of input from receptors, other t h a n those w i t h afferents i n the above nerves, were capable of s t i m u l a t i n g vasopressin release  after  haemorrhage.  I n the  present  experiments changes i n vasopressin i n rabbits w i t h sectioned aortic depressor nerves and vagus nerves were not observed. C a r o t i d sinus pressure w a s m a i n t a i n e d constant at 100 m m H g i n these rabbits therefore it was possible that the carotid sinus baroreceptors were capable of o v e r r i d i n g a n y stimulus for the release of immunoreactive vasopressin caused by other volume sensitive receptors.  Vasopressin Response to Volume Expansion: In previous experiments performed on anesthetized dogs it w a s demonstrated that volume  expansion  (+ 4  to  +20  mL/kg)  produced  significant decreases  in  plasma  vasopressin and there w a s a linear correlation between the change i n blood volume and the l o g a r i t h m of p l a s m a vasopressin (Ledsome et a l . 1985).  These decreases i n p l a s m a  vasopressin i n response to a n increase i n blood volume i n the dog were likely to have been the result of increased input from a t r i a l and a r t e r i a l receptors.  I n the rabbits used i n the  present experiments, i n the absence of aortic baroreceptor input, volume expansion did not induce a n y changes in the p l a s m a concentration of vasopressin suggesting t h a t i n this preparation  increased  input  vasopressin concentration.  from  atrial  receptors  was  not  capable  of  decreasing  V o l u m e expansion i n the rabbits, w i t h intact aortic depressor  nerves, decreased the p l a s m a concentration of vasopressin only after the vagus nerves were cut. A l t h o u g h the increase i n a r t e r i a l pressure i n response to volume expansion w a s s i m i l a r before and after vagotomy, a r t e r i a l pressure was higher after vagotomy.  An  appropriate level of a r t e r i a l pressure m a y have to be achieved before the additional aortic baroreceptor input had a significant effect on vasopressin release.  These results support  the hypothesis that the decrease i n vasopressin due to volume expansion w a s not caused  135  by s t i m u l a t i o n of a t r i a l receptors.  Cardiovascular Responses to Blood Volume Changes The  data reported i n chapter 2 showed t h a t haemorrhage consistently decreased  a r t e r i a l pressure and r i g h t a t r i a l pressure, whereas volume expansion increased a r t e r i a l pressure and right a t r i a l pressure. The  magnitude of the changes i n a r t e r i a l pressure  and r i g h t a t r i a l  pressure  m e a s u r e d 10 minutes after haemorrhage were unaffected b y b i l a t e r a l vagotomy or b y aortic barodenervation (tables I & I V ) .  T h i s w a s demonstrated i n figure 18 (bottom panel)  by the fact that M A P w a s restored to pre-haemorrhage levels w i t h i n 10 minutes i n rabbits w i t h no aortic baroreceptors or a t r i a l receptors.  T h i s recovery could have been due to  restoration of volume from the extracellular space, a change i n v a s c u l a r capacitance or buffering b y other receptors. One  minute  after  the  haemorrhage,  however,  right  atrial  pressure  was  significantly lower i n the aortic barodenervated rabbits (table V ) . T h i s suggests t h a t the aortic baroreceptors m a y contribute to the i m m e d i a t e buffering of a t r i a l pressure although it w a s not possible to demonstrate a significant effect of aortic baroreceptors on the immediate buffering of M A P .  The p a r t i a l recovery of R A P at 10 minutes, seen i n the  aortic barodenervated rabbits w a s further support for the hypothesis that mechanisms independent of baroreceptors were contributing to recovery from haemorrhage. H a e m o r r h a g e i n the rabbits w i t h intact aortic depressor nerves was accompanied by  significant elevations of vasopressin whereas i n the rabbits w i t h sectioned aortic  depressor nerves there w a s no change i n vasopressin d u r i n g haemorrhage.  Since the level  of M A P measured 10 minutes after haemorrhage w a s s i m i l a r i n rabbits w i t h and without intact aortic depressor nerves  (table I, I V ) this casts  doubt on the  role played b y  vasopressin as a pressor agent i n the recovery of M A P after haemorrhage. V a s o p r e s s i n has been shown to help m a i n t a i n M A P under conditions of blood  136  volume depletion (Laycock et a l . 1979, P a n g et al. 1983), however, there were several differences between these studies and the present study.  L a y c o c k et a l . (1979)  were  c o m p a r i n g the M A P change w i t h haemorrhage between Brattleboro (vasopressin deficient) rats and L o n g E v a n s control rats. and the  p l a s m a vasopressin  The fall i n M A P d u r i n g haemorrhage was attenuated  concentration  (measured  by bioassay)  was significantly  greater i n the L o n g E v a n s r a t s as compared to the Brattleboro rats. P a n g et a l . (1983) demonstrated  that administration of a vasopressin  antagonist  ^(CH^gTyKMeJAVP)  altered the regional distribution of cardiac output w h i c h accompanied haemorrhage of 20% of the blood volume i n anaesthetized rats. T h e y concluded that vasopressin contributed to the maintenance  of M A P and blood flow distribution after  haemorrhage.  (1983)  measure  in  did  not  plasma  levels  of  vasopressin  the  rats  P a n g et a l . during  these  haemorrhages. A d m i n i s t r a t i o n of vasopressin i n n o r m a l conscious a n i m a l s did not significantly elevate M A P unless the p l a s m a vasopressin concentration w a s supraphysiological (Cowley 1985).  I n the present study haemorrhage of 20% and 30% of the blood volume increased  p l a s m a vasopressin  concentration to 40 p g / m L .  It w a s  unlikely that the  levels of  vasopressin achieved b y haemorrhage i n these rabbits were sufficient to contribute to a n elevation of M A P . N e i t h e r haemorrhage nor volume expansion had a n y effect on heart rate.  Heart  rate was unaffected by bilateral vagotomy suggesting that i n the anesthetized rabbit w i t h a constant carotid sinus pressure of 100 m m H g there was m i n i m a l tonic v a g a l inhibition of heart rate. T h i s was i n agreement w i t h the results from the d a t a reported i n C h a p t e r 1.  Interaction Between High and Low Pressure Receptors T h a m e s and S c h m i d (1981) were the first to clearly indicate a n interaction between the high a n d low pressure receptors i n the release of vasopressin.  I n anaesthetized dogs  T h a m e s and S c h m i d (1981) showed that the increased release of vasopressin i n response  137  to v a g a l cold block w h i c h w a s present  at carotid sinus pressure  of 50 m m H g ,  was  abolished b y concomittant moderate carotid sinus baroreceptor s t i m u l a t i o n . W h e n carotid sinus baroreceptors  were m a x i m a l l y  s t i m u l a t e d d u r i n g v a g a l cold block the  concentration of vasopressin w a s decreased.  These experiments suggested  plasma that  the  ultimate release of vasopressin w a s the result of a n interaction between afferent inputs from a t r i a l receptors and carotid sinus baroreceptors.  F u r t h e r support for this hypothesis  came from S h a r e (1965) who reported t h a t left a t r i a l distension i n dogs abolished the rise i n vasopressin concentration w h i c h accompanied carotid occlusion.  Share (1965) did not  measure the carotid sinus pressure d i s t a l to the occlusion therefore it w a s impossible to quantify the relationship between carotid sinus pressure concentration.  and the p l a s m a vasopressin  A l t h o u g h T h a m e s and S c h m i d (1981) measured the carotid sinus pressure  i n their preparation the changes i n carotid sinus pressure were large (from 50 m m H g to 135 m m H g and from 50 m m H g to 200 m m H g ) therefore one could not describe the precise interaction between  the two stimuli for vasopressin release.  T h e results from  the  experiments presented i n Chapter 3 were designed to investigate the relationship between k n o w n steady  state levels of carotid sinus pressure  designed to unload the a t r i a l receptors.  and two levels of haemorrhage  T h e aortic baroreceptor i n p u t w a s eliminated b y  sectioning the aortic depressor nerves i n a l l the rabbits.  Results of these studies showed  t h a t m i l d haemorrhage (10% blood volume) w a s ineffective i n s t i m u l a t i n g a release of vasopressin i n aortic barodenervated rabbits even at a carotid sinus pressure of 60 m m H g . T h i s demonstrated  t h a t i n rabbits w i t h m i n i m a l inhibition of vasopressin release b y  sinoaortic baroreceptors,  mild  unloading of the  a t r i a l receptors  could not cause  an  increased release of vasopressin. I n several species i t has been shown t h a t haemorrhage did not increase vasopressin unless greater t h a n  10% of the blood volume had been  removed ( H e n r y et a l . 1968, Goetz et a l . 1974, L a r s s o n et a l . 1978).  In conscious goats  haemorrhage of 12 m L / k g (10% of the B V ) did not change p l a s m a vasopressin whereas haemorrhage of 16 m L / k g resulted i n significant increases i n vasopressin (Larsson et a l .  138  1978). H e n r y et a l . (1968) demonstrated that non-hypotensive haemorrhage (10% of the blood volume) significantly increased p l a s m a vasopressin i n only 3 out of 11 dogs.  In  h u m a n s there was no measureable change i n blood pressure or p l a s m a vasopressin w h e n 10% of the blood volume w a s removed (Goetz et a l . 1974).  P r e v i o u s l y R a n k i n et a l .  demonstrated that haemorrhage of 10% of the blood volume caused significant release of vasopressin i n the anaesthetized rabbit.  These experiments differed from the  present  experiments i n that the rabbits had intact carotid sinuses rather t h a n carotid sinuses perfused at constant pressure.  Since unloading of the carotid sinus baroreceptors  can  increase the p l a s m a concentration of vasopressin (Share and L e v y 1962, C l a r k and S i l v a 1967) perhaps the additional reduction i n the s t i m u l u s from carotid sinus baroreceptors i n these intact rabbits augmented the response of p l a s m a vasopressin to haemorrhage of 10 % of the blood volume. H a e m o r r h a g e of 2 0 % of the blood volume caused variable (and not statistically significant) increases i n vasopressin i n the aortic barodenervated rabbits. T h i s was true at carotid sinus pressure baroreceptors haemorrhage.  of 6 0 m m H g and 120 m m H g indicating that the carotid sinus  were not e x e r t i n g a n inhibitory effect on a n y vasopressin response It was expected that at low C S P and i n aortic barodenervated  to  rabbits,  conditions would be o p t i m a l for release of vasopressin i n response to haemorrhage.  The  absence of consistent changes i n vasopressin under these conditions m a k e it unlikely that a t r i a l receptors were contributing to the inhibition of release of vasopressin. findings  were  contrary  to  the  findings of T h a m e s  and  Schmid  (1981).  These Although  statistically there was no change i n vasopressin the large increase i n vasopressin seen i n a few animals suggested the presence of a non-vagal, high threshold m e c h a n i s m for the release of vasopressin after haemorrhage.  N o additional evidence for the existence of such  a m e c h a n i s m was seen w h e n blood volume w a s reduced b y 30% i n rabbits w i t h carotid sinus pressure at 60 m m H g (Chapter 4).  Receptors sensitive to changes i n blood volume  have been found i n the mesenteric ( G a m m o n and B r o n k 1935), renal ( K o s t r e v a et a l .  139  1981) and hepatic ( L a u t t 1983) circulation.  There was no direct evidence that these  volume sensitive receptors could alter the p l a s m a concentration of vasopressin, however, some electrophysiological studies have indicated that stimulation of afferent fibres i n the r e n a l nerves could alter the a c t i v i t y of neurosecretory neurons i n the supraoptic nuclei (Calaresu and Ciriello 1981).  T h i s w a s indirect evidence t h a t receptors w i t h afferents i n  the r e n a l nerves could control vasopressin release.  Subsequent studies, however, have  suggested t h a t the stimulus for activation of these r e n a l afferents is l i k e l y to be chemical i n nature ( D a y and Ciriello 1985). The results from the experiments i n C h a p t e r 3 have shown t h a t volume expansion of neither 10% nor 20% of the blood v o l u m e altered p l a s m a concentration of vasopressin i n the aortic barodenervated rabbits. B a c k g r o u n d carotid sinus pressure had no effect on this response to volume expansion. I n C h a p t e r 2 it w a s shown t h a t i n rabbits w i t h sectioned vagus  nerves  and  carotid  sinus  pressure  held  at  100  mmHg,  volume  expansion  significantly decreased vasopressin w h e n the aortic depressor nerves were intact.  This  suppression of vasopressin was attributed to loading of the aortic baroreceptors since it could be abolished by sectioning the experiments  seen  in  Chapter  3  aortic depressor  would  support  the  nerves.  T h e results from  contention  that  intact  the  aortic  baroreceptors were necessary for the suppression of vasopressin i n response to volume expansion since i n aortic barodenervated rabbits, despite increased s t i m u l i from a t r i a l receptors, there was no change i n vasopressin d u r i n g volume expansion. The increased vasopressin associated w i t h haemorrhage was thought to be due to w i t h d r a w a l of afferent input from sinoaortic baroreceptors rather t h a n a t r i a l receptors i n h u m a n and non-human primates ( A r n a u l d et a l . 1977, G i l m o r e et a l . 1982).  When  haemorrhage was not accompanied b y significant decreases i n a r t e r i a l pressure there w a s no change i n p l a s m a vasopressin i n both monkeys and m a n (Goetz et a l . 1974, Gilmore and Z u c k e r 1980, Goldsmith et a l . 1984). A s was mentioned earlier i n the discussion, dogs and cats depend more on low pressure receptors i n the control of vasopressin release.  140  Zucker and G i l m o r e (1975) demonstrated  the presence  of stretch or volume sensitive  receptors i n the left a t r i a of monkeys. T h e y also showed that i n comparison w i t h the dog, the receptors i n the a t r i a of the monkey were significantly less sensitive to changes i n a t r i a l pressure.  T h i s difference i n receptor sensitivity between dogs and p r i m a t e s m a y  have been due to a shift from quadrupedal to u p r i g h t or semi-upright posture as postulated by Gilmore et al. a t r i a , i n the sensitivity.  (1980).  m o n k e y , as  A n alternate explanation m a y be that the s m a l l e r size of the compared  to the  dog, w a s  responsible  for the  decreased  H i c k s et al. (1986) showed there w a s a n inverse relationship between a t r i a l  volume receptor sensitivity and left a t r i a l size. T h e present experiments have shown t h a t i n rabbits without changing input from aortic baroreceptors,  volume expansion  did not change  vasopressin either at a high or low carotid sinus pressure.  the  p l a s m a concentration of  Therefore i n this preparation  there did not appear to be a significant interaction between the a t r i a l receptors and the carotid sinus baroreceptors i n the control of p l a s m a vasopressin.  Atrial Receptors, Aortic Baroreceptors and Vasopressin Release In the experiments reported i n C h a p t e r 4 a n attempt was made to identify a role for  the  a t r i a l receptors  i n the  response  haemorrhage (30% of the blood volume).  of p l a s m a  vasopressin  to  a  more  severe  I n rabbits w i t h intact aortic baroreceptors  and  m i n i m a l input from carotid sinus baroreceptors ( C S P 60 m m H g ) the vasopressin response to haemorrhage  w a s compared i n rabbits w i t h and without input from a t r i a l receptors.  H a e m o r r h a g e increased p l a s m a concentration of vasopressin before and after section of the vagus nerves, however the elevation of vasopressin following vagotomy was attenuated. T h i s agreed w i t h the results obtained by W a n g et a l . (1983) who found that i n conscious dogs the increased release of vasopressin i n response to haemorrhage attenuated following cardiac denervation.  was significantly  Since cardiac denervation interrupted  afferent  fibres from both the a t r i a and ventricles W a n g et a l . (1986) repeated the experiment, but  141  this time they denervated only the ventricles. T h e increase i n vasopressin i n response to haemorrhage was significantly attenuated  as compared to s h a m operated control dogs.  T h e y did not speculate on the anatomical or physiological characteristics of the proposed v e n t r i c u l a r receptors.  It has been well documented t h a t there are ventricular receptors  w h i c h are localized to the posterior w a l l of the left ventricle and are stimulated b y intracoronary injection of v e r a t r i n e (Zucker et a l . 1983) and cryptenamine (Thames et a l . 1980), howevwe, s t i m u l a t i o n of these veratrine sensitive receptors caused an attenuation of the rise i n vasopressin elicited b y haemorrhagic hypotension. Therefore, these receptors could not have been responsible for the attenuated rise i n vasopressin seen i n the study reported b y W a n g et a l . (1986). In the present study there were two possible explanations for the attenuation of the haemorrhage induced rise i n vasopressin w h i c h followed v a g a l section. Receptors w i t h afferents i n the vagus nerve m a y have been activated b y haemorrhage increased release of vasopressin.  and caused a n  R e m o v a l of the vagus nerves would have removed this  s t i m u l a t o r y input and thereby attenuated the response. These receptors m i g h t have been located i n the ventricles ( W a n g et a l . 1986) or possibly i n other v a s c u l a r beds w h i c h have receptors w i t h v a g a l afferents.  Gattone et a l . (1986) have s h o w n t h a t w h e n horseradish  peroxidase w a s injected into the kidney it was transported to the nodose ganglion i n the rat.  This suggested that receptors i n the kidney have n e u r a l connections w i t h i n the vagus  nerve. D a y and Ciriello (1981) have s h o w n that stimulation of chemosensitive receptors i n the kidney significantly increased the firing rate of single units recorded i n the supraoptic nuclei of the h y p o t h a l a m u s , thus implicating these r e n a l receptors i n the release of vasopressin.  There was no evidence that these renal receptors could be activated b y a  physiological stimulus and therefore their involvement i n the present study remained only speculative. An haemorrhage  alternative  explanation  for  the  attenuated  vasopressin  response  to  stems from the evidence showing that unloading of the a t r i a l receptors  142  caused increased p l a s m a concentration of vasopressin i n dogs. afferents  e a r r i n g information from a t r i a l receptors  stimulated release  of vasopressin.  Therefore r e m o v a l of  could attenuate the  haemorrhage  A l t h o u g h this was a possible explanation for our  findings it w a s unlikely , since none of the other experiments described i n this w o r k provided evidence w h i c h could lead to a change dependent on v a g a l afferents.  i n p l a s m a vasopressin  which  was  143  Role of Vasopressin in Cardiovascular Control T h e experiments reported i n this thesis have demonstrated t h a t the carotid sinus baroreceptors  and  aortic  baroreceptors  vasopressin i n the anaesthetized rabbit.  contribute  to  the  control of the  release  of  N o evidence was obtained for a contribution to  vasopressin control by the a t r i a l receptors therefore this section of the discussion dealt w i t h the relationship between the sinoaortic baroreceptors and vasopressin release. It w a s shown that r e l a t i v e l y low total baroreceptor input w a s required to achieve m a x i m a l inhibition of vasopressin release.  A t the p l a s m a osmolality present i n the  experiments described (300-310 mosmol/kg) m a x i m u m baroreceptor inhibition decreased p l a s m a vasopressin to a p p r o x i m a t e l y 10 p g / m L .  Therefore i n the conscious rabbit,  at  n o r m a l a r t e r i a l pressure (80-100 m m H g ) it is likely that baroreceptor inhibition of the release of vasopressin would be almost m a x i m a l and volume expansion would not be expected to produce a n y further inhibition of vasopressin.  D u r i n g haemorrhage  arterial  pressure w o u l d have to be reduced b y a large amount before a n y elevations i n vasopressin would be seen. T h e daily fluctuations i n blood volume or blood pressure w h i c h m i g h t occur in a n a n i m a l are probably not sufficient to stimulate pronounced changes i n the level of vasopressin i n the conscious rabbit.  I n the present experiments vasopressin rose to 40  p g / m L i n response to haemorrhage of 30% of the blood volume. L e v e l s of vasopressin of this magnitude would not be expected to elevate M A P , especially i n the presence of intact arterial baroreceptors (Cowley, 1982). I n light of these observations vasopressin m a y not be involved i n buffering falls i n arterial pressure unless the reduction i n a r t e r i a l pressure results i n a pronounced rise i n vasopressin. In future experiments it would be of interest to examine the relationship between arterial baroreceptor activity and p l a s m a vasopressin at a lower p l a s m a osmolality. A t a lower p l a s m a osmolality, p l a s m a vasopressin concentration should be reduced to the range in w h i c h changes i n u r i n a r y concentration m i g h t be expected, that is 1-6 p g / m L , (Bie, 1980).  Changes i n a r t e r i a l baroreceptor a c t i v i t y m i g h t then influence the excretion of  144  water b y the kidney and thus contribute to body fluid volume control. I n the n o r m a l rabbit such a m e c h a n i s m m a y be of more importance t h a n a direct role of vasopressin i n the control of M A P through a change i n vascular resistance and capacitance. Despite the lack of a contribution towards the n o r m a l control of M A P , vasopressin has been shown to p l a y a n important role i n the development and maintenance of some forms of experimental hypertension Krukoff  and  Calaresu  1984,  ( M o h r i n g et a l . 1977, Share  Brooks et  al.  1985,  Chiu  and  and Corfton  McNeill  1984,  1985).  mechanisms behind the role of vasopressin i n these pathophysiological conditions u n k n o w n but m a y involve sensitization of the baroreceptors  The are  (Cowley et al 1974), or a n  interaction w i t h central cardiovascular neurons ( K r u k o f f and C a l a r e s u 1984), or increased v a s c u l a r responsiveness to vasopressin (Hoffman et a l . 1977).  145  REFERENCES A a r s , H . 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