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Renal hypersensitivity to vasopressin in congestive heart failure : significance of endothelin receptor… Wong, Bonita P. H. 1997

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RENAL HYPERSENSITIVITY TO VASOPRESSIN IN CONGESTIVE HEART FAILURE: SIGNIFICANCE OF ENDOTHELIN RECEPTOR REGULATION IN THE INNER MEDULLARY COLLECTING DUCT by BonitaPH. Wong B.Sc, The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1997 © Bonita PH. Wong, 1997 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 M£D/C/A/£I The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Plasma vasopressin (AVP) and endothelin (ET) levels are often elevated in congestive heart failure (CHF) and have been linked to edema formation commonly observed in this clinical setting. The mechanisms by which these two hormones contribute to edema remain obscure. In vivo and in vitro studies were designed to further understand the role of AVP in CHF and to examine its interaction with ET receptors. Clearance studies were performed to compare the renal excretory function in UM-X7.1 cardiomyopathic (CM) hamsters (280 to 300 days old) and age-matched healthy controls. Both groups had well-maintained glomerular filtration rates throughout the experiments. Exogenous administration of AVP (0.3 ng-kg'^ min"1) had no effect on any of the measured clearance parameters in the CM animals but markedly reduced the fractional excretion of sodium ( F E N A ) and water (FEH2O) in the controls by 40 and 46%, respectively. Combined infusion of a V i antagonist and a V2 agonist at the same dose similarly decreased F E N A and FEH20 in the healthy animals. However, the CM group exhibited an attenuated response in all of the measured hemodynamic and clearance parameters even though their cAMP production was five-fold higher than that of normal animals. Additional studies support the notion that basal salt and water reabsorption in CHF was maximal, which would account for the lack of response to infusions of AVP or combined Vi antagonist and V2 agonist. Nonselective blockade with the Vi and V2 antagonists 0.3 ng-kg^ -min"1 produced natriuresis and diuresis in the CM hamsters ( F E N A : 4.8 + 0.6 vs. 7.9 + 1.1%, p<0.05; FEH2O-' 1.5 + 0.2 vs. 2.2 + 0.3%, p<0.05) but did not affect fluid reabsorption in the normal hamsters. Profuse diuresis in the diseased animals may be partially attributed to an alteration in V 2 ii receptor signaling as reflected by decreased urinary cAMP levels. Hence, increased basal cAMP synthesis in the kidney potentially impairs salt and water excretion in the pathophysiological state. Altered regulation of other hormonal systems might have contributed to the apparent AVP hypersensitivity in decompensated heart failure. Previous studies have indicated E T inhibits the actions of AVP within the kidney. Whether the reverse relationship exists and what the implications are in CHF have not been investigated extensively. Accordingly, the mechanisms that reduce E T receptors ( E T A and E T B subtypes) in inner medullary collecting duct (IMCD) were determined to show how AVP controls E T function at the receptor level. Competitive binding experiments were performed to examine the effects of a AVP signaling pathway on E T receptor binding. Overnight incubation of rat IMCD cells with AVP significantly reduced the maximal binding capacity, B m a x , of E T . Activation of adenylate cyclase by forskolin decreased the total E T receptor binding and preferentially reduced E T B receptor density by -42% with no effect on the E T A subtype. Involvement of the PKA pathway in E T B receptor downregulation was strongly implicated by the observation that a cAMP analogue, Rp-cAMPS, blocked the inhibitory influence of AVP on ET-1 binding. The data further indicate that the IQ values of the E T receptors were decreased significantly and demonstrate that AVP reduced E T receptor density and increased the affinity of the existing receptors. Altogether, the competitive binding experiments suggest the following novel idea: AVP controls sodium and water reabsorption by activating its receptors and downregulating E T B receptors via a cAMP-dependent pathway. Since E T A and E T B subtypes have different actions, changes in their distribution in vivo would affect normal hemodynamics, natriuresis, and diuresis. Hence, AVP-induced heterologous regulation of E T B receptors may result in salt and water retention, exacerbating the condition of congestive heart failure. iii TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS ACKNOWLEDGMENTS CHAPTER 1 INTRODUCTION 1.1 Congestive Heart Failure 1.1.1 Initial adaptations to myocardial injury 1 1.1.2 Development of heart failure 3 1.1.3 Neurohumoral activation in congestive heart failure 4 Vasoconstrictor systems 4 Vasodilator systems 5 Loss of counterregulatory vasodilating mechanisms 6 1.1.4 Renal adaptations: salt and water retention 6 1.1.5 Consequences of excessive hemodynamic and neurohumoral activation 7 1.2 Vasopressin 1.2.1 Biosynthesis and degradation 10 1.2.2 Osmotic and non-osmotic regulation of vasopressin release 11 1.2.3 Vasopressin receptors 12 1.2.4 Role of V2 receptor in salt and water reabsorption 13 1.2.5 Regulation of hydroosmotic response to vasopressin 18 1.2.6 Vasopressin in congestive heart failure 19 1.3 Endothelin 1.3.1 Endothelin isopeptides and receptors 21 ii iv vii ix x iv 1.3.2 Regulation of endothelin receptors 22 1.3.3 Renal effects of endothelin 25 1.3.4 Endothelin in salt and water homeostasis 28 1.3.5 Endothelin in congestive heart failure 29 1.4 Functional interactions between vasopressin and endothelin 30 1.5 Hypothesis 34 1.6 Objectives and rationale 38 CHAPTER 2 MATERIALS AND METHODS 2.1 In Vivo Experimental Design 2.1.1 Animals 40 2.1.2 Clearance studies 42 Surgical preparation 42 Protocol 1: Time control 43 Protocol 2: Renal responses to exogenous vasopressin 43 Protocol 3: Renal responses to exogenous V\ antagonist and V2 agonist 43 Protocol 4: Renal responses to exogenous Vt and V2 antagonists 43 Blood and urine analysis 43 Inulin clearance: determination of glomerular filtration rate 44 2.2 In Vitro Experimental Design 2.2.1 Animals 44 2.2.2 Tissue preparation 44 Isolation of inner medullary collecting duct (IMCD) cells 44 Drug treatments of isolated IMCD cells 47 Preparation of homogenate 48 2.2.3 Competitive binding assays 48 Analytic methods 49 2.3 Reagents 49 2.4 Statistics 49 CHAPTER 3 RESULTS 3.1 In Vivo Studies 3.1.1 Time control studies 50 3.1.2 General characteristics of cardiomyopathic hamsters 50 3.1.3 Renal responses to exogenous vasopressin or a Vt antagonist with a V2 agonist 55 3.1.4 Renal responses to exogenous V; and Vi antagonists 62 3.1.5 Urinary cAMP measurements 62 3.2 In Vitro Studies 3.2.1 Time control studies 69 3.2.2 Effects of vasopressin and forskolin on endothelin-1 binding to IMCD 69 3.2.3 Effect of Rp-cAMPS on endothelin-1 binding to IMCD 75 3.2.4 Changes in endothelin receptor binding 80 CHAPTER 4 DISCUSSION 4.1 Cardiomyopathic hamsters as an experimental model of congestive heart failure 86 4.2 Systemic and renal hemodynamics 87 4.3 Clearance responses 90 4.4 Generation of adenosine 3', 5'-cyclic monophosphate in congestive heart failure 91 4.5 Heterologous regulation of endothelin receptors by vasopressin 93 4.6 Implications of endothelin receptor downregulation in congestive heart failure 95 4.7 Perspectives 97 4.8 Future directions 97 CHAPTER 5 SUMMARY 99 CHAPTER 6 CONCLUSIONS 101 REFERENCES 102 APPENDIX 117 vi LIST OF FIGURES Figure 1.1 Adaptive mechanisms that lead to salt and water retention in congestive 8 heart failure Figure 1.2 Role of V2 activation in water transport in the collecting duct 15 Figure 1.3 Proposed mechanism for vasopressin-induced docking of vesicles containing 17 aquaporin-2 at the luminal membrane of the collecting duct Figure 1.4 Endothelin-B receptor regulation 23 Figure 1.5 Sites of endothelin action along the nephron 26 Figure 1.6 Heterologous regulation of vasopressin and endothelin actions 3 1 Figure 1.7 Proposed mechanisms for vasopressin hypersensitivity in congestive heart 35 failure Figure 2.1 In vivo experimental design 45 Figure 2.2 In vitro experimental design 46 Figure 3.1 Comparison of heart weight/body weight in normal and cardiomyopathic 52 hamsters Figure 3.2 Renal function of normal and cardiomyopathic hamsters 53 Figure 3.3 Fractional excretion of water in cardiomyopathic hamsters 54 Figure 3.4 Relationship between renal function and fractional excretion of water in 56 cardiomyopathic hamsters Figure 3.5 Relationship between heart weight/ body weight ratio and basal urinary 57 cAMP levels in normal and cardiomyopathic hamsters Figure 3.6 Comparison of basal urinary cAMP levels in normal and cardiomyopathic 58 hamsters Figure 3.7 Fractional excretion of sodium following V 2 receptor activation with and 60 without a Vi antagonist in normal and cardiomyopathic hamsters Figure 3.8 Fractional excretion of water following V 2 receptor activation with and 61 without a Vi antagonist in normal and cardiomyopathic hamsters vii Figure 3.9 Fractional excretion of sodium following Vi and V2 receptor blockade in 65 normal and cardiomyopathic hamsters Figure 3.10 Fractional excretion of water following Vi and V 2 receptor blockade in 66 normal and cardiomyopathic hamsters Figure 3.11 Percent increase in urinary cAMP levels following selective V 2 activation in 67 normal and cardiomyopathic hamsters Figure 3.12 Percent decrease in urinary cAMP levels following V 2 blockade in normal 68 and cardiomyopathic hamsters Figure 3.13 Summary of the relative change in urinary cAMP levels following V 2 activation 70 and blockade in normal and cardiomyopathic hamsters Figure 3.14 Time control: effect of overnight incubation on endothelin receptors 71 Figure 3.15 Representative competitive displacement curve for 125I-ET-1 binding to IMCD 72 cells with vasopressin treatment Figure 3.16 Effect of vasopressin on total endothelin receptor density 73 Figure 3.17 Effect of vasopressin on endothelin receptor subtypes 74 Figure 3.18 Representative competitive displacement curve for 125I-ET-1 binding to IMCD 76 cells with forskolin treatment Figure 3.19 Effect of forskolin on endothelin receptor density 77 Figure 3.20 Effect of Rp-cAMPS on endothelin receptor density 78 Figure 3.21 Blockade of vasopressin-induced endothelin receptor regulation 79 Figure 3.22 Effects of vasopressin and forskolin on endothelin receptor affinity 81 Figure 3.23 Effects of vasopressin and forskolin on E T B receptor affinity 82 Figure 3.24 Effect of Rp-cAMPS on endothelin receptor affinity 83 Figure 3.25 Representative scatchard plot for ET B receptors in untreated and 84 vasopressin-treated inner medullary collecting duct cells Figure 3.26 Representative scatchard plot for E T B receptors in untreated and 85 forskolin-treated inner medullary collecting duct cells viii LIST OF TABLES Table 1.1 Causes of cardiac muscle dysfunction 2 Table 2.1 Grading system for severity of congestive heart failure in cardiomyopathic 41 hamsters Table 3.1 Hemodynamic and renal excretory function of normal golden Syrian hamsters 51 Table 3.2 Hemodynamic and renal excretory function of cardiomyopathic and control 59 hamsters in response to exogenous vasopressin Table 3.3 Hemodynamic and renal excretory function of cardiomyopathic and control 63 hamsters in response to exogenous Vi antagonist and V 2 agonist Table 3.4 Hemodynamic and renal excretory function of cardiomyopathic and control 64 hamsters in response to exogenous Vi and V 2 antagonists Table 7.1 Reported kinetic properties of endothelin receptors in the renal medulla 117 i x LIST OF ABBREVIATIONS ACE Angiotensin converting enzyme AG Agonist Angll Angiotensin II ANP Atrial natriuretic peptide ANT Antagonist AQP2 Aquaporin 2 ATL Ascending thin limb AVP Arginine vasopressin Bmax Maximal binding capacity cAMP Adenosine 3', 5'-cyclic monophosphate CCD Cortical collecting duct cGMP Guanosine 3', 5'-cyclic monophosphate CHF Congestive heart failure CM Cardiomyopathic DAG Diacylglycerol EDRF Endothelium-derived relaxing factor EDTA Ethylenediaminetetraacetic acid ET Endothelin E T A Endothelin receptor subytpe A E T B Endothelin receptor subtype B E T C Endothelin receptor subytpe C F E N 3 Fractional excretion of sodium FEH20 Fractional excretion of water GFR Glomerular filtration rate IMCD Inner medullary collecting duct n»3 Inositol 1,4,5-trisphosphate Dissociation constant K f Ultrafiltration coefficient MAP Mean arterial pressure X mRNA Messenger ribonuclei acid NDI Nephrogenic diabetes insipidus NE Norepinephrine NO Nitric oxide PDE Phosphodiesterase PGC Glomerular capillary pressure P f Osmotic water permeability coefficient PGE 2 Prostaglandin E 2 PKA Protein kinase A PKC Protein kinase C PLC Phospholipase C PNa Plasma sodium ppET-1 Preproendothelin-1 PVN Paraventricular nuclei RAAS Renin-angiotensin-aldosterone system RBF Renal blood flow SEM v Standard error of the mean SNGFR Single nephron glomerular filtration rate SON Supraoptic nuclei TAL Thick ascending limb UCAMP Urinary cAMP uv Urinary flow uN av Urinary sodium excretion Vasopressin receptor V i v 2 Vasopressin receptor V 2 VAMP Vesicle-associated membrane protein xi ACKNOWLEDGMENTS I am grateful to my supervisor Dr. Norman Wong for the opportunity to work in his laboratory and for his continual patience and support. I would also like to thank Dr. Eric Wong for his helpful advice, Alice Fok for her technical assistance and encouragement, and Edward Mak for the iodination of 1 2 5I-ET-1. xii CHAPTER 1 INTRODUCTION 1.1 Congestive Heart Failure Congestive heart failure (CHF) is a circulatory disorder that primarily originates from myocardial muscle dysfunction (130). Table 1.1 lists some conditions that may impair the heart's ability to pump adequate quantities of blood to the peripheral organs. The initial adaptive response to cardiac injury is the activation of hemodynamic and neurohormonal mechanisms that augment contractility and maintain cardiac output; however, these compensatory systems become maladaptive following prolonged utilization. As heart failure evolves, circulatory flow inevitably becomes insufficient to properly oxygenate body tissues. Peripheral vasoconstriction and sodium retention subsequently ensue to maintain the effective circulating fluid volume. Unfortunately, endogenous mechanisms aimed to restore cardiac function ultimately exert adverse effects that exacerbate the pathophysiological state of CHF. This stage represents a shift from the compensatory to the decompensatory state of heart failure. 1.1.1 Initial adaptations to myocardial injury Myocardial injury reduces cardiac output but may be well-tolerated at first because of central and peripheral compensatory mechanisms. Since the failing heart ejects blood ineffectively, the end-diastolic volume increases. Lengthening of cardiac muscle fibres thus enhances contraction according to the Frank-Starling principle. Baroreceptors in the carotid sinus and aortic arch also sense a reduction in arterial pressure during heart failure. They then transmit signals via cranial nerves IX and X to the hypothalamus and medulla in the brain to enhance sympathetic tone, which promotes a positive inotropic state. As a result of these beneficial hemodynamic and neurohumoral forces, cardiac function is restored towards normal and heart failure is asymptomatic. 1 Table 1.1. Causes of cardiac muscle dysfunction (Ref. 15). • Hypertension • Myocardial infarction/ischemia • Cardiomyopathy • Cardiac dysrhythmias • Heart valve abnormalities • Pericardial effusion/myocarditis • Pulmonary embolus/pulmonary hypertension • Renal insufficiency • Spinal cord injury • Aging 2 1.1.2 Development of heart failure The transition from asymptomatic to clinical heart failure begins when compensatory hemodynamic and neurohumoral systems introduce deleterious physiological effects. Prolonged cardiac dilatation stretches the sarcomeres to their maximal length such that additional increases in cardiac filling pressures do not improve the pumping ability of the injured heart. Moreover, cardiac distension leads to thinning, necrosis, and fibrosis of the ventricular wall as well as structural and functional alterations of atrial receptors, which would otherwise regulate sympathetic activity (66). With consistent activation of the sympathetic nervous system, the heart eventually becomes desensitized to P-adrenergic stimuli as a result of receptor downregulation or uncoupling from adenylate cyclase (13). At the same time, cardiac function is also compromised as peripheral blood vessels become more responsive to a-adrenergic stimuli and vascular resistance is increased (45). Consequently, the failing heart is burdened with additional hemodynamic stress caused by increases in afterload. In response to increased cardiac load, myocardial stretch, and trophic factors such as norepinephrine (NE) and angiotensin II (Ang II), structural remodeling of the heart occurs. However, this adaptation further aggravates the pathophysiological condition. Because myocytes are terminally differentiated, they only increase in length (dilate) and thickness (hypertrophy) (27). Cardiac remodeling may indeed be necessary to accommodate the workload on the heart, but it is also associated with an increase in energy expenditure. Because wall thickening impairs oxygen diffusion, the energy supply available to the myocardial cells becomes diminished. Heart failure advances as cardiac output becomes insufficient to meet the body's metabolic demands. Thus, sustained hemodynamic and neurohumoral activation loses beneficial effects and progressively, worsens cardiac function. 3 1.1.3 Neurohumoral activation in congestive heart failure Following the onset of heart failure, the cardiovascular system is directed to maintain adequate perfusion pressures by systemic vasoconstriction and sodium retention. Various neurohumoral forces are involved and are categorized into vasoconstrictors (catecholamines, angiotensin II, vasopressin, and endothelin) and vasodilators (atrial natriuretic peptide, prostaglandins, and endothelium-derived relaxing factor). The delicate balance of these opposing systems governs the physiological response and determines the transition from compensatory to decompensatory states of CHF. Vasoconstrictor systems The sympathetic nervous system is activated early in heart failure. Its peripheral effects include arterial vasoconstriction and positive cardiac chronotropy and inotropy. As well, the kidney is richly innervated with sympathetic nerves (115). Enhanced NE levels in CHF readily stimulate renin release, thereby contributing to salt reabsorption and volume expansion. It is noteworthy that sympathetic stimulation amplifies the actions of other vasoconstrictor systems. That is, the effects of NE, Ang II, vasopressin (AVP) are additive and synergistic (33). The renin-angiotensin-aldosterone system (RAAS) is also an active mechanism, as reflected by the apparent increases in renin secretion in the initial stages of CHF (129). Renin converts angiotensinogen, which is mostly produced in the liver, to angiotensin I. The subsequent cleavage of angiotensin I by angiotensin converting enzyme (ACE) results in Ang II formation. As a potent vasoconstrictor, Ang II is perhaps most important at the efferent arteriole in the glomerulus where it is largely responsible for maintaining glomerular filtration rate (GFR) in CHF. Additional actions of Ang II include potentiation of catecholamine activity (25), reduction of the glomerular filtration surface area (74), and stimulation of proximal sodium reabsorption (146). Ang IT may also be converted to angiotensin III, which then stimulates the zona glomerulosa of the adrenal glands to produce aldosterone (74). In the distal nephron, aldosterone stimulates sodium reabsorption and potassium secretion by targeting Na7K+ or NaTEt transport. Unlike healthy subjects, CHF patients accumulate excess sodium in the body and are unable to escape the salt-retaining effects of aldosterone even after a few days (17). This is because aldosterone degradation in the liver is reduced during heart failure. Clearly, the RAAS has diverse functions with effector sites in the vasculature and kidney. Vasopressin (AVP) and endothelin (ET) are two other major vasoconstrictor systems. Their contributions to the development of CHF will be discussed further in subsequent sections. Vasodilator systems Atrial natriuretic peptide (ANP) is a natriuretic hormone produced by the atria and ventricles when atrial pressures increase. It binds to receptors located in the glomeruli, loop of Henle, and inner medulla (25, 120). ANP induces salt excretion by increasing GFR and decreasing the medullary concentration gradient established by countercurrent multiplication (52). In addition, ANP directly alters tubular reabsorption by inhibiting Na +/H + exchange and Na +/P04 cotransport (60). ANP is also known to antagonize Ang II effects at the proximal tubule and to inhibit oxygen consumption required for Na+ transport through cellular increases in cGMP in the collecting duct (58, 61). Prostaglandins are vasodilators that are synthesized in the endothelial cells of arterioles, mesangial cells, proximal tubule, and collecting duct in response to renal hypoperfusion and to vasoconstriction produced by NE, Ang II, and AVP (141). Although their primary function is to maintain renal blood flow (RBF) by decreasing the vascular resistance at the afferent arteriole, prostaglandins also limit urine concentrating ability (128, 138). Prostaglandin E 2 (PGE2) has been 5 demonstrated to inhibit basal and AVP-mediated sodium and water reabsorption in the thick ascending limb (TAL) and collecting duct, respectively (151, 65, 121). Loss of counterregulatory vasodilating mechanisms Under normal conditions, vasoconstrictive effects are limited by vasodilatory effects. For example, ANP tends to induce natriuresis and diuresis by directly affecting glomerular and tubular function and indirectly by inhibiting NE, renin, and AVP release and actions (8, 138). Endothelium-derived relaxing factor (EDRF) is known to offset the vasoconstrictive effects of ET (100). However, these counterregulatory actions are blunted in heart failure (25, 100). Cody et al demonstrated that exogenous ANP failed to induce any changes in sodium excretion in patients with heart failure. Abassi et al later confirmed that the renal response to ANP is attenuated in CHF rats (1, 25). Moreover, it has been reported that hyponatremia is evident when the cardiac ejection fraction is decreased to less than 15% (134). This may be attributed to reduced ANP delivery to the kidney, end-organ resistance, or an inability to overwhelm the effects of salt-retaining systems in the disease state. In the absence of counterregulatory mechanisms normally induced by vasodilators, vasoconstrictor systems become unopposed and their actions are amplified. As a result, sodium retention by the kidney ensues and ultimately leads to volume expansion. 1.1.4 Renal adaptations: salt and water retention During CHF, diminished cardiac output reduces the fraction of blood volume distributed to the kidneys (155). Unless heart failure is severe, GFR is maintained even when the ultrafiltration coefficient (Kf) is reduced. Vasoconstrictors such as NE, Ang II, and AVP have a greater effect on the efferent than the afferent arterioles such that the filtration fraction increases (75, 155), The consequent elevation in glomerular capillary pressure (PGC) offsets other factors 6 that would otherwise decrease GFR in CHF. When perfusion pressure decreases, mechanoreceptors and chemoreceptors in the kidney also respond by modifying urinary sodium and water excretion. Specifically, the juxtaglomerular apparatus in the kidney monitors RBF and GFR. It detects the sodium and chloride concentrations at the macula densa and regulates the release of renin, which influences tubular sodium reabsorption. Perhaps enhanced fluid reabsorption along the nephron is the major contributor to volume expansion in CHF (8). In fact, Hostetter et al found rats with mild to moderate infarcts have an impaired response in sodium and water excretion following a saline infusion (69). As already mentioned, RBF is decreased while GFR is relatively maintained in CHF. Increased filtration fraction leads to low hydrostatic pressure and high oncotic pressure within the peritubular capillary. Hence, Starling forces greatly favor solute and water transport from the proximal tubule (155). Several other animal models of heart failure also exhibit enhanced fractional fluid reabsorption at the proximal tubule and an inability to decrease sodium reabsorption after a saline load (23, 75, 87). Sodium reabsorption appears to be increased in the distal nephron as well. Schneider et al found that sodium excretion was reduced even though proximal reabsorption was normal in dogs with arteriovenous fistulas (144). More recent studies indicated that the loop of Henle was the site of enhanced reabsorption (105, 154). 1.1.5 Consequences of excessive hemodynamic and neurohumoral activation Decreased myocardial performance initially activates neurohumoral activity in an effort to increase cardiac contractility and to restore cardiac output. Unfortunately, excessive stimulation of vasoconstrictor systems and concomitant loss of counterregulatory vasodilating actions introduce physiological risks. Figure 1.1 summarizes the compensatory events leading to salt and 7 Figure 1.1 Adaptive mechanisms that lead to salt and water retention in congestive heart failure. Low-output heart failure reduces the effective circulating volume and activates compensatory neurohumoral and renal mechanisms. Baroreceptors in the carotid sinus and aortic arch enhance sympathetic tone in response to the perceived volume depletion. Increased renal nerve activity therefore favors vasoconstriction, renin release from the kidney, and potentiation of other vasoconstrictor mechanisms. Alterations in renal hemodynamics include decreased renal blood flow. The glomerular filtration rate (GFR) remains relatively unchanged in mild heart failure, thereby increasing the filtration fraction and fluid reabsorption in the proximal tubule. In severe heart failure, GFR decreases and thus reduces salt and water excretion. Vasodilatory systems become attenuated as heart failure progresses and no longer counterbalance endogenous vasoconstrictor systems that are initially activated to improve cardiac output. Hence, excessive salt and water retention results and heart failure shifts from a state of compensation to decompensation as the actions of vasoconstrictors introduce deleterious physiological effects. ANP, atrial natriuretic peptide; EDRF, endothelium-derived relaxing factor; RAAS, renin-angiotensin-aldosterone-system; AVP, vasopressin; ET, endothelin. See text for further discussion. 8 9 water retention in CHF. Fluid retention by the kidney eventually leads to venous congestion and pulmonary edema. Decreased blood flow to the skeletal muscle results in exercise intolerance in patients with CHF. This is accompanied by abnormalities in hematologic, hepatic, pancreatic, and biochemical functions (15). Clearly, CHF is a complicated disorder that progresses over time and affects multiple organ systems. 1.2 Vasopressin 1.2.1 Biosynthesis and degradation The vasopressin (AVP) gene (~2000 bp) is located in close proximity to oxytocin on human chromosome 20 and expressed in the adrenal medulla, ovary, testis, thymus, sensory ganglia, and predominantly in the hypothalamus (143). The AVP product is a nonapeptide hormone with a six-membered ring and a tail of three amino acids that is synthesized as a preprohormone called propressophysin in the supraoptic nuclei (SON) and paraventricular nuclei (PVN) within the hypothalamus. A hydrophobic signaling sequence connects the AVP sequence at the TV-terminus to its carrier protein neurophysin, which is followed by a glycoprotein at the C-terminus (143). Although its role is unclear, neurophysin is physiologically significant because Brattleboro rats, whose neurophysin sequence exhibits a deletion mutation, fail to produce normal AVP products and therefore suffer polyuria. Abramow et al suggested that neurophysin regulates the proteolysis of AVP and itself from the precursor (2). The entire propressophysin is packaged into secretory granules that are then transported down the axons to the posterior pituitary. AVP and neurophysin separate following proteolysis but are stored in the neurohypophysis until secretion. Two independent AVP pools exist at the axon terminals: a readily releasable pool and a 10 comparably larger storage pool. A V P and its carrier are exocytosed upon sodium and calcium influx following depolarization of the axon. Plasma A V P levels are governed by secretion and degradation. A V P has a half-life of 5 to 10 minutes in humans, and its clearance in dogs has been reported to be biphasic with half-lives of 1.4 and 4.1 minutes (168). Sites of A V P degradation include the liver, intestine, and kidney (113). Renal clearance of A V P is dependent on the balance of plasma bound A V P and the freely filtered form (147). 1.2.2 Osmotic and non-osmotic regulation of vasopressin release In 1947, Verney proposed the existence of central osmoreceptors, which have now been located in the anterior hypothalamus. Robertson et al also showed that A V P release is very sensitive to plasma osmolality in vivo (135, 165). When the plasma osmolality falls below the threshold (-280 mOsm/kg) for example, A V P secretion is almost undetectable. Above this level, osmolality and A V P concentration exhibit a linear relationship such that a 1% change in plasma osmolality increases plasma A V P by approximately 1 pg/ml, which is sufficient to alter water clearance (51). Systemic hemodynamics are also important in fluid volume homeostasis, acting in part through the non-osmotic regulation of A V P (145). Baroreceptors located in the atria, ventricle, aortic arch, and carotid sinus are known to detect blood volume. They send afferent signals via the vagus and glossopharyngeal nerves to the nucleus solitaris in the medulla, which then relays post-synaptic messages to the SON and P V N for appropriate A V P release. Increased stretch of the volume sensors inhibits A V P secretion, whereas decreased stretch effects the opposite response. Non-osmotic stimuli are considered less potent in altering A V P release than osmotic 11 stimuli since arterial pressure or blood volume is reduced at least 5 to 10% before circulating A V P significantly increases (51). However, the body preferentially protects itself over blood volume loss when osmotic and non-osmotic stimuli transmit conflicting messages. During hypovolemia, non-osmotic regulation takes precedence and favors AVP release and thirst in spite of hypoosmolar conditions. Cowley demonstrated that perceived decreases in blood volume resets the osmotstat or threshold for osmoregulation such that more AVP is released for any given osmolality (28). Failure to suppress abnormal AVP secretion may be the underlying cause of hyponatremia in some pathophysiological states. 1.2.3 Vasopressin receptors AVP exerts physiological function by binding to seven membrane-spanning receptors that are coupled to different intracellular second messengers. The V i receptor activates the phosphoinositide pathway to mobilize cytosolic calcium (114). It has two subtypes: V u is predominantly expressed in the liver, platelets, smooth muscle cells, glomerular mesangial cells, vasa recta, and medullary interstitial cells, whereas Vib or V 3 is found in the anterior pituitary (85). AVP also stimulates adenylate cyclase through the V 2 receptor, which has been localized to the ascending thin limb (ATL), TAL, and collecting duct (3, 78). It is of interest that AVP induces both Ca^ " mobilization and cAMP production via V 2 receptors only in the collecting ducts (39). That AVP sensitivity is cell-type specific reflects the existence of varying V 2 subtypes or receptor coupling abilities (21, 79). In fact, Firsov et al recently identified two V 2 mRNA variants, which result from alternative splicing, in the rat kidney (44). Unlike the longer form (V^), the shorter V2s transcript lacks a nucleotide for encoding the last transmembrane domain and its product does not elevate cAMP levels. Whether V 2 S exhibits any physiological significance is unknown. 12 However, both V2 spliced variants co-exist along the nephron and are susceptible to homologous downregulation. V i and V 2 receptors may be expressed in the same cells. In fact, binding studies and molecular analysis have identified a minor population of V i a receptors in the collecting ducts (3, 44). Therefore, it is possible that Vi and V 2 signaling pathways interact. Based on their experiments using the toad bladder, SchlondorfF et al suggested that phorbol esters and V i activation may influence AVP-induced water transport by decreasing intracellular cAMP levels (142). However, their conclusion is controversial in light of recent findings that showed V i a potentiates V2-induced cAMP production in Chinese hamster ovary cells by a PKC- and Ca^-dependent process (95). Possible mechanisms for Via-mediated effects include enhanced G s protein coupling to the V 2 receptor or to adenylate cyclase. Such interactions between V i and V 2 receptors within the mammalian kidney have yet to be confirmed. 1.2.4 Role of V 2 receptor in salt and water reabsorption The generation of cAMP has been associated with changes in renal reabsorption of sodium, urea, and water via specific proteins on the apical membrane (124). V 2 receptors stimulate the Na+/K72Cl" cotransporter at the TAL in rabbit and rodents but not dog and human (2). The mechanism probably involves a cAMP-dependent pathway that enhances potassium conductance across the luminal membrane. As well, Kleyman et al demonstrated sodium channel expression at the luminal membrane of A6 cells is increased in the presence of AVP and forskolin (94). Besides its contribution to the hypertonicity of the medullary interstitium, AVP also increases the number of water channels (aquaporins) inserted in the luminal membrane to stimulate water reabsorption in the distal nephron segments. At the renal collecting tubules, AVP 13 specifically regulates Aquaporin 2 (AQP2), which mediates water transport and is impermeable to other small solutes (148). AQP2 is mostly contained within the intracellular granules of principal cells under normal hydration. These granules are anchored to the cytoskeleton and constitute the reserve pool. A minority of AQP2 is releasable and is docked at the luminal membrane in preparation for immediate exocytosis upon AVP stimulation (63). According to the membrane shuttle hypothesis, AVP stimulation governs the cycling of water channels between the cytoplasm and luminal membrane. In the absence of AVP, water transport in the collecting duct is inhibited (Figure 1.2). Urine becomes hypotonic since basal osmotic water permeability (Pf) is only about 20 um/s (139). During the dehydrated state and plasma AVP elevation, AQP2 is heavily expressed at the apical membrane to facilitate water diffusion. The Pf increases within 20 seconds and varies from 200 to 1000 Ltm/s (102). Consequently, the kidney excretes a concentrated urine. Upon binding to the basolateral V 2 receptors, AVP activates adenylate cyclase and converts ATP to 3', 5'-cyclic monophosphate (3', 5'-cAMP), whose cytoplasmic level depends on phosphodiesterases (36). Activation of PKA facilitates the phosphorylation of a specific site (Ser 256) located in the C-terminus of AQP2 (101). This process favors the AQP2 transcription in the nucleus and protein translocation to the apical membrane. In 1978, Taylor et al first suggested that cytoskeletal components are central to water reabsorption in the collecting duct (161). Reorganization of actin filaments and spectrin allows the release of reserved AQP2 from the cytoplasm. The precise mechanisms by which AQP-containing granules detach from the cytoskeleton are not fully understood, but vesicle release may also require PKA phosphorylation of the anchoring filaments (63). Finally, docking and exocytosis of the preformed water channels at the luminal membrane of the collecting duct ensue by a process that is similar to neurosecretion at the nerve terminal and chromaffin cells. 14 Figure 1.2 Role of V2 activaton in water transport in the collecting duct. In the absence of vasopressin (AVP), the collecting duct is impermeable to water and dilute urine is excreted. Presence of AVP facilitates V2 receptor activation, which then stimulates cAMP production. Subsequent activation of protein kinase A (PKA) favors transcription of aquaporin 2 (AQP2) and insertion of AQP2-containing vesicles into the luminal membrane of the collecting duct. Passive water reabsorption through AQP2 results in concentrated urine. See text for further discussion. 15 X AQP-2 containing vesicle V A M P (cellubrevin/ synaptobrevin) Syntaxin Inside Cell Membrane Lumen Figure 1.3. Proposed mechanism for vasopressin-induced docking of vesicles containing aquaporin-2 (AQP2) at the luminal membrane of the collecting duct. Vesicle-associated membrane protein (VAMP) mediates fusion of the vesicle and cell membrane via a syntaxin protein. NEM-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) may also be important components of the attachment complex. See text for further discussion. 17 Docking of the AQP-2 containing vesicles to the luminal membrane is a regulated process involving vesicle-associated membrane proteins (VAMPs) and their target membrane receptors (Figure 1.3). Specifically, cellubrevin has been located on the intracellular vesicles and the apical membrane of the IMCD (64, 46). In rat inner medulla, Jo et al also identified synaptobrevin which is responsible for the fusion of AQP2-containing endosomes with each other (86). These two VAMPs bind to various isoforms of syntaxin, a membrane receptor, on the vesicles and luminal membrane of the IMCD. Docking of the vesicles facilitates the expression of AQP2 proteins at specific sites on the luminal membrane. Water transport is mediated once AQP2 is inserted into the membrane, and it is terminated when AQP2 is endocytosed. 1.2.5 Regulation of hydroosmotic response to vasopressin AVP-stimulated sodium and water reabsorption is regulated by a number of factors. For example, adrenergic CL2 agonists inhibit the hydroosmotic response to AVP in rat cortical collecting duct (CCD) by diminishing adenylate cyclase activity (99). Prostaglandins have also been reported to inhibit NaCl reabsorption by AVP in the medullary TAL and collecting duct via a similar mechanism (65, 76, 153). In cortical collecting tubules, PGE 2 inhibits AVP-stimulated osmotic water reabsorption (121). Moreover, intracellular calcium levels have been associated with water permeability in the collecting duct, but their effects remain controversial. Humes et al found that verapamil, which blocks calcium entry, elevates cAMP synthesis but inhibits AVP response in toad urinary bladder (70). Charbardes et al proposed that calcium stimulates prostaglandin production, which in turn inhibits the action of AVP (22). However, Teitelbaum and Berl found that decreased intracellular calcium enhances AVP-induced cAMP formation in rat IMCD by a direct effect on adenylate cyclase and that the process is not prostaglandin-dependent (163). Dillingham et al concluded that the observed physiological response to lowered cytosolic 18 calcium levels results from the modification of post-cAMP events that reduce the hydraulic conductivity stimulated by AVP (34). Ca-calmodulin may also be potentially important in AVP action because specific blockers of calmodulin inhibit water flow in the toad urinary bladder and the collecting tubule; however, the mechanisms involved are not known (11,35, 104). 1.2.6 Vasopressin in congestive heart failure Plasma AVP levels are often abnormally high in patients and experimental animal models with severe CHF (131, 133, 157). They may be partially attributed to elevated AVP expression in the hypothalamus as a result of increased biosynthesis or mRNA stability (92). Other mechanisms involved include impaired hypothalamic osmoreceptors or left atrial receptors that would otherwise inhibit AVP secretion. Because acute changes in blood volume and pressure are known to reset the osmostat (135), the inadequate perfusion pressure sensed in CHF possibly lowers the threshold and leads to inappropriate AVP secretion (38, 158). The inverse relation between plasma AVP levels and osmolality during cardiac dysfunction supports the idea that osmoregulation is abnormal in this pathological state (133). In 1986, Goldsmith and colleagues found that the relative decrease in plasma AVP was greater in normal subjects than patients with heart failure following an oral water load (55). Despite the augmented plasma AVP levels, a group of CHF patients responded normally to volume expansion by forming a dilute urine while other CHF patients retained excess water because their urine osmolality remained unchanged. This additional evidence reveals a complex view of elevated AVP concentrations in the pathogenesis of CHF, suggesting individual renal responses are variable. Hence, appropriate salt and water excretion in CHF may depend on whether the patient develops tubular AVP resistance, which may result from W2 receptor downregulation or antagonism of post-receptor events. 19 Previous studies indicate AVP has a significant role in hemodynamics in heart failure as well. Administration of exogenous AVP at concentrations observed in CHF produces symptoms in normal subjects. Goldsmith and coworkers found that increasing normal plasma AVP by 50% in healthy humans significantly increased peripheral vascular resistance and concomitantly depressed cardiac output and stroke volume (56). Furthermore, Yared et al reported that AVP-induced vasoconstriction is pronounced in CHF (174). In rats pretreated with an ACE inhibitor, AVP blockade caused a significant decrease in afterload in the myocardial infarcted animals but not the normal group. The events leading to enhanced AVP effects on the peripheral vasculature remain undefined but may result from a dysfunction in opposing systems or altered sensitivity of the baroreceptor reflex in CHF. Nonetheless, AVP deserves equal attention as other potent vasoconstrictors that are activated in CHF. Goldsmith et al found AVP levels correlated with plasma renin activity in CHF patients (54). Moreover, Amolda et al showed that AVP and RAAS contribute equally to the development of low-output failure in rabbits because blockade of the separate systems improved cardiac output and peripheral vascular resistance to a similar extent (6). Additional studies indicate endogenous AVP is potentially important in CHF. V i receptor blockade alone increased cardiac output while V 2 blockade caused diuresis in CHF dogs (123). The same study showed that a combined administration of V i and V 2 antagonists produced additive effects. Mulinari et al reported similar results in rats with impaired ventricular dysfunction (119). Accordingly, a clear understanding of AVP responses on the systemic and renal vasculature is crucial in the pathogenesis of CHF. 20 1.3 Endothelin 1.3.1 Endothelin isopeptides and receptors The endothelin ( E T ) family was first identified by Yanagisawa et al and consists of 21-amino acid peptides (-2.5 kDa) with three known isotypes: E T - 1 , ET -2 , and ET -3 (80, 173). All have polar side chains attached to a hairpin configuration held by disulfide linkages between Cys1-Cys15 and Cys3- Cys11 residues; however, the highly conserved and hydrophobic COOH-terminus is the portion which confers bioactivity (151). Sarafotoxins, extracted from the venom of Atractaspis engaddensis, share this conservative sequence and are E T agonists. The three E T isotypes primarily differ in their potencies and binding affinities. For example, the E T A receptor activates the phosphoinositol/ Ca + + pathway and preferentially binds to ET -1 and ET -2 with greater affinity. The E T B receptor stimulates nitric oxide (NO) production and binds to all three E T isotypes and sarafotoxins with nearly equal affinities (159). Recently, an ET-3-specific receptor subtype called ETc was isolated and cloned from cultured Xenopus laevis dermal melanophores and has been identified in cultured bovine endothelial cells (42, 89, 140). Accordingly, there may be other E T receptors present in the body. E T receptors are widely distributed in the endothelium, central and peripheral nervous systems, as well as the liver, heart, gut, adrenal, and eye. They are perhaps most abundant in the kidney, where immunoreactive E T is observed in arterioles, peritubular capillaries, capillary loops, and mesangial cells of the glomeruli (169). Karet et al showed that two-thirds of the total E T binding occurs in the human medulla (88). The same study found E T A receptors exist primarily in renal arteries and in afferent and efferent glomerular arterioles while the E T B subtype is evenly distributed on epithelial cells and accounts for 65% of the total specific ET-1 binding in the human kidney. 21 1.3.2 Regulation of endothelin receptors Prolonged exposure of an agonist may cause an attenuated or diminished response at its receptor (homologous desensitization) or at another receptor type (heterologous desensitization). Mechanisms of receptor regulation include internalization, degradation and inactivation, G-protein uncoupling, and transcriptional control. Figure 1.4A shows numerous factors regulate E T B receptors (103); however, the specific events involved are still undefined. Studies on other serpentine receptors may provide valuable insights. For instance, the p-adrenergic receptor desensitization and resensitization are well-documented (62, 150). At low agonist concentrations (nanomolar), PKA phosphorylation of the third intracellular loop uncouples the p-adrenergic receptor from heterotrimeric G-proteins and consequently inhibits intracellular signaling. At high agonist concentrations (micromolar), regulation of the P-adrenergic receptor involves phosphorylation of the cytoplasmic tail by p-adrenergic receptor kinase (PARK). Such modification facilitates the binding of P-arrestin to the C-terminus of the receptor, which then promotes receptor uncoupling and internalization (43). The latter mechanism is unlikely in ET receptor regulation because deletion mutations of putative phosphorylation sites on the cytoplasmic tail of the human E T A receptor have not been shown to affect the rate of agonist-induced desensitization (31, 32). Koshimizu et al also found at least ten of the twelve phosphorylation sites on the C-terminus of the human E T B are irrelevant to homologous desensitization (98). Nonetheless, these observations do not exclude the potential role of Ser/Thr phosphorylations in the intracellular loops in the uncoupling of ET receptors to their G-proteins. It is of interest that both ET receptor subtypes have potential PKC-dependent phosphorylation sites [Ser/Thr-Xo-3-Arg/Lys] (4, 140). However, only the E T B receptors contain a consensus sequence [Arg/Lys-Arg/Lys-X-Ser/Thre] that is specific for PKA phosphorylation and is located 22 Figure 1.4 Endothelin-B (ETB) receptor regulation. A: Factors that specifically upregulate (+) and downregulate (-) ET B receptors. B: ET B receptor belongs to the G-protein coupled seven-transmembrane receptor family. Presence of a ET agonist promotes G-protein coupling to the third intracellular loop of the E T B receptor and the initiation of signal transduction. Protein kinase A (PKA) phosphorylation at the consensus sequence [Arg/Lys-Arg/Lys-X-Ser/Thre] has been proposed to interfere with G-protein coupling. See text for further discussion. 23 A) C-type natriuretic peptide Angiotensin II Basic fibroblast growth factor (+) ET B Receptor (-) cAMP Catecholamines B) ET B RECEPTOR ACTIVATION ET B RECEPTOR DESENSITIZATION outside inside outside inside COOH COOH 24 proximally to the site proposed for G-protein coupling in the third cytoplasmic loop (140). This particular motif is absent in E T A (4). Regulation of E T B receptors may therefore involves a process similar to that for $-adrenergic receptors at low agonist concentrations (Figure 1.4B). Several studies have implicated the involvement of protein kinases in ET receptor regulation. Durieu-Trautmann et al demonstrated that forskolin and dibutyryl cAMP decrease the binding capacity of ET receptors in rat astrocytoma C6 cells (37). Asada et al later concluded that downregulation of the E T B receptor mRNA in ROS 17/2 rat osteosarcoma cells involves a PKA-dependent pathway (7). Their findings suggest that ET induces homologous regulation of its receptors by accumulating cAMP directly through adenylate cyclase activation or indirectly through a PGE2-dependent pathway. Recent studies with microdissected rat CCD cells confirm the relevance of PKA in E T B but not E T A receptor desensitization (160). An alternative pathway appears to modulate ET receptors. Roubert et al showed that exposure to Ang II and AVP downregulate ET receptors in vascular smooth muscle cells (137). Because similar results were obtained with phorbol esters, a PKC-dependent regulatory pathway likely exists. Additionally, Cozza and Gomez-Sanchez have reported that PKC activation decreases surface ET receptor numbers by receptor internalization (29). The observation that PKA and PKC modulate ET receptors suggests the existence of multiple regulatory mechanisms, which may be specific to cell type or receptor subtype. 1.3.3 Renal effects of endothelin In the kidney, ET predominantly has a paracrine and/or autocrine function with multiple sites of action along the nephron as shown on Figure 1.5 (151). Binding to E T A receptors alters renal hemodynamics by changing both variables of the single nephron glomerular filtration rate (SNGFR). ET-1 constricts the efferent arteriole more effectively than the afferent, thereby 25 Figure 1.5 Sites of endothelin (ET) action along the nephron. ET controls fluid homeostasis at multiple sites in the kidney. In addition to its effects on renal hemodynamics and ultrafiltration coefficient ( K f ) , ET inhibits sodium reabsorption in the proximal tubule (PT) via a mechanism independent of E T A and ET B receptors. ET regulation of sodium transport in the distal nephron may depend on the activation of specific receptor subtypes which have been shown to have opposing actions on amiloride-sensitive sodium channels in A6 cells (47). ET also causes natriuresis and diuresis via E T B by inhibiting the Na7K+-ATPase and AVP action. GL, glomerulus; TAL, thick ascending limb; CD, collecting duct. See text for further discussion. 26 27 elevating the PGC- It also affects the K f , which decreases when mesangial cell contraction reduces the available filtering surface area. ET also exerts direct tubular actions that diminish sodium and water reabsorption and can increase urinary flow irrespective of renal hemodynamics. 1.3.4 Endothelin in salt and water homeostasis E T is an important hormone in the regulation of volume fluid homeostasis. Previous studies reported E T was antinatriuretic because of its vasoconstrictive properties and activation of renin and aldosterone release. However, recent evidence indicates that E T enhances fractional sodium excretion by affecting tubular reabsorption. For example, E T may induce natriuresis through activation of atrial and brain natriuretic peptides or inhibition of the Na7K+-ATPase on the basolateral side of the nephron (177). Alternatively, Clavell and colleagues suggested that a mechanism independent of E T A and E T B activation decreases sodium reabsorption at the proximal tubule (24). Gallego and Ling also found that ET-1 regulates the amiloride-sensitive sodium channel expressed in A6 distal nephron cells (47). At subpressor doses (100 pM), ET -1 activates the E T B receptor and inhibits sodium transport by increasing the mean channel closed time. In contrast, sodium reabsorption is enhanced at higher ET-1 concentrations (10 nM) which prolong the opening of the sodium channel via E T A receptors. The functional response in Na+ transport may ultimately be related to the existing circulating E T concentration and activation of either E T A or E T B . ET also affects body fluid volume by controlling free water clearance through ET B receptors, probably as a compensatory mechanism for its effects on renal hemodynamics (41, 164). Its actions specifically target the antidiuretic response of AVP in the collecting duct such that diuresis occurs even when RBF and GFR are reduced (24). The specific mechanisms by which ET causes diuresis are discussed in Section 1.4. 28 1.3.5 Endothelin in congestive heart failure Circulating ET levels are augmented two- to four-fold in CHF and correlate with the severity of this disorder (112, 167). Mechanisms for this striking increase may result from decreased ET degradation and clearance or enhanced levels of the ET precursor (59). As well, in vitro studies have shown that Ang II stimulates ET gene expression in endothelial cells (77). This finding is particularly interesting because RAAS is a major vasoconstrictor system that is activated during CHF. Therefore, Ang II may be partially responsible for the elevation in ET levels observed in heart failure. Other stimuli for ET synthesis and release include increased atrial and venous pressures, hypoxia, inadequate arterial volume, and catecholamines (103). Elevation of ET levels initially occurs as an adaptive response to improve preload and cardiac output. Chronic exposure to increased ET concentrations, however, may worsen the pathophysiological state. ET potentiates the activity of both the sympathetic nervous system and RAAS, thereby increasing aldosterone production and leading to salt retention. As a mitogenic factor, ET stimulates the expression of immediate early genes such as c-fos and egr-l that may contribute to ventricular hypertrophy (103). Moreover, Kiowski et al found that ET causes vasoconstriction in severe CHF patients, and its plasma concentration correlates directly with the degree of pulmonary hypertension and vascular resistance but inversely with cardiac output (93). Based on their studies with chronic heart failure dogs, Shimoyama et al similarly concluded that ET affects systemic hemodynamics (149). Intravenous administration of a nonselective ET receptor antagonist, bosentan, reduced systemic vascular resistance and improved cardiac output. Clearly, ET has the potential to manifest many symptoms associated with CHF. However, the functional significance of high plasma ET levels still requires further investigation. In an experimental dog model of heart failure, Calderone et al found that basal 29 inositol phosphates and phospholipase C (PLC) activities are reduced despite high circulating ET concentrations (16). They believed that these changes in the ET signaling pathway reflect tissue desensitization in response to elevated ET levels in the pathophysiological state. Loffler et al showed that diminished ET reactivity in CHF rabbits is attributed to a reduction in ET receptor density in the heart and kidney (108). Whether ET directly contributes to the progression of CHF or is simply a marker of severity of disease remains unanswered. 1.4 Functional interactions between vasopressin and endothelin Neurohumoral responses are highly complicated. Additive effects among angiotensin, AVP, and NE have been demonstrated in in vivo studies and probably result from interactions of post-receptor events (82). Some evidence for the integrated actions of ET and AVP have been reported and are summarized on Figure 1.6. Imai et al (77) demonstrated that AVP induces the gene expression of preproendothelin-1 (ppET-1) in endothelial cells via the V i receptor; however, it is uncertain whether AVP increases ET synthesis or decreases mRNA degradation. Because the human ET-1 gene contains AP-l/jun binding sites in its 5'- region, the induction of ppET-1 mRNA expression by a potential PKC-dependent mechanism is possible (81). Other investigators have also implied that ET and AVP functions are interrelated because these hormones are coexpressed in some cells. Yoshizawa et al identified both ET-1 and AVP in rat neurophypophyseal axons, and Kaufmann et al found a correlation between levels of these two hormones in humans with various pathophysiological conditions (90, 176). That ET-1 stimulates AVP release in rat hypothalamus via E T A receptors further indicates that ET exerts paracrine and/or autocrine control on AVP (175). Mosqueda-Garcia and colleagues provided additional insights into the physiological responses arising from ET and AVP interactions (118). 30 Figure 1.6 Heterologous regulation of vasopressin (AVP) and endothelin (ET) actions. AVP increases mRNA expression of preproET-1 (ppET-1). ET enhances AVP release from the neurohypophysis via E T A receptors. In the collecting duct, ET antagonizes AVP action through V2 receptors by reducing cAMP generation and osmotic water permeability (Pf). Mechanisms may involve direct inhibition of adenylate cyclase or indirect regulation secondary to protein kinase C (PKC) and protein kinase G (PKG). See text for further discussion. 31 3 2 Microinjection of ET-1 into the subfornical organ of the brain concomitantly increased plasma AVP levels and blood pressure but decreased heart rate in rats. Interestingly, the cardiovascular changes were abolished with prior infusion of an E T A or V i antagonist. These findings reaffirm that E T A activation mediates vasoconstriction and additionally suggest that it stimulates release or synthesis of AVP which then contributes to the hemodynamic effects of ET-1. In vivo studies have also shown that exogenous ET prevented normal AVP responses in dogs (53). ET selectively inhibits AVP-induced cAMP accumulation in the collecting duct by a process independent of phosphodiesterases, cyclooxygenase, and intracellular Ca + 2 (164). Edwards et al also reported that ET-1 inhibits AVP-stimulated increases in Pf and cAMP accumulation in rat IMCD via E T B receptor activation (41). Using cultured rat inner medullary collecting tubules, Teitelbaum demonstrated that PKC activation inhibits cAMP accumulation by AVP stimulation through a Gi-dependent pathway (162). Kohan et al confirmed these findings and further indicated that ET counteracts AVP-induced water flow via E T B receptors by a process involving G; coupling and subsequent PKC activation (97). Moreover, Garcia et al have suggested that NO decreases AVP-stimulated water permeability in the rat CCD by stimulating guanylate cyclase (48). The consequent increase in cGMP activates protein kinase G (PKG), which alters intracellular cAMP levels and therefore decreases Pf. Furthermore, inhibition of PKG was shown to attenuate the inhibitory effect of NO on AVP-mediated water diffusion at the IMCD. The pathways by which PKG affects cAMP accumulation are unresolved but may involve the modulation of cAMP stability, AVP receptor, stimulatory G-protein, or adenylate cyclase. Because E T B binding produces an EDRF, presumed to be nitric oxide (NO), via a L-arginine pathway (40), it cannot be excluded that ET may antagonize AVP in the collecting duct via a NO-dependent pathway. 33 While ET-1 and AVP have been suggested to influence the development of CHF, the precise mechanisms involved remain unclear. Numerous experiments have focused on the individual hormone actions rather than the interactive roles of ET and AVP under normal and pathophysiological states. Hence, mutual regulation of these two hormones and its consequences in salt and water retention associated with CHF should be explored in detail. 1.5 Hypothesis Renal hypersensitivity to AVP in CHF has been previously demonstrated in in vitro studies (109). Isolated IMCD cells from CM hamsters produced comparatively more cAMP than control cells in the presence of AVP. This effect was consistent and concentration-dependent. Forskolin pretreatment mimicked these results, further supporting that V 2 signaling is enhanced in the disease state. The significance of these findings may be relevant under physiological conditions. It is recognized that elevation of plasma AVP concentrations develop in the early stages of heart failure in CM hamsters (68, 156). Figure 1.7 shows the proposed mechanisms for the enhanced response to AVP and its potential effects in CHF. Under pathological conditions, elevation of circulating AVP levels may increase receptor binding. Activation of V 2 receptors in the renal collecting ducts may cause adenylate cyclase to generate cAMP more effectively. These events may be augmented by V i receptors which potentiate V2-induced production of cAMP by a PKC-dependent pathway (95). The subsequent stimulation of PKA would enhance AQP2 transcription and insertion into the luminal membrane of the collecting duct to facilitate passive water reabsorption. In addition to increased AVP receptor signaling, AVP hypersensitivity in CHF may evolve from diminished activity of counterregulatory mechanisms. ET inhibits sodium and water reabsorption in the renal medulla via 34 Figure 1.7 Proposed mechanisms for vasopressin (AVP) hypersensitivity in congestive heart failure. Increased circulating AVP directly promotes salt and water retention by activating V 2 receptors. Stimulation of adenylate cyclase produces cAMP, and subsequent activation of protein kinase A (PKA) increases aquaporin 2 (AQP 2) transcription and protein expression at the luminal membrane of the collecting duct. Passive water transport through AQP2 occurs and results in water retention. AVP binding to Vi receptors also potentiates V2-induced cAMP production via a PKC-dependent pathway. In addition, AVP may contribute to salt and water retention indirectly by limiting counterregulatory forces. It may exert heterologous regulation of ET B receptors which contain a consensus sequence specifically for PKA phosphorylation in the third intracellular loop. Modification of ET B receptors may interfere with G-protein coupling so that E T B no longer inhibits Na+ reabsorption and AVP-induced increases in cAMP levels and osmotic water permeability (Pf). While ET B receptors become downregulated, binding to E T A receptors would be augmented when plasma endothelin (ET) levels increase in congestive heart failure. Hence, E T A activation would further contribute to salt and water retention by promoting Na+ reabsorption through amiloride-sensitive Na+ channels. See text for further discussion. 35 36 E T B receptors, specifically targeting the amiloride-sensitive Na+ channels and AVP-induced cAMP accumulation and Pf in the collecting duct (41 , 47 , 164). Overcoming the inhibitory influence of E T would promote fluid retention by AVP. In fact, previous studies have reported E T receptor downregulation in the heart and kidney of experimental CHF animal models (108, 171). In particular, renal distribution of E T receptor subtypes in the CM hamsters was abnormal with marked reductions in E T B and not E T A receptors (171). As such, renal tubular function would be altered because E T tends to exert opposing effects via distinct second messenger systems. A predominance of E T A receptors may overwhelm the natriuretic and diuretic effects of E T B . Mechanisms of renal E T B regulation in the pathophysiological state is unknown; however, a recent study suggested that AVP and E T are mutually antagonistic (160) . It is of particular interest that only the B-subtype of E T receptors exhibits a consensus sequence for PKA phosphorylation in the third intracellular loop. Coincidentally, V2 receptors, which activate the PKA pathway and are responsible for urine concentration, are the dominant AVP receptor subtype in the IMCD. Given these circumstances, AVP modulation of E T B function by receptor phosphorylation is probable and would involve a regulatory process similar to agonist-induced P-adrenergic desensitization. Altogether, this thesis proposes the following novel idea: Abnormal volume homeostasis in CHF may result from AVP hypersensitivity to its receptors and heterologous regulation of E T receptors. In the absence of counterregulatory forces normally induced by E T , the hydroosmotic response to AVP ultimately impairs salt and water excretion in CHF. 3 7 1.6 Objectives and rationale The current study was undertaken to understand the pathogenesis of abnormal fluid retention in decompensated heart failure. It comprises in vivo and in vitro experiments, which were aimed to determine the contributions of AVP to volume homeostasis in the setting of CHF. Additionally, the study investigates whether AVP-mediated mechanisms modulate ET receptor regulation and discusses their potential relevance in edema formation. Elevation of plasma AVP is often observed in CHF, but its physiological effects remain obscure. Therefore, renal clearance experiments were performed to compare the excretory function of normal and cardiomyopathic (CM) hamsters after infusions of AVP, a Vi antagonist with a V 2 agonist, or V i and V2 antagonists. Blood and urine samples were collected for measurements of hematocrit, plasma electrolyte analysis, GFR, and urine flow. Production of cAMP, a second messenger of V 2 receptors in the collecting duct, was also determined to ascertain if the hydroosmotic response to the drug administrations was AVP-dependent. The apparent AVP hypersensitivity in CM animals observed in in vivo studies prompted further investigation of the active mechanisms. E T causes salt and water excretion via E T B receptors (41, 177). Accordingly, the potential for AVP-induced heterologous regulation of E T receptors was examined. Competitive binding assays were conducted using rat IMCD cells pretreated with AVP or forskolin, which specifically activates adenylate cyclase, to determine if there were inhibitory effects on E T B binding capacity. To confirm the involvement of the AVP-mediated PKA pathway, a cAMP analogue (Rp-cAMPS) was employed in an attempt to prevent E T B downregulation by AVP. By selectively regulating E T B receptors, AVP may limit the natriuretic and diuretic actions of E T and simultaneously enhance its hydroosmotic response. 38 It is anticipated that the findings of this study are crucial in interpreting the events leading excessive fluid retention in CHF. The complex neurohumoral interactions between AVP and ET had been previously neglected but deserve further attention. Understanding the direct and indirect AVP actions may help develop therapeutic strategies that specifically induce free water clearance, which would benefit patients with hyponatremia. 39 CHAPTER 2 METHODS 2.1 In Vivo Experimental Design 2.1.1 Animals Male cardiomyopathic (CM) Syrian hamsters (CHF 148) between 280 to 300 days old of the UM-X7.1 strain and age-matched healthy controls (CHF 147) were employed for the present studies (Canadian Hybrid Farms, King County, Canada). These animals were housed in a climate-controlled environment at 22°C. They were provided with standard laboratory chow (Ralston Purina Co., St. Louis, MO) and had free access to drinking water. The CM line of Syrian hamsters were derived from the cross-breeding of healthy hamsters with the BIO 14.6 strain of CM hamsters (71). Initially, focal myocardial lesions are observed and are followed by degeneration of cardiac muscle and cardiac hypertrophy. As heart failure progresses, enlargement of the liver, spleen, pancreas, and intestines may be evident. Pulmonary edema, hypernea, and cyanosis also occur. Because these CM hamsters animals exhibit a gradual onset of the disease and symptoms commonly found in human CHF, they represent a suitable experimental model for this study. The degree of CHF in the CM hamsters is categorized according to a previously established scale that is summarized in Table 2.1 (9, 18). Grade 0 represents normal conditions while Grade 1 is mild heart failure with minimal subcutaneous edema and no measurable exudates in the body cavities. Moderate heart failure (Grades 2 and 3) is characterized by diffuse subcutaneous edema and hemorrhage and necrosis of the liver. In the advanced stage of Grade 3, the animals also have pulmonary congestion and less than 2.5 ml of exudates in the chest and abdominal cavities. Grade 4 represents severe heart failure in which edema of the entire body is evident and measurable exudates exceed 2.5 ml. 40 Table 2-J. Grading system for severity of congestive heart failure in cardiomyopathic hamsters. Grade of CHF Subcutaneous edema Ascites Pleural effusions Pericardial effusions Grade 1 : Mild minimal none none none Grade 2: Moderate diffuse mild none none Grade 3: Advanced moderate diffuse <2.5 ml <2.5 ml none Grade 4: Severe severe; present in entire body >2.5 ml >2.5ml present 41 2.1.2 Clearance studies Surgical preparation The hamsters were first anesthetized with Inactin (25 mg/kg, i.p). Tracheotomy was performed immediately, and polyethylene tubing (PE 160) was inserted to ease ventilation in the anesthetized animals. The left carotid artery and right jugular vein were catheritized with polyethylene tubings (PE 50) for blood sampling and intravenous infusions, respectively, and the bladder was cannulated for timed urine collections. Throughout the experiments, their body temperature was maintained around 37°C by a warmed operating table and heating lamp. A priming dose of 0.9% saline containing 3H-labeled inulin was administered after completion of surgery, and a constant infusion of 0.0144 ml/min using a compact infusion pump (Harvard Apparatus Co., Inc., Millis MA) was initiated to maintain a steady level of hydration. The hamsters were allowed to stabilize for 60 minutes before two 30-minute baseline clearances were obtained. This was followed by a half-hour lead-in period during which drug infusions began for the subsequent determination of two 30-minute experimental clearances. Data were averaged over the pre- and post-drug clearances. The mean arterial pressure (MAP) was monitored using a pressure transducer connected to a Gilson recorder (Middleton, WI) throughout the studies. Blood was also sampled into heparinized capillary tubes at the midpoint of each clearance period for hematocrit and plasma osmolality, sodium, and inulin measurements. Standard equations were used to determine the GFR and fractional excretion of sodium and water (FE N a and FEmo, respectively). The hamsters were sacrificed at the completion of each experiment, and their heart and liver weights were measured. 42 Protocol 1: Time control Time control clearances were determined using male golden Syrian hamsters (n=9), which were infused with 3H-inulin-saline (vehicle) only to demonstrate that the animals remained stable for the duration of the studies. After one hour of equilibration, blood and two 60-minute urine samples were collected at 30-minute intervals and analyzed. Protocol 2: Renal responses to exogenous vasopressin Normal and CM hamsters (n=6 each) were surgically prepared as described in Section During the baseline clearance periods, the animals received 0.9% 3H-inulin saline only. A half-hour infusion of 3H-inulin-saline containing [Arg8]-vasopressin (AVP) at 0.3 ng-kg^ -min"1 elapsed before two 30-minute post-drug periods began. Protocol 3: Renal responses to exogenous Vi antagonist and V2 agonist Normal and CM hamsters (n=6 each) were studied as in Protocol 2. However, a Vi receptor antagonist (ViANT; [Phenylac1, D-Tyr(Et)2, Lys6, Arg8, des-Gly9]-vasopressin) and a V 2 receptor agonist (V 2AG; [Deamino-Cys1, Val4, D-Arg8]-vasopressin; dVDAVP) were simultaneously infused into the animals at 0.3 ng-kg^ -min"1 after the baseline clearance periods. Protocol 4: Renal responses to exogenous Vj and V2 antagonists Normal and CM hamsters (n=6 each) were studied as described in Protocol 2 except the ViANT was infused with a V 2 receptor antagonist (V2ANT; [Propionyl1, D-Tyr(Et)2, Val4, Abu6, Arg8'9]-vasopressin) into the animals at 0.3 ng-kg^ -min"1 after the baseline clearance periods. Blood and urine analysis Hematocrit was measured with a micro-hematocrit reader (Phillips-Drucker, Astoria Oregon, USA). Plasma and urine samples were analyzed for sodium and potassium levels using a IL943 flame photometer (Instrumentation Laboratory, Lexington Mass.). Urinary cAMP 43 measurements were determined by radioimmunoassay. Inulin clearance: determination of glomerular filtration rate Ten microlitres of plasma or urine were transferred to scintillation vials, and 5 ml of Scinti Verse were then added. A Beckman LS 6500 Multipurpose Scintillation Counter (Fullerton, CA) was used to measure the radioactivity. Figure 2.1 summarizes the experimental approach in the clearance studies described above. 2.2 In Vitro Experimental Design The approach to completing competitive binding assays is illustrated in Figure 2.2. 2.2.1 Animals Male Wistar rats weighing 250 to 300 g were obtained from Charles River Breeding Laboratories (Wilmington, MA). The animals were first injected with sodium pentobarbital (50 mg/kg, i.p.). Their kidneys were removed after the onset of anesthesia and dissected along the longitudinal axis to extract the papillary regions. 2.2.2 Tissue preparation Isolation of inner medullary collecting duct (IMCD) cells The papillary tissues were minced in 1 ml of 37°C RPMI-1640 medium containing collagenase (1.5 mg/ml). They were then digested in 4 ml of the same collagenase mixture at 37°C. After 30 minutes, the tissues were resuspended with a pasteur pipette, and digestion was terminated by adding 4 ml of 37°C RPMI-1640 medium with 10% bovine calf serum (BCS). The sample was centrifuged at 1000 rpm for 3 minutes, and the resulting pellet was mixed in the buffer prior to an one-hour incubation at 37°C. The mixture was again centrifuged at 1000 rpm for 3 44 Normal Hamsters Cardiomyopathic Hamsters C L E A R A N C E S T U D I E S PHASE I 0.9% Saline-vehicle PHASE II Drug Infusions Vasopressin Vi Antagonist + V2 Agonist V i + V2 Antagonists Blood and Urine Samples - GFR, urine volume and sodium - Hematocrit, plasma electrolytes Figure 2.1 In vivo experimental design. Summary of procedures for and analyses of clearance studies performed on normal and cardiomyopathic hamsters. 45 IMCDs of Left Kidney IMCDs of Right Kidney Vehicle Vehicle Vasopressin Vasopressin Forskolin L Rp-cAMPS + Vasopressin ET Receptors ET B Receptors K d and B m a x ::i Figure 2.2 In vitro experimental design. Summary of procedures for and analyses of competitve binding studies with isolated rat inner medullary collecting duct (EMCD) cells. 46 minutes, after which the supernatant was aspirated and the pellet was resuspended in 37°C RPMI-1640 medium (lml/lOOfig protein) containing isobutylmethylxanthine (50 \iVml). A 10-minute incubation at 37°C followed. A sample of the IMCD preparation was sent to the Division of Anatomic Pathology, University of British Columbia, for histological identification. The observed cells were low columnar and lacked a brush border. Histological sections stained by specific mouse monoclonal antibodies were also positive for epithelial membrane antigen (EMA) (Dako, Santa Barbara, CA) and low molecular weight cytokeratin (LMWCK) (Enzo, New York) but were negative for high molecular weight cytokeratin (HMWCK) staining (Enzo, New York). These findings confirm the cells in the sample were of collecting duct origin. Drug treatments of isolated IMCD cells Time control experiments were first performed to ascertain ET-1 binding properties remained consistent following the overnight incubation and that any alteration in ET receptor kinetics demonstrated in subsequent studies using treated IMCD cells were drug-induced. IMCD cells isolated from both kidneys of each rat were incubated in RPMI-1640 medium containing 10% BCS at 37°C for 17 hours. In additional experiments, IMCD cells of one kidney from each animal were pretreated with either 10"8 to 10"5 M of [Arg8]vasopressin (AVP) or 10"6 M of forskolin in RPMI-1640 medium containing 10% BCS and incubated under the same conditions as for the time controls. Where the effect of a specific PKA inhibitor was investigated, IMCD cells were pretreated with 10"6 M of Rp-cAMPS, which is the Rp diastereoisomer of cAMP, for 2 hours at 37°C prior to the overnight incubation with AVP. The IMCD cells from the contralateral kidney constitute the control groups and were treated under the same conditions except for the addition of AVP, 47 forskolin, or Rp-cAMPS. Samples were collected following the incubation period and stored at -80°C for competitive binding assays. Preparation of homogenate The tissues were homogenized on ice with a Caframo stirrer (Wiarton, Ontario) at setting 8 for 2 minutes prior to performing competitive binding assays. The homogenate was then centrifuged at 1500 rpm for 5 minutes to remove nuclei and debris, and its supernatant was further sedimented at 15000 rpm for 15 minutes at 4°C. Thereafter, the pellet was resuspended in 5.0 ml of 1% Tris-Tyrode buffer and sonified briefly with a Branson Sonifier (Danbury, C T ) . Two 50 u.1 aliquots of each purified tissue sample were collected for protein measurements, which were determined by the Lowry method. 2.2.3 Competitive binding assays The maximal binding capacity (Bmax) and dissociation constant (Ka) of E T receptors were determined from competitive binding experiments. ET-1 was radiolabeled with 1 2 5I using the Chloramine-T method (72). The homogenate (100 ul) prepared from Section was mixed with increasing cold ET -1 concentrations (0.39, 0.78, 1.56, 3.13,, 25.0, 50.0, and 100 ng per 20 ul), and 100 ul of 1 2 5I-ET-1 (-100 000 cpm) was later added to each tube. In studies that specifically examine the E T B receptor, the homogenate was preincubated with an E T A antagonist BQ 123 (100 nM) for 10 minutes prior to cold ligand addition. Labeled and unlabeled ET-1 were allowed to compete for receptor sites on the cellular membranes at 22°C for 30 minutes, after which reactions were terminated by adding 50 ul of 10% normal rabbit serum, 100 ul of goat anti-rabbit, and 1 ml of 6% polyethylene glycol (m.w. 8000). Subsequently, the samples were centrifuged at 3000 rpm for 30 minutes. Receptor-bound radioactivity was separated from free ligands by vacuum suction and counted in a LKB 1275 minigamma counter (Wallac, Finland). 48 Analytic Methods Specific binding was the difference between the binding with buffer (total binding) and 100ng/20ul of unlabeled ligand (nonspecific binding). This determined the actual amount of 1 2 5I-ET-1 bound to receptors and eliminated irrelevant radioactive counts. The computer-based LIGAND program (Munson and Rodbard 1980) derived the dissociation constant (Ka) and maximal binding capacity (Bmax). The ratios of ET receptor subtypes in the IMCD were calculated by comparing changes in Bmax values for competitive ET-1 binding with or without an E T A antagonist. 2.3 Reagents Inactin was purchased from Byk Gulden Konstanz (Germany). The Vi and V 2 antagonists, V 2 agonist, ET-1, and the selective E T A antagonist ( B Q 123) were all obtained from Bachem Bioscience (Bubendorf, Switzerland). The specific PKA inhibitor (Rp-cAMPS) was purchased from Calbiochem Biochemicals (San Diego, CA). 3H-inulin-methoxy was purchased from NEN Dupont (Boston, MA) and 1 2 5I from Mandel Scientific Company Ltd. (Guelph, ON). Scinti Verse was obtained from Fisher Scientific and bovine calf serum (BCS) from Hyclone Laboratories, Inc. (Logan, Utah). All other reagents were from Sigma Chemicals (St. Louis, MO). 2.4 Statistics Except where indicated, reported values in text, figures, and tables are the means + SE (SEM) of the number of experiments indicated by n. Paired and unpaired Student's / tests were used to determine differences within the same group as appropriate, and analysis of variance was used to compare two or more groups. Results were considered significant if p < 0.05. 49 CHAPTER 3 RESULTS 3.1 In Vivo Studies 3.1.1 Time control studies Data from normal golden Syrian hamsters (n=9) show the animals remained stable for the duration of the experiments. Table 3.1 indicates infusion of 0.9% saline-vehicle had no effect on the hematocrit, which was 49 + 3% in Phases I and IT. Moreover, the renal excretory function in these animals was unchanged throughout the studies. All of the measured clearance parameters remained the same despite a slight decrease (8.8%) in MAP. 3.1.2 General characteristics of cardiomyopathic hamsters The CM hamsters employed in the experiments were 280 to 300 days old. Based on their clinical signs, they were categorized in the moderate stage of CHF except for one animal which had severe heart failure. Although their body weight was lower compared to the age-matched control (126 + 1 vs. 158 + 1 g, p<0.01, n=22), Figure 3.1 indicates that the CM hamsters exhibited cardiac hypertrophy because their heart to body weight ratio was significantly higher than normal (4.03 + 0.09 vs. 3.38 + 0.06 mg/g, p<0.01). Renal excretory function may be related to the heart to body weight ratio, which reflects the severity of CHF. Figure 3.2 demonstrates that the normal group generally had higher GFR than the CM hamsters. Nevertheless, GFR seemed well-maintained in the early stages of CHF when the heart to body weight ratio was between 3.5 and 4.0 mg/g; however, further cardiac enlargement was associated with depressed GFR. Because a decline in GFR is indicative of renal failure, some of the CM hamsters might have suffered kidney dysfunction as well. Figure 3.3 illustrates a linear relationship between the fractional excretion of water (FEH2o) and heart size in the CM animals (r=0.87, p<0.01, n=19), suggesting an association in tubular reabsorption. Water 50 Table 3.1. Hemodynamic and renal excretory function of normal golden Syrian hamsters (n=9). Phase I Phase U MAP (mmHg) 137+17 125 ± 16* Hematocrit (%) 49 ± 3 49 ± 3 GFR (ml-min'^ kg1) 2.5 + 0.5 2.9 ±0 .5 U V (ul/min) 7.8 ±1 .5 6.6 ± 0.9 UNaV (u.mol/min) 1.7 ±0 .2 1.5 ±0 .4 P N a (mM) 138 + 2 139 ± 2 F E N a (%) 4.1 ±0 .8 3.6+1.1 FEH20 (%) 2.9 ± 0 . 4 2.1 ±0 .4 Values are expressed as mean ± S E M . * p<0.05 vs. baseline. M A P , mean arterial pressure; GFR, glomerular filtration rate; UV, urine flow; U>j a V, urinary sodium flow; P > j a , plasma sodium; ¥E^a, fractional sodium excretion; FEH2O, fractional water excretion. 51 6 T 5 + r 4 + o o 8 I I 3 + 2 + 1 + 0 Normal Cardiomyopathic Figure 3.1 Comparison of heart weight/body weight ratio (mg/g) in normal and cardiomyopathic hamsters (n=22). 52 1.0 I 0.5 a o 2.5 A Normal • C d^iomyopathic A • A A A • A A " • A A A 3.0 3.5 4.0 4.5 5.0 5.5 Heart Weight/Body Weight (mg/g) Figure 3.2 Renal function of normal and cardiomyopathic hamsters. The basal glomerular filtration rate (ml/min) tends to decrease as the heart to body weight ratio (mg/g) increases. n=22 for control group; n=21 for cardiomyopathic group. 53 12 x 10 + •Normal A Cardiomyopathic 8 + y = 5.53x-18 6 + m 1*4 4 + 2 + # • A A * A • 4 0 3.0 3.5 4.0 4.5 5.0 5.5 Heart Weight/Body Weight (mg/g) Figure 3.3 Fractional excretion of water (FEH2O) in normal and cardiomyopathic (CM) hamsters. Trendline is fitted to data from the CM group and shows that the heart to body weight ratio (mg/g) correlates positively with the FEH2O (%) in the diseased hamsters (r=0.87, p<0.01;n=19). 54 clearance was greater in the presence of decreased glomerular filtration. As Figure 3.4 illustrates, a negative linear relation between the FEH2O and GFR exists in the disease state (r=-0.53, p<0.05, n=19). This observation may be attributed to changes in the production of cAMP, which is the V 2 receptor second messenger required for water transport in the collecting duct. Figure 3.5 indicates that urinary cAMP levels decreases exponentially as heart failure becomes more severe in the CM hamsters (r=0.78, p<0.01; n=16). The control group did not exhibit this particular trend. In the present studies, the averaged basal urinary cAMP concentration (UCAMP; Figure 3.6) and plasma osmolality (Posm) in the CM hamsters were maintained at normal levels (UCAMP: 8.9 + 1.6 vs. control 10.5 ± 1.5 nmole/ml, ns; Posm: 293 + 3 vs. control 293 + 6 mosm/kg, ns; n=18). 3.1.3 Renal responses to exogenous vasopressin or a V t antagonist with a V 2 agonist Administration of AVP to the CHF hamsters had no effect on the MAP and clearance parameters (Table 3.2). However, normal hamsters exhibited a reduction in the FEmo (4.6 + 1.0 vs. 2.5 + 0.5%, p<0.05, n=6) in the absence of any changes in systemic and renal hemodynamics. The urinary sodium excretion (LWV) and fractional excretion of sodium (FENa) also decreased by 40% from baseline during AVP infusion but did not reach statistical significance (p=0.066 and p=0.057, respectively). The effects of an exogenous V 2 agonist on renal clearance were studied in another set of normal and CM hamsters. A V i antagonist was administered with the V 2 agonist to block the effects of endogeous AVP, whose release may be enhanced by anesthetics. As Figures 3.7 and 3.8 show, the control group following the combined infusion of a V i antagonist and a V2 agonist, which decreased salt and water excretion to a similar extent as AVP. While the CHF hamsters exhibited an attenuated response in all of the measured hemodynamic and clearance parameters, the normal hamsters became antinatriuretic and antidiuretic following selective V2 receptor 55 12 x 10 + • Normal • Cardiomyopathic 8 + 0 s-O CN 35 w to 6 + y=-2.27x+8.3 4 + • • • 2 + 0 1.0 • • y=-0.43x+3.43 + + + 1.5 2.0 2.5 3.0 GFR (ml»min"1»kg'1) 3.5 4.0 Figure 3.4 Relationship between renal function and fractional excretion of water (FEH2O) in normal and cardiomyopathic ( C M ) hamsters. Red trendline is fitted for data from the C M group and shows that the basal glomerular filtration rate (ml-min'-kg1) correlates negatively with the FEH2O in the diseased animals (r= -0.53, p<0.05; n=19). Blue trendline indicates that the negative correlation is absent in the normal group (r=-0.21, ns). 56 3.0 3.5 4.0 4.5 5.0 5.5 Heart W e i g h t / B o d y Weight (mg/g) Figure 3.5 Relationship between heart to body weight ratio (mg/g) and basal urinary cAMP levels (nmole/ml) in normal and cardiomyopathic hamsters. Exponential decrease is fitted to data from cardiomyopathic hamsters. r=0.61, p<0.01; n=16. 57 30 T 25 + f 20+ i 15 + A A 10+ 5+ 0 i A A A A I Normal A I A A A A Cardiomyopathic Figure 3.6 Comparison of basal urinary cAMP levels (nmole/ml) in normal and cardiomyopathic hamsters. n=18 for control group; n=17 for cardiomyopathic group. 58 Table 3.2. Hemodynamic and renal excretory function of cardiomyopathic and control hamsters in response to exogenous AVP (n=6). CONTROL GROUP CHF Baseline AVP Baseline AVP MAP (mmHg) 123 ± 9 115 + 8 111 ± 9 97 ± 13 Hematocrit (%) 50 ± 2 50 + 2 49 ± 2 47 ± 2 GFR (ml-min^kg"1) 1.5 ± 0.1 1.5 + 0.1 2.7 ± 0.2 n 2.3 ±0.5 UV (ul/min) . 6.1 + 1.5 5.4+1.3 6.1 ±0 .9 6.0+1.4 UNOV (|j.mol/min) 3.5 + 0.9 2.1+0.5 2.5 ±0 .8 1.7 ±0 .4 PNa(mM) 140 + 2 138+1 145 ± 2 148 ± 5 F E N A (%) 6.7+1.3 4.0 + 0.9 5.2+ 1.6 4.6 + 1.2 FEH20 (%) 4.6+1.0 2.5 ±0 .5* 2.1 ± 0 . 5 f 2.6 ±0 .7 Values are expressed as mean + SEM. * p<0.05 vs. baseline;f p<0.05 vs. control; n p<0.01 vs. control. 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O S •« i) ai 5 f> "S £ o cu O S ° • SP S 3, cd , <U "5 < 00 cj cd W 61 activation. Table 3.3 shows that the GFR remained stable in the drug clearance periods for both animal groups; however, significant decreases in urinary flow rate (UV), UwaV, F E N a , and FEH2o were evident only in the healthy hamsters. 3.1.4 Renal responses to exogenous Vi and V 2 antagonists To reveal any differences in endogenous AVP activity between normal and CHF hamsters, Vi and V 2 antagonists were simultaneously infused into the animals to block the respective receptors. The hemodynamic and renal responses are summarized in Table 3.4. In both groups of hamsters, the MAP decreased by -15% from baseline (p<0.05). Interestingly, the hematocrit and all other measured clearance parameters were unaffected in the normal animals. In contrast, the CHF hamsters demonstrated a 7.7% decrease in their hematocrit and significant increases in sodium and water excretion. While the GFR and plasma protein and sodium levels remained steady, the UV and U N 3 V significantly increased following the infusions of the AVP antagonists (p<0.01). Figures 3.9 and 3.10 illustrate that the mean baseline levels of F E N a and FEmo were increased by 32 and 39%, respectively, with Vi and V 2 receptor blockade in the CM hamsters. 3.1.5 Urinary cAMP measurements To ascertain whether the renal responses were V2-dependent, changes in cAMP levels after selective V 2 activation and blockade were determined in the urine of normal and CM hamsters. Based on the consistent increases in cAMP excretion, Figure 3.11 suggests that administration of a V 2 A G elevated the production of the second messenger from baseline in both groups of animals but had a greater influence on the CM hamsters. Figure 3.12 further supports the idea that V 2 hyperesponsiveness occurs in pathological conditions. Nonselective blockade by Vi and V 2 antagonists reduced urinary cAMP by up to 43% in the CM hamsters and up to 20% in 62 Table 3.3. Hemodynamic and renal excretory function of cardiomyopathic and control hamsters in response to exogenous Vi antagonist (ANT) and V 2 agonist (AG) for n=6. CONTROL GROUP CHF Baseline Vj ANT + V 2 A G Baseline V i ANT + V 2 AG MAP (mmHg) 154 + 6 149 ± 7 100 ± 7 n 90 + 5 Hematocrit (%) 55 + 1 54+1 4 8 ± 3 f 49 + 3 Plasma Protein (g/dl) 6.0 + 0.9 5.4 + 0.8 5.0 + 0.2 4.9 + 0.3 GFR (ml-mm^kg"1) 2.2 + 0.3 2.03 + 0.3 2.5 + 0.4 2.0 ±0 .4 UV ((il/min) 10.0+1.9 5.3+ 1.0* 5.1 + 1.3f 5.9 ± 1.1 UNaV (p.mol/min) .3.0 + 0.4 1.3 + 0.1*** 2.9 + 0.8 2.6 ±0 .2 P N a (mM) 137 + 2 134+1 138 + 3 135 ± 4 F E N a (%) 6.9+1.1 3.9 + 0.7** 6.8 + 2.0 6.9 ± 1.3 FEH20 (%) 3.1+0.6 1.8 + 0.4* 2.7 + 0.8 2.6 ±0 .5 Values are expressed as mean ± SEM. * p<0.05 vs. baseline; **p<0.01 vs. baseline; ***p<0.005 vs. baseline;1 p<0.05 vs. control; n p<0.01 vs. control. MAP, mean arterial pressure; GFR, glomerular filtration rate; UV, urine flow; U N a V, urinary sodium flow; PN a, plasma sodium; FE N a , fractional sodium excretion; FEmo, fractional water excretion. 63 Table 3.4. Hemodynamic and renal excretory function of cardiomyopathic and control hamsters in response to exogenous Vi and V 2 antagonists (ANT) for n=6. CONTROL GROUP CHF Baseline V! & V 2ANT Baseline V i & V 2 ANT MAP(mmHg) 131 +9 112 + 5* 119 ± 5 102 ± 5 * Hematocrit (%) 55 ± 1 55 + 1 5 2 ± l f 48 + 1** Plasma Protein (g/dl) 5.7 + 0.2 5.6 + 0.2 5.6 ±0 .2 5.1 ±0 .3 GFR (ml-min -^kg1) ' 1.9 + 0.3 2.0 ± 0 . 4 2 . 6 ± 0 . 2 t 3.2 ±0 .5 UV (uJ/min) 4.0 + 0.6 4.7 ±0 .5 4.8 ± 0.8 8.1 ±0.6*** U N 3 V (u,mol/min) 1.1+0.2 1.3 ± 0 . 2 2 . 2 ± 0 . 3 f t 4.0 ±0.5** PNa(mM) 135 + 2 137 ± 1 140 ± 2f 136 + 2 F E N a (%) 3.2 + 0.8 3.6 ±0 .7 4.8 ±0 .6 7.9 ± 1.1* F E H 2 O ( % ) 1.6 + 0.4 1.9 ±0 .4 1.5 ± 0 . 2 2.2 ±0 .3* Values are expressed as mean ± S E M . * p<0.05 vs. baseline; **p<0.01 vs. baseline; ***p<0.005 vs. baseline; 1 p<0.05 vs. control; f t p<0.01 vs. control. M A P , mean arterial pressure; GFR, glomerular filtration rate; UV, urine flow; U N a V, urinary sodium flow; P N A , plasma sodium; F E N A , fractional sodium excretion; FEH2O, fractional water excretion. 64 C/3 OQ CN > + (S3 o ! 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I '3 < % s aj « 5 W o CU O 75 « .52 ' * 5 E C/3 WH * C C C w 3 .2 § a — [ I . £ cd CN ^H ^ — o ,—i O c/a « e 3 § c . — cs CS t i . CJ ^> CS CJ 66 350T 50 Normal Cardiomyopathic Figure 3.11 Percent increase in urinary cAMP levels following selective V 2 activation in normal and cardiomyopathic hamsters. Animals were infused with 0.9% 3H-inulin saline during baseline clearance periods and either vasopressin (AVP) or a Vi receptor antagonist ( V i A N T ; Phenylac1, D-Tyr(Et)2, Lys6, Arg8, des-Gly9]«vasopressin) with a V 2 agonist (V 2AG; dVDAVP) at 0.3 ng'kg'-min'during experimental clearances. Individual experiments are shown (n=6). 67 110 T * 50 + 40--30 Normal Cardiomyopathic Figure 3.12 Percent decrease in urinary cAMP levels following V 2 blockade in normal and cardiomyopathic hamsters. Animals were infused with 0.9% 3H-inulin saline during baseline clearance periods and a Vi receptor antagonist ( V i A N T ; Phenylac1, D-Tyr(Et)2, Lys6, Arg8, des-Gly9]'vasopressin) with a V 2 receptor antagonist ( V 2 A N T ; Propionyl1, D-Tyr(Et)2, Val4, Abu6, Arg8,9]-vasopressin) at 0.3 ng-kg'-min"1 during experimental clearances. Individual experiments are shown (n=6). 68 the controls. On average, the diseased animals produced almost five-fold as much U C AMP than the controls following V 2 stimulation (164 + 56 vs. 34 + 12%, p<0.05, n=6). As well, antagonism of the V 2 receptors affected the CM group more significantly as well, reducing the cAMP generation by 25 + 4 % compared to only 8.5 + 3.8 % in the control. Figure 3.13 summarizes these results. 3.2 In Vitro Studies 3.2.1 Time control studies Isolated IMCD cells from right and left kidneys of each rat were incubated at 37°C for 17 hours prior to performing competitive binding studies to ensure ET receptor kinetics remained constant in the absence of AVP, forskolin, or Rp-cAMPS treatment. Figure 3,14 illustrates that the incubation period and conditions did not affect ET-1 binding. Total ET receptor density between each pair of kidneys was equal (3171 + 506 vs. 3671 + 466 fmol/mg protein, n=4). Similarly, no change was observed in the densities of ET receptor subtypes (ETA: 1255 + 400 vs. 1430 + 319 fmol/mg protein; ET B : 1917 + 202 vs. 2241 + 367 fmol/mg protein). Receptor affinity (Ka) was likewise stable following the overnight incubation (Total ET receptor: 2.0 + 0.7 vs. 2.5 + 0.7nM;ETB: 1.0 ±0 .7 vs. 1.3+0.1 nM). 3.2.2 Effects of vasopressin and forskolin on endothelin-1 binding to IMCD Overnight incubation of AVP (10"8 to 10'5 M) consistently decreased ET-1 binding in IMCD cells. Figure 3.15 shows a representative competitive displacement of 125I-ET-1 by increasing concentrations of unlabeled ET-1 in the presence and absence of AVP and the E T A antagonist, B Q 123. The AVP-induced effects on ET-1 binding resulted from a reduction of the total ET receptor density (Bmax) in IMCD cells with parallel decreases of ~25 to 31% in E T A and E T B receptor subtypes (Figures 3.16 and 3.17). 69 03 c t - g cs o c • c "° o c3 C/3 ' 5 o SO 03 <N > + + 09 " f i O < > + m o CxD =5 ~ Q -2 cd 00 § « 3 0 a > « 70 EH O +•* Oh <U < u rt m H W o cx 1) o < H pq to 2 .s «-<3> CD s « 9- ^ o cx to <l> rt H W 13 43 2 (insjojd 8UI/|OUIJ) 71 Figure 3.15 Representative competitive displacement curve for I-ET-1 binding to inner medullary collecting duct (IMCD) cells with vasopressin (AVP) treatment. Isolated rat IMCDs were preincubated for 17 hours at 37°C with or without 10"6 M of AVP, and binding assays were performed with 150 pM of 125I-ET-1 and 0 to 0.1 pM of unlabeled ET-1 at 22°C for 30 minutes. Specific binding to E T B receptors were determined in the presence of an E T A antagonist (100 nM), BQ 123. 72 o c o u C/3 C/3 OH O CO 03 > • • O 0> 03 cu CO 2 oo I o o o o oo o o o o o © o o o o o o 1* o o o o o o o o o •55 60 +| (upjcud 8ui/|oug) X B m g 73 o o U o c o U -S CA cn i -Cu o c/5 > m m H H '35 C/3 I -o cn > < < w m • • s © o cn cn <U «-< CX O cn > o oo • o o o o S O o o o o o o o o o © o o CN XBUI, o o o (upjojd 9ui/[0ug) x m u g c d 4—' GO GO ed CU i s a. ^ C TJ o <u TJ O 4> 3 warn r, , V a o c cu o « GO ~ c d <U > o 'S oo.SP .23 C c/3 <u NH g>2 74 Because AVP primarily activates the PKA pathway via V 2 receptors in IMCD, the second messenger cAMP may also be important in ET receptor regulation. To determine such a potential role, IMCD cells were incubated with forskolin (10"* M), which directly activates adenylate cyclase. Forskolin mimicked the inhibitory influence of AVP on ET-1 binding, as shown by the displacement curves on Figure 3.18. It decreased the Bmax of ET receptors from 6099 + 1319 to 3939 + 672 frnol/mg protein (p<0.05, n=6). Particularly, Figure 3.19 indicates that forskolin treatment affected E T B receptor density only (5029 + 998 vs. 2941 + 674 fmol/mg protein, p<0.005) but did not alter E T A receptors at all (1070 + 393 vs. 998 + 224 fmol/mg protein, ns). 3.2.3 Effect of Rp-cAMPS on endothelin-l binding to IMCD Involvement of the PKA pathway in ET receptor regulation was strongly implicated when the IMCD cells were incubated with a cAMP analogue, Rp-cAMPS, and then with AVP at lO^M. Rp-cAMPS clearly prevented AVP-induced ET receptor downregulation. As Figure 3.20 shows, the total ET receptors (5890 + 840 fmol/mg protein, n=5) were significantly more abundant in cells preincubated with Rp-cAMPS and AVP than with AVP alone (4114 + 800 fmol/mg protein, p<0.01) and were comparable to previously reported control levels (5438.65 + 1318 fmol/mg protein, n=22). Furthermore, Figure 3.21 illustrates Rp-cAMPS was effective in reversing the inhibitory influence of AVP on E T B receptors (3155 + 724 vs. 4589 + 734 fmol/mg protein, p<0.01) and not E T A (958 + 239 vs. 1300 + 239, ns). It is noteworthy that pretreatment of IMCDs with Rp-cAMPS alone did not affect ET-1 binding (total ET receptors: 2760 + 518 vs. 2843 + 538 frnol/mg protein, ns; ET B : 2372 + 491 vs. 2336 + 492 frnol/mg protein, ns). Hence, Rp-cAMPS affected AVP-stimulated and not basal regulation of surface ET receptors. 75 Control A Control+ BQ123 -°- Forskolin • Forskolin+ BQ123 0 CQ 1 H m T 3 JL> <U Xi P3 —J *S o x -9 -8 Log [ET] (M) Figure 3.18 Representative competitive displacement curve for 125I-ET-1 binding to inner medullary collecting duct (IMCD) cells with forskolin treatment. Isolated rat IMCDs were preincubated for 17 hours at 37°C with or without 10"6 M of forskolin, and binding assays were performed with 150 pM of 125I-ET-1 and 0 to 0.1 uM of unlabeled ET-1 at 22°C for 30 minutes. Specific binding to ET B receptors were determined in the presence of an E T A antagonist (100 nM), BQ 123 76 0 1 o o O • • CO M O cx o rt CQ H W cn CL a> o w rt < H W c/5 - = cn o a, D O <D rt H w o H (uisjoad Sui/[oug) X B U I g <L> 77 m 00 • — ft O 00 03 > • -x-•X-00 O ft t j a> rt m H LU oo O •*-< ft CJ to rt < H UJ ft UJ o H 2 cj a. C N O 4> , o jq o o p — f j j c CQ + 1 cS O <U CN O (uisjojd §ui/{oug) xma 78 g o O . PQ 7000 T 6000 + 5000 + 4000 + 3000 + 2000 1000 0 ET Receptors I E T B Receptors E T A Receptors CONTROL AVP AVP + Rp-cAMPS Figure 3.21 Blockade of vasopressin (AVP)-induced endothelin receptor regulation. Isolated rat inner medullary collecting duct (IMCD) cells were preincubated with 10"6 M of AVP for 17 hours at 37°C or Rp-cAMPS for 2 hours before AVP treatment prior to competitive binding of ET-1. Distribution of E T A and ET B receptor subtypes was determined with an E T A antagonist (100 nM), BQ 123. Maximal binding capacity (Bmax) values were derived by the LIGAND program. Values are mean ± SE. n=22 for control; n=5 each for AVP and AVP + Rp-cAMPS treatments. *Significant difference (p<0.05) from Bmax following AVP treatment. 79 3.2.4 Changes in endothelin receptor binding Scatchard transformations were used to elucidate additional changes in ET-1 binding in the presence of AVP or forskolin. As shown in Figure 3.22, 10'8 to 10"5 M of AVP or IO-6 M forskolin consistently increased the affinity of the total ET receptors. Specifically, the K<j for E T B receptors tended to decrease and reflects the increased affinity of this subtype to the ET-1 ligand (Figure 3.23). PKA blockade with Rp-cAMPS also inhibited AVP-induced changes in the Kd values, which were comparable to control levels and significantly higher than in IMCD cells pretreated with AVP only (Figure 3.24). Figures 3.25 and 3.26 are representative scatchard plots for E T B receptors in AVP- and forskolin-treated IMCD cells. Results from the treated cells consistently showed that the x-intercept, B r a a x , decreased and the slope, -1/Ka, increased from control. Taken together, the data suggest that a PKA-dependent mechanism affected ET-1 binding through alterations in receptor density and affinity. 80 o to c o U o C/3 J -o t i n • o C/3 s— o J s i t •9 o • o .5 s CA c 5 - t 81 £9 M 3 T 5 CU B cu .S «i -o c CU 3 O co cn 2 - <2 p o cfi ^ 1- <— O u B . P 4> ft. - < a? s 2 c 2 •8£ I | CO jjj O eC 3 TJ c o e c3 o •5 9 0 a 1 .ap S 1 / 3 CO CN cu «« .t! TJ * s ~° CO cu SI w to u. .s °* CO cU t o S cu O *c « & © S 8 ° w C/3 It' | i | E co « H 1 CO UJ — _ c 3 E C+H 3 O o cu « 3-s D Crt * UJ S va. £ > 8 o 2 w Q co y IT C N rn o 3 TJ c (PVu) p ^I Ii U H U ? i M 2 c o H 9 UJ 5 C*H < . O Q oo g TJ « .s s 1-81 cu o * O >». V S c o o E o cfa o o o •c a. 82 (Wu) p }[ 83 0 H 1 1 1 1 ' 1 1 H 1 1 1 1 0 0.20 0.40 0.60 0.80 1.00 1.20 Bound (nM) Figure 3.25 Representative scatchard plot for E T B receptors in untreated and vasopressin (AVP)-treated inner medullary collecting duct (IMCD) cells. Isolated IMCD cells were preincubated with or without 10"6 M of A V P for 17 hours at 37°C prior to competitive binding of ET-1. The L I G A N D program was used to derive the maximal binding capacity (B m a x ) and dissociation constant (Kj) of the E T B receptors. 84 0.20 -, • Control A Forskolin 0.20 0.40 0.60 0.80 1.00 Bound (nM) Figure 3.26 Representative scatchard plot for E T B receptors in untreated and forskolin-treated inner medullary collecting duct (IMCD) cells. Isolated IMCD cells were preincubated with or without lO^M of forskolin for 17 hours at 37°C prior to competitive binding of ET-1. The LIGAND program was used to derive the maximal binding capacity (Bm a x) and dissociation constant (Kd) of the E T B receptors. 85 CHAPTER 4 DISCUSSION Reported plasma AVP elevations in CHF patients and experimental models have led to the speculation that AVP participates in the pathogenesis of impaired water excretion in the disease state (54, 83, 156). Hence, the purpose of this thesis was to further understand the significance of AVP in CHF and to examine its interaction with ET. The results reveal that basal sodium and water reabsorption is maximal in CM hamsters, thus accounting for the attenuated renal response to exogenous AVP or a selective V 2 agonist. Additional studies support that these animals exhibit AVP hypersensitivity in the kidney because they generated significantly more cAMP than the control group after V 2 activation. This enhanced response to AVP in CHF might have evolved from increased receptor activity and/or suppression of another endocrine system, ET, which would otherwise antagonize AVP-stimulated water flow in the collecting duct. 4.1 Cardiomyopathic hamsters as an experimental model of congestive heart failure The CM hamsters employed in this study develop progressive cardiomyopathy with distinguishable characteristics at different stages of the disease. Signs of the dystrophic state include cardiac hypertrophy, a lower than normal body weight, pleural effusions, and generalized edema (71, 156). Dilation of the right atrium and ventricles accompanied by calcium accumulations in the ventricles and other tissues have also been reported (9, 71). It is postulated that calcium overload induces necrosis of cardiac cells (126). As in CHF patients, abnormal regulation of neurohumoral systems may be present in CM hamsters. Horvath et al had first speculated that hypothalamic secretion of AVP is increased in CM hamsters (68). As well, Sved et al found that elevated circulating AVP concentrations in these animals are apparent at 5 months of age during compensated heart failure and almost double 86 by 11 months during decompensated heart failure (156). Because the CM hamsters in the present study were aged between 280 to 300 days, their plasma AVP levels were likely inappropriately high. The events leading to this phenomenon are unclear but are probably independent of osmotic or hypovolemic stimuli. Data from the present studies show that the plasma of the CM animals was not hyperosmotic and arterial blood pressure was generally lower but not significantly different from control. Instead, mechanisms might have involved an enhancement of AVP secretion in response to non-osmotic mechanisms, an inability to suppress release, increased synthesis, or decreased metabolism and degradation. Both hematocrit and plasma protein concentration of the CM hamsters at baseline levels also tended to be lower than normal, suggesting hemodilution from fluid retention. Because the cardiomyopathic hamsters exhibit many of the signs and symptoms present in patients with CHF, they represent a suitable experimental model for studying edema formation characteristic of the disorder. 4.2 Systemic and renal hemodynamics AVP is a potent vasoconstrictor (116, 117), but the MAP was unaltered in both healthy and CM hamsters following the administration of AVP. This observation may be explained in several ways. First, it is well-accepted that V i activity has a minor role in the maintenance of vascular tone under normal conditions (12, 14, 49). Vasoconstriction induced by acute AVP infusions might have been offset by the baroreflex mechanism or other neuroendocrine systems in the control group. As well, several studies support the notion that V 2 receptor activation produces prostaglandins, kinins, or NO that opposes V i effects in the systemic and renal circulations (91, 107, 123). Smith et al previously reported that chronic elevations of plasma AVP do not induce hypertension unless water intake is concomitantly increased (152). Hence, lack of a pressor 87 response in the CM hamsters, which supposedly have elevated endogenous plasma AVP concentrations, was not unexpected (156). Moreover, the increase in circulating AVP after short-term infusions of this hormone was probably minor relative to the chronically high baseline levels and was therefore insufficient to cause significant hemodynamic changes in the CM hamsters. Alternatively, compensation for AVP-induced vasoconstriction might have included end-organ resistance as a result of V i receptor or second messenger regulation. These mechanisms would have prevented major changes in systemic hemodynamics during acute AVP infusion. The hemodynamic effects of combined V i antagonist and V 2 agonist administration are more complex. It remains controversial whether basal V i activity contributes to the maintenance of vascular tone under normal and heart failure conditions because selective V i antagonism does not always lower arterial blood pressure (133). Nicod et al found that V i inhibitors caused pronounced vasodilation in patients with severe heart failure and raised AVP levels but not in patients with moderate heart failure (125). The present data indicate that V 2 activation during V i blockade tended to reduce the MAP in both normal and CHF hamsters. Although the hemodynamic effect was greater in the pathological state than in control, it was not statistically significant. Absence of marked vasodilation in the two animal groups may be attributed to increased fluid retention after the infusion of a V 2 agonist or the activation of other pressor systems, such as the RAAS and sympathetic nervous system, that might have compensated for any hemodynamic change during V i antagonism (83, 132). Involvement of additional pressor agents in the regulation of MAP may be particularly important during heart failure. In fact, Manolis et al demonstrated that suppression of NE augmented AVP release in patients with CHF, suggesting that AVP has a secondary role in hemodynamic control (111). Moreover, Cowley proposed that AVP sensitizes the baroreceptors through V 2 receptors (28). Hence, administration 88 of a V 2 agonist might have partially ameliorated the heart failure-induced impairment of the baroreflex mechanism and favored vasoconstriction in the CM hamsters. This would have counterbalanced the anticipated vasodilation by a Vi antagonist. Finally, it is noteworthy that AVP antagonists are not always effective in Vi blockade and decreasing peripheral vascular resistance. Creager et al found some Vi antagonists only induced a depressor response in the presence of extremely high plasma AVP and may even increase arterial pressure in some patients with normal levels of this hormone (30). The role of V 2 receptors in hemodynamic control remains inconclusive. Liard has previously shown that Vi antagonism of physiological AVP caused a greater decrease in peripheral vascular resistance than nonselective Vi and V 2 antagonism in dogs with heart failure (106). This suggests V 2 activation induces vasodilation directly or indirectly. Interestingly, the current results indicate normal and CM hamsters became hypotensive during the simultaneous infusion of Vi and V 2 inhibitors. Fluid loss coupled with Vi antagonism might have overwhelmed the compensatory vasoconstriction by Ang II and NE during AVP blockade. However, it should be recognized that both hamster groups were euvolemic despite V2-induced diuresis because plasma protein and sodium were essentially unaltered from baseline and hemoconcentration was not evident. A potentially important cardiovascular role of V 2 receptors remains to be elucidated. Renal hemodynamics were relatively unaltered throughout the experiments as well. The GFR was well-maintained within each normal and CM hamster following AVP infusion alone. This indicates that AVP action at the afferent and efferent arterioles in these animals was minimal. The GFR tends to decline with the severity of heart failure (8, 155). Therefore, a steady GFR during Vi antagonism shown by the data also reflects mild to moderate rather than severe heart failure in the CM hamsters. With steady renal hemodynamics throughout the study, the observed 89 changes in salt and water excretion following various drug infusions were directly associated with tubular function. 4.3 Clearance responses Infusions of AVP alone or a V i antagonist with a V 2 agonist into the control hamsters caused pronounced antinatriuresis and antidiuresis with the FEna and FEH2O decreasing by 40-46%. AVP reduces renal medullary blood flow through the vasa recta by V i receptors, thereby establishing an osmotic gradient for V2-induced water diffusion from the lumen into the hypertonic medullary interstitium. Blockade of V i receptors may have increased renal medullary blood flow and induced slight "washout" of the osmotic gradient. However, the simultaneous infusion of a V 2 agonist would have promoted Na+ and urea transport via cAMP-dependent pathways in the distal nephron (124, 166). Therefore, it is likely that the marked reduction in sodium and water excretion in the control hamsters was a consequence of increased tubular reabsorption. In contrast to the normal hamsters, the same drugs had no renal effect in the CM animals. Such a blunted response might have resulted from maximal sodium and water reabsorption already induced by high endogenous AVP concentrations under the stressed conditions of CHF. Perhaps a rate limiting step exists downstream of V 2 receptor activation. The data may reflect the inefficiency of the membrane shuttling system. Trafficking of AQP2-containing vesicles from the cytoplasm to the luminal membrane of the collecting duct is a complicated process that involves cytoskeletal components and docking proteins, all of which may be under tight regulation (64, 161). Alternatively, an exhausted supply of releasable or stored AQP2, as a result of increased channel insertion into and limited endocytosis from the luminal membrane, may ensue in the 90 presence of elevated plasma AVP in CHF. Xu et al have recently demonstrated that AQP2 mRNA and protein are increased in rats with compensated and decompensated heart failure following coronary artery ligation (172). The number of AQP2 proteins was increased by approximately 50%. Moreover, Ma et al confirmed that the kidneys of CHF rats express abnormal AQP2 concentrations, which are particularly high in the renal cortex (110). Thus, the cellular machinery may be operating maximally during CHF so that slight elevations of plasma AVP from acute infusions would not have elicited additional renal responses. Differences between the renal responses to infusions of Vi and V 2 antagonists in normal and CM hamsters suggest that the kidney is hypersensitive to AVP in CHF. Under these conditions, the CHF animals became natriuretic and diuretic as their F E N 3 and FEH2O increased by 65 and 47%, respectively, from baseline values. However, the same excretory parameters were unchanged in three of the six control hamsters. In the remaining three healthy animals, endogenous AVP activity was minimal since the Vi and V 2 antagonists reduced FEna and FEH2O by less than 20%. Clearly, the physiological importance of AVP in CM hamsters is different from normal. The enhanced basal AVP response in the CM hamsters might have evolved from increased AVP receptor density or affinity, alterations in signal transduction and transcriptional control, or the alleviation of inhibitory mechanisms on AVP action during the development of CHF. 4.4 Generation of adenosine 3', 5'-cyclic monophosphate in congestive heart failure Determination of urinary cAMP levels additionally supports the idea that the kidney is hypersensitive to AVP in CHF. Basal levels of excreted cAMP decreased exponentially with the severity of heart failure. Nonetheless, the percentage change in cAMP generation after selective 91 V 2 activation in the CM hamsters was increased by almost five-fold greater than in the control animals. This corroborates with the abnormally high rate of cAMP formation by isolated IMCDs of CM hamsters following AVP and forskolin treatments (109). Hence, the anticipated fluid retention in the CM animals in response to exogenous AVP or a selective V 2 agonist was likely impeded by events downstream of adenylate cyclase activation and cAMP synthesis. During the infusion of V i and V 2 antagonists, significantly lower cAMP levels were detected in the urine of CM hamsters only. In the control group however, urinary cAMP was unchanged from baseline in half of the tested animals. The remaining healthy hamsters displayed a minor decrease (up to 20% only) in urinary cAMP relative to the CM strain (up to 43%). These observations reaffirm that endogenous AVP activity at the V 2 receptor, as reflected by increased levels of cAMP, is enhanced in the CM hamsters. Discrepancies in AVP-induced cAMP accumulation between normal and CM hamsters may be associated with the stability of this second messenger. A potential role for phosphodiesterases (PDE) in regulating the cAMP-mediated actions of AVP has been previously reported. Methylxanthine compounds, such as theophylline, inhibit cAMP breakdown and potentiate AVP action (57, 127). Moreover, elevated PDE activity was suggested as the cause of nephrogenic diabetes insipidus (NDI), leading to AVP resistance in the kidney and polyuria (67, 84). Coffey et al also showed that PDE inhibition in NDI mice facilitates increased cAMP accumulation and insertion of water channels into the luminal membrane of the collecting duct, thereby restoring the hydroosmotic response to AVP (26). That abnormal PDE activity may also be associated with water retention in CHF should be considered. Decreased breakdown of cAMP would likely enhance the activity of this second messenger and prolong the V 2 receptor signaling pathway, thus favoring antidiuresis. 92 4.5 Heterologous regulation of endothelin receptors by vasopressin Previous reports indicate that cAMP specifically downregulates E T B receptors (7, 160). Heterologous regulation of ET by other neurohumoral factors which activate the cAMP-PKA axis have also been examined briefly. For example, Takemoto et al showed that AVP desensitizes ET B receptors in rat CCD (160). The present study produced similar results. Overnight incubation of rat IMCD with AVP (10"8 to 10"5 M) consistently reduced ET-1 binding with the ET B receptor density decreasing to a comparable extent as the total ET receptors. In addition, data from competitive binding experiments confirm that cAMP has an important regulatory role in AVP-induced E T B downregulation since direct activation of adenylate cyclase with forskolin affected E T B and not E T A receptors. The ability of Rp-cAMPS, a competitive cAMP analogue, to prevent the inhibitory influence of AVP on E T B further supports the postulate that a PKA-dependent pathway is involved. That the cAMP-PKA axis selectively reduced ET B receptor density suggests distinct regulatory mechanisms exist for ET receptor subtypes. This is not surprising because only the E T B subtype possesses a PKA-activated phosphorylation sequence TV-terminal to the proposed G-protein-binding site (RKKSGMQIALNDHLKQRR) in the third intracellular loop (160). The corresponding domain in the E T A receptor is occupied by two consensus sequences for PKC phosphorylation. Hence, amino acid differences between the two ET receptors provide the basis for separate regulatory pathways. The observed AVP-induced changes in E T B receptors may be attributed to both short- and long-term mechanisms. AVP likely bound to V 2 receptors in the IMCD cells and stimulated cAMP production. Activation of PKA subsequently initiated Ser/Thr phosphorylation in the third intracellular loop of nearby E T B receptors. This in turn uncoupled the E T B receptor from heterotrimeric G-proteins and prohibited further signal transduction, thereby causing rapid 93 receptor desensitization. Koshimizu and colleagues have already speculated that phosphorylation of the intracellular loops of the ET B receptors is potentially important in the desensitization of human ET B receptors (98). Their proposal is plausible since PKA phosphorylation of the (3-adrenergic receptor, which is also a G-protein coupled seven-transmembrane receptor, causes desensitization at low agonist concentrations (nanomolar) (43). However, site-directed mutagenesis of the particular serines and threonines is necessary to ascertain the PKA-activated phosphorylation motif is indeed involved in ET B regulation. Because the cells were pretreated with AVP or forskolin for 17 hours, long-term regulation of ET receptors involving degradation, inactivation, and internalization, as well as transcriptional control, is likely important. Receptor internalization might have followed desensitization since the ET B receptor density was reduced and was accompanied by an increase in receptor affinity, which could represent a compensatory mechanism for decreased ET binding sites. These observations are in agreement with a previous report, which supports that ET receptor downregulation results after an 18 hour exposure to ET-1 in rat mesangial cells (10). In addition, AVP might have influenced ET B gene expression. Asada et al demonstrated that cAMP began to decrease E T B mRNA in rat osteosarcoma cells at 6 hours and its effects were maximal at 24 hours (7). Exposure to isoproterenol and forskolin downregulated ET receptors in a similar biphasic manner within the same time span in rat astrocytoma C6 cells (37). Therefore, the results from this study indicate that heterologous regulation of ET by AVP might have involved shortening the half-life of ET B through post-translational receptor phosphorylation and/or decreasing receptor mRNA. 94 4.6 Implications of endothelin receptor downregulation in congestive heart failure Alterations in ET receptor density are apparent in heart failure. Loffler et al demonstrated that ET receptors are reduced by 61% in the kidney of CHF rabbits (108). Wong et al confirmed this observation in CM hamsters (171). In particular, the ET B subtype was downregulated by -56% whereas E T A remained unaffected. The pathways responsible for this phenomenon are unknown; however, decreased ET binding capacity has been associated with parallel increases in plasma ET (19, 108). Elevated circulating ET concentrations in CHF may arise from decreased clearance and metabolism or increased secretion, which would induce homologous receptor downregulation. Alternatively, it is conceivable that heterologous regulation by other neurohumoral factors accounts for the reduction in ET receptors during heart failure. Because CM hamsters exhibit AVP hypersensitivity in vivo and AVP selectively modulates ET B receptors in vitro, there may be a potential role for AVP in the altered distribution of ET receptor subtypes. Since E T A and ET B generally elicit opposing physiological functions, selective downregulation of E T B would likely perturb normal ET actions within the kidney irrespective of the mechanisms involved. It is well-established that E T B receptors cause natriuresis and diuresis (96). Zeidel et al showed that ET-1 reduced sodium reabsorption by inhibiting the Na7K+-ATPase secondary to PGE 2 production via E T B activation in rabbit IMCD cells (177). Alternatively, ET B -induced stimulation of PLC and dihydropyridine-sensitive Ca^ channels may respectively enhance PKC and intracellular Ca^ levels, which inhibit sodium reabsorption from the tubular fluid (96). Gallego and Ling further demonstrated that E T B receptors prolong the mean closed time of amiloride-sensitive Na+ channels in A6 distal nephron cells whereas E T A receptors prolong the mean open time to augment sodium transport from the lumen (47). Thus, the apparent natriuretic response to ET depends on the balance of E T A and E T B receptor subtype activation. 95 Additionally, E T B is known to regulate water excretion by directly targeting AVP action in the kidney. E T B associates with a pertussis toxin-sensitive inhibitory G-protein and activates a PKC-dependent mechanism that inhibits adenylate cyclase and the subsequent synthesis of cAMP (97, 162). Because NO interferes with cAMP accumulation and reduces Pf in the IMCD (48), the possibility that E T B inhibits water reabsorption via a NO-dependent pathway cannot be excluded. Regardless of the events upstream of cAMP generation, reduced PKA phosphorylation causes free water clearance as a result of decreased AQP2 mRNA expression and protein insertion of AQP2 to the luminal membrane of the collecting duct. In short, ET B activation blocks AVP-induced increases in cAMP accumulation and water flux at the IMCD. Clearly, an altered distribution of ET receptors may potentially affect renal hemodynamics and tubular reabsorption. With a reduction in E T B binding sites in CHF, ET may be unable to exert normal physiological functions unless increases in affinity of the remaining E T B are able to compensate for the receptor downregulation. A predominance of E T A receptors would overwhelm the natriuretic and diuretic responses normally promoted by ET B . For example, E T A action would favor systemic and renal vasoconstriction. A reduction in RBF would increase the filtration fraction at the glomeruli, ultimately enhancing proximal tubular reabsorption. The functions of E T A receptors in the collecting duct are not fully understood; however, studies with Chinese hamsters ovary cells showed that transfected bovine E T A caused cAMP accumulation to increase upon exposure to ET-1 (5). Thus, it is reasonable to expect elevated cAMP levels in the presence of high ET A : E T B s and AVP-mediated water flow would be favored under these conditions. As CHF progresses, GFR also decreases and results in increased salt and water conservation. Altered distribution of ET receptor subtypes may therefore contribute to the 96 pathogenesis of decompensated heart failure that is characterized by abnormal salt and water retention. 4.7 Perspectives With decreased E T B binding sites in CHF, the E T B signaling pathway would be disrupted and ET would be less effective as a natriuretic and diuretic agent despite elevated circulating levels of this peptide. In addition, its inhibitory actions on AVP activity in the kidney would diminish. Because AVP release is inappropriately suppressed for any given plasma osmolality in CHF, hyperactivity at the level of V 2 receptors may ensue. Generation of cAMP and stimulation of AQP2 expression and protein insertion into the luminal membrane in the renal collecting ducts would no longer be susceptible to ET regulation under the stressed conditions of CHF. Particularly, AVP-induced Pf would increase, allowing the passive diffusion of water from the lumen into the hypertonic renal medullary interstitium. As heart failure advances, cardiodepression is accompanied by further decreases in the effective circulating fluid volume. This would provide a non-osmotic stimulus for AVP release and perpetuate the vicious cycle of unopposed salt and water reabsorption in the kidney. Ultimately, renal AVP hypersensitivity would lead to the development of fluid retention commonly observed in CHF. 4.8 Future directions Excessive neuroendocrine activation is indicative of poor prognosis in patients with heart failure (111). Inhibition of AVP action has been demonstrated to be beneficial in humans with severe CHF and experimental animals (30, 49, 119). The results of this thesis further suggest that functional interactions between AVP and ET modulate fluid volume homeostasis and may be related to the development of hyponatremia in a number of disorders. To conclusively define the 97 implications under normal and pathological conditions, additional studies directed at investigating the two endocrine systems are warranted. First, V 2 receptor characterization and circulating levels of AVP should be determined along with AQP2 mRNA and protein expression in heart failure. Immunohistochemical techniques should also be applied to localize the distribution of cytoplasmic and membrane-bound AQP2. It would be of interest to determine the turnover rate of AQP2 to explore possible mechanisms of enhanced water retention in decompensated heart failure. Finally, the use of selective ET agonists and antagonists should be examined to confirm their regulation of AVP function. Because various neurohumoral forces are simultaneously activated in CHF, it is crucial to consider both the individual and interactive roles of each hormonal system in the treatment of this disorder. Perhaps an E T B agonist and an AVP antagonist may be used in conjunction with currently prescribed drugs such as ACE inhibitors, E T A antagonists, diuretics, and cardiac inotropic agents for congestive heart failure. The present study has initiated new, exciting approaches in the understanding salt and water regulation. The data suggest that alternative directions for CHF treatment based on specific interventions with AVP and ET systems may be forthcoming and will aid the development of a more effective therapeutic strategy. 98 CHAPTER 5 SUMMARY Experiments were performed to elucidate the functional significance of AVP in CHF. Exogenous AVP infusion had no effect in CM hamsters with moderate heart failure but significantly decreased sodium and water clearances in age-matched healthy controls. Similar results in fluid excretion were also observed following selective V 2 receptor activation; however, it is noteworthy that simultaneous V i antagonist and V 2 agonist administration produced a five-fold higher increase in cAMP levels in the CM hamsters relative to control animals. The blunted physiological change in salt and water conservation under pathophysiological conditions probably resulted from exhaustion of cellular machinery downstream of cAMP generation. That marked natriuresis and diuresis, accompanied by decreased cAMP levels, was present in CM but not in healthy animals during nonselective V i and V 2 blockade further supports the renal action of endogenous AVP is enhanced in CHF. The apparent AVP hypersensitivity during heart failure likely involved the loss of counterregulatory mechanisms; thus, whether AVP induces heterologous regulation of ET receptors was explored in this study. Competitive binding studies revealed that ET receptor expression was susceptible to AVP regulation. Overnight incubation of rat IMCD cells with AVP consistently affected ET-1 binding as a result of decreased E T A and E T B receptor densities. As AVP primarily exerts its actions via a PKA pathway in the IMCD, the effects of direct adenylate cyclase activation were determined. Interestingly, pretreatment with forskolin reduced the maximal binding capacity of E T B receptors but had no effect on the E T A subtype. Selective regulation of ET B receptors by a cAMP-dependent mechanism was later confirmed in cells preincubated with a cAMP analogue, Rp-cAMPS, which specifically abolished AVP-induced 99 downregulation of E T B receptors. As well, the dissociation constant for ET-1 binding was consistently decreased in the presence of AVP and forskolin, suggesting that higher ET B receptor affinity might have been a compensatory response to the decrease in binding sites. Taken together, AVP specifically regulates ET B receptor density via a cAMP-dependent pathway. This study provides evidence for AVP hypersensitivity, which may be attributable to augmented V 2 receptor activity, in heart failure. Additionally, it raises the speculation that AVP may exert heterologous regulation of ET B receptors that would otherwise offset excessive antinatriuresis and antidiuresis. In the absence of these counterregulatory forces, AVP actions are amplified, contributing to the pathogenesis of CHF. 100 CHAPTER 6 CONCLUSIONS The results from this study strongly indicate that renal AVP hypersensitivity contributes to edema formation in CHF. Data support antinatriuresis and antidiuresis under pathophysiological conditions are associated with enhanced V 2 receptor activity, which may be attributed to a loss of counterregulatory mechanisms. 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Endothelin, a peptide inhibitor of Na7K+-ATPase in intact renal tubular epithelial cells. Am. J. Physiol. 257:C1101-C1107, 1989. 116 APPENDIX 3 w PU 2 0 0 0 0 00 c 'S e CU > CU C U E o o CO CN J S — • f a I § .5 r - H c i cu f— CU r - H S » c CO <: H W 00 oo 00 c -5 c o <N "S CO C/3 CU C U E o U a x ffl '2 C CO 0 3 5 _« m cu ~ H o ,H W CU l oo -s •£ |-S c o ' § l_ 3 -*-» CO NO CO Cu co ^ C/3 ^ c/3 i E ^ • ~« *r\ U . CM cu — o 1 * -CO Ui (N h cu -3 cu ^ * C/3 » o i/-i 00 -c -5 -3 '> c > —, g § w • 2 E i - H C O I— CS u , cU — 3 Cu "S * C/3 U 3 ~3 CU s 3 c cu u . 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