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Atrial natriuretic peptide in aging rats : evidence for altered processing, secretion and receptor binding Kao, Jonathan 1990

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ATRIAL NATRIURETIC PEPTIDE IN AGING RATS: EVIDENCE FOR ALTERED PROCESSING, SECRETION AND RECEPTOR BINDING by JONATHAN KAO B.Sc, University of Toronto, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medicine) We accept this thesis as conforming to the required standard Urs Steinbrecher Dr. Jean Shapiro Dr. Simon Rabkin D r . Ralph Keeler Dr. Steve Pelech THE UNIVERSITY OF BRITISH COLUMBIA March, 1990 © Jonathan Kao, 1990 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. ^ Medicine Department of The University of British Columbia Vancouver, Canada Date A p r i l 30, 1990 DE-6 (2/88) ABSTRACT The recently discovered atrial natriuretic peptide (ANP) has potent diuretic, natriuretic and hypotensive effects, and is believed to be involved in the maintenance of sodium homeostasis in both normal and pathological conditions. The mammalian aging process is associated with a host of abnormalities that include, among others, a compromised ability to regulate sodium homeostasis. There are reports that demonstrate a positive correlation between plasma ANP levels and age in man; accordingly, the aim of this study was to examine whether age-related sodium imbalance is associated with disturbances in the homeostasis of ANP. Specifically, the intracellular storage, processing and secretion of ANP from the atrium was studied and associated with circulating ANP concentrations and ANP receptor binding kinetics. Studies were conducted with four groups of male Wistar rats designated as 1-, 3-, 10-, and 20-month-old. 24-hour renal clearances were conducted to assess age-related changes in renal functions. GFR and U N a V increased steadily from 1 to 10 months of age and decreased in the 20-month-old, while fractional excretion of water ( F E ^ ) and sodium (FE„a) declined initially (from 1 to 10 months) and then rose in the 20-month-old group. Circulating ANP levels in the rats was significantly correlated with the increase in age (N = 147, r = 0.59, p < 0.0005). Atria of the animals were isolated and superfused with a modified Langendorff apparatus. The spontaneous release of ANP increased from 1 to 3 months, and steadily decreased after 3 months. The results indicate that ANP secretion increases with maturation and thereafter declines with advancing age. - ii -ANP concentrations in the right and left atria were also quantified. The results revealed that atrial ANP content increased from 1 to 3 months and decreased progressively with age. There was a positive correlation between the rate of ANP release and atrial ANP content (N= 42, r=0.50, p<0.01), suggesting that the release of ANP from the right atrium was associated with the atrial content. The concurrence of a reduction in ANP secretion but with elevation in plasma ANP concentration in the aged (20-month-old) rats, suggests that there may be an impairment in renal clearance of ANP. It was established that the main molecular species present in the atrium was y-ANP and that this was unaffected by age as assessed by reverse-phase high performance liquid chromatography (RP-HPLC) coupled with radioimmunoassay. The molecular forms of ANP secreted by the atrium consisted of predominantly a-ANP, with a smaller amounts of y-ANP. y-ANP constituted only 1% of the total secreted ANP in the 1-, 3-, or 10-month-old rats, but up to 8% was detected in 20-month-old rats. Although both a-ANP and y-ANP were present in the circulation, the ratio of y-ANP/a-ANP increased significantly with age. The concentration of y-ANP in the plasma of the 20-month-old rats was two- to three-fold higher than In the two younger groups (1- and 3-month-old). These data imply that the post-transcriptional processing of prohormone y-ANP to active a-ANP is altered with age. Radio-ligand binding experiments were carried out using glomerular ANP receptors to determine whether the age-related alterations in plasma ANP levels has an effect on the binding of ANP to its target tissues. Both the receptor density (Bmax) and the equilibrium dissociation constant (kj increased from 1 to 3 months but decreased from 3 to 20 months. - iii -Collectively, these results suggest that: 1) Aging affects atrial ANP content and consequently influences the release of ANP from the isolated atria. 2) The processing of prohormone y-ANP to active a-ANP is modified with age. 3) Plasma levels of ANP increase with age, which may result in down-regulation of ANP receptor density and increased efficacy in receptor binding affinity. These may represent the compensatory responses. - iv -TABLE OF CONTENTS PAGE ABSTRACT ii TABLE OF CONTENTS v LIST OF FIGURES viii LIST OF TABLES xi ACKNOWLEDGEMENTS xii 1.0 INTRODUCTION 1.1 Historical Aspects of ANP 1 1.2 Molecular Structure of ANP 2 1.3 Release of ANP 4 1.4 ANP Receptors 6 1.5 Intracellular Signals Mediating ANP Action 8 1.6 Physiology of ANP 9 1.6.1 Effects on Vascular Smooth Muscle 9 1.6.2 Effects on Renal Hemodynamics and Sodium Excretion 10 1.6.3 Effects on Blood Pressure and Blood Volume . . 11 1.6.4 Effects on Renin-Angiotensin-Aldosterone System 12 1.7 Pathophysiological Implications 13 1.8 Aging 14 2.0 MATERIALS AND METHODS 2.1 Materials 19 2.1.1 Rats 19 - v -2.1.2 Chemicals 19 2.2 Methods 19 2.2.1 24-Hour Clearance Studies 19 2.2.2 Measurement of Plasma ANP Levels 20 2.2.3 Spontaneous Release of ANP 21 2.2.4 Determination of Atrial ANP Content 21 2.2.5 HPLC Analysis of ANP Molecular Species 23 2.2.5.1 Plasma Samples 24 2.2.5.2 Perfusates 24 2.2.5.3 Atrial ANP 24 2.2.6 Radioimmunoassay for ANP 24 2.2.6.1 Preparation of Radiolabeled ANP . . . 24 2.2.6.2 Radioimmunoassay 25 2.2.7 Receptor Studies 27 2.2.7.1 Preparation of Glomerular Microsomes 27 2.2.7.2 Receptor Binding Assay 28 2.3 Data Analysis 30 3.0 RESULTS 3.1 Physical Characteristics of the Rats 31 3.2 Effect of Age on Renal Excretory Function 31 3.3 Radioimmunoassay of ANP 35 3.4 Effect of Age on Plasma ANP Levels 35 3.5 Effect of Age on ANP Secretion Rate 38 3.6 Effect of Age on Atrial ANP Content 43 - vi -3.7 Effect of Age on Molecular Species of Circulating ANP 46 3.8 Effect of Age on Molecular Species of Secreted ANP 51 3.9 Effect of Age on Molecular Species of Atrial ANP 51 3.10 Effect of Age on Glomerular Receptor Binding Characteristics 57 3.10.1 Optimal Conditions for Radiolabelled Receptor Assay 57 3.10.2 Competition and Scatchard Plots 57 4.0 DISCUSSION 4.1 Effect of Age on Renal Function 64 4.2 Effect of Age on Plasma ANP Levels 65 4.3 Effect of Age on ANP Secretion 67 4.4 Effect of Age on Atrial ANP Content 68 4.5 Effect of Age on Molecular Species of Circulating ANP 70 4.6 Effect of Age On Molecular Species of Secreted ANP . . . . 73 4.7 Effect of Age on Molecular Species of Atrial ANP 75 4.8 Effect of Age on Glomerular ANP Receptor Binding Characteristics 75 5.0 SUMMARY 83 6.0 REFERENCES 86 - vii -LIST OF FIGURES FIGURE PAGE 1 Schematic representation of the processing of y-ANP into N-terminal y-ANP fragment (10K), N-peptide, and a-ANP (3K), the active circulating form 3 2 Schematic overview of ANP synthesis, storage, release, response, and manipulation 5 3 Schematic representation of the modified Langendorff apparatus used for perfusing atria 22 4 Chromatographic separation of the 1 2 5I-ANP and 1 2 5I peaks in the labelling of ANP with iodine-125 26 5 Growth curve of the male Wistar rats 32 6 Representative standard curve of a-ANP. B/Bo, Bound to free ratio 36 7 Recovery of a-ANP added to a pool of rat plasma 37 8 Effect of age on circulating ANP levels 39 9 Relationship between plasma ANP levels and age 40 10 Effect of age on the rate of ANP secretion from isolated right atrium 41 11 Summary of the amount of ANP released from each age group 42 12 Effect of age on atrial ANP content 44 13 Relationship between amount of ANP released and the atrial ANP content 45 14 Elution positions of synthetic a-ANP and 0-ANP, and purified y-ANP 47 15 Representative RP-HPLC profiles of the plasma IR-ANP. A) 1-month-old B) 3-month-old C) 10-month-old D) 20-month-old 48 - viii -16 Schematic illustrations of individual plasma concentrations of a-ANP and y-ANP in the total plasma IR-ANP concentration 49 17 Schematic illustrations of average percentages of a-ANP and y-ANP in the total plasma IR-ANP concentration 50 18 Representative RP-HPLC profiles of the perfusate IR-ANP. A) perfusate from 1-month-old rats. B) perfusate from 3-month-old rats. C) perfusate from 10-month-old rats. D) perfusate from 20-month-old rats 52 19 Schematic illustrations of individual perfusate concentrations of a-ANP and y-ANP in the total perfusate IR-ANP concentration 53 20 Schematic illustrations of average percentages of a-ANP and y-ANP in the total perfusate IR-ANP concentration 54 21 Representative RP-HPLC profiles of right auricular extracts of the four age groups of rats. A) extract from a 1-month-old rat. B) extract from a 3-month-old rat. C) extract from a 10-month-old rat. D) extract from a 20-month-old rat 55 22 Representative RP-HPLC profiles of left auricular extracts of the four age groups of rats. A) extract from a 1-month-old rat. B) extract from a 3-month-old rat. C) extract from a 10-month-old rat. D) extract from a 20-month-old rat 56 23 Line graph showing the effect of incubation time on the binding of 1 2 5I-ANP to membrane fraction from rat kidney in each age group 58 24 Line graph showing effect of the amount of receptor proteins on the specific binding of 1 2 5I-ANP 59 25 Representative competitive inhibition plot of 1 2 5I-ANP binding to membrane fractions from rat kidneys in each age group 60 26 Representative Scatchard plot of 1 2 5I-ANP binding to membrane fractions from rat kidneys in each age group 62 - ix -27 Summary of the receptor binding - equilibrium dissociation constant (KJ and maximum binding capacity (Bm J 63 28 Summary of possible age-related alterations in the homeostatic balance of ANP in aged rats 84 - x -LIST OF TABLES T A B L E P A G E I Age, body and organ weights i n the four age groups of rats 33 II Summary of 24-hour renal clearance data 34 - xi -ACKNOWLEDGEMENT I would like to first thank Dr. Norman Wong for allowing me to work in his laboratory, and for his continual support as a teacher, inspirer, and friend. I would also like to thank him for his financial sponsorship that enabled me to complete my graduate studies. I would like to thank Dr. Eric Wong for his continual friendship and for his excellent and pertinent advice and technical expertise that assisted me in this project. I would like to thank Alice Chan and Edward Mak for their continual friendship and technical assistance. I would like to thank David Ridout and Silvia Isakson for their technical assistance. - xii -1.0 INTRODUCTION 1.1 Historical Aspects of ANP The recent discovery of atrial natriuretic peptide (ANP) is the result of a lengthy and intensive search for an extrarenal substance or mechanism that has long been suspected to be involved in the regulation of sodium and fluid balance. As early as 1952, Peters (1952) had postulated the existence of a mechanism that would respond to increase arterial volume with corresponding increases in the renal excretion of salt. Five years later, Smith (1957) suggested that the cardiac atria participate in sodium-volume regulation. Based on this speculation, Gauer, Henry and Seiker (1961) later demonstrated that a distention of the heart's right atrium caused both a diuresis and natriuresis. In 1956, Kisch (1956) reported the presence of endocrine-like granules in tissue of the cardiac atria, which were characterized by Jamieson and Palade (1964). However, more than ten years passed before it was observed that granularity of the atrial cells undergoes changes in response to sodium or volume deprivation and adrenalectomy (Cantin et al., 1975; De Bold et al., 1978). It was not until 1981 that the significance and relevance of these atrial granules was demonstrated, de Bold and his colleagues (1981) found that infusing rat atrial tissue extracts into rats induced an impressive natriuresis and a reduction in arterial pressure. They also observed a significant rise in haematocrit. Such effects were not observed when ventricular extracts were infused into rats. - 1 -When other investigators (Keeler, 1982; Trippodo et al.,1982; Sonnenberg and Cupples, 1982) confirmed de Bold's findings, the existence of a potent natriuretic and vasorelaxant (Currie et al.,1983; Kleinert et al.,1984) substance in the heart's atrial granules was confirmed. Research based on these findings became intense, making atrial natriuretic peptide the most aggressively pursued peptide discovered to date. 1.2 Molecular Structure of ANP Soon after the confirmation of ANP as a natriuretic peptide, the isolation, purification, sequencing, and chemical synthesis was accomplished. The active form of ANP in the circulation is a 28-amlno acid peptide (a-ANP) in both man and animals (Miyata et al.,1987). This peptide is synthesized in man as the C-terminus of a 151 amino acid polypeptide (in rat 152 amino acid). perpro-ANP, encoded on chromosome 4 (Lewicki et al.,1986). Post-translational cleavage of the hydrophobic leader sequence generates a 126 amino acid prohormone, pro-ANP (ANP^^g) or y-ANP, which is stored in granules within the atrial myocyte (Kangawa et al., 1984b) (Fig. 1). It appears that immediately before, during, or promptly after release, y-ANP is cleaved by a processing enzyme to yield the active C-terminal peptide a-ANP (ANP^^g) . The amino acid sequence of human a-ANP is same to rat a-ANP, except a single substitution of isoleucine for methionine at position 12 (Kangawa et al., 1984a). a-ANP contains a 17-amino acid disulphide ring and a Phe-Arg sequence at the carboxy-termlnal, which are required for its biological activity (Currie et al., 1984; Sugiyama et al., 1984). Its half-life in the circulation is short, ranging from about 30 seconds to about 2.5 minutes (Katsube et al., 1986; Luft et al., 1986; Yandle et al., 1986). The N-terminal 98 amino acid fragment, - 2 -peptide 99 \ #126 •s-sH r-hANP o c— s - s h a-hANP N-Peptide (10K N-terminal fragment) , ( j 2 8 iS Si i i 'S s« 2 8 / % 1 /9-hANP Fig. 1 Schematic representation of the processing of y-ANP into N-terminal y-ANP fragment (10K), N-peptide, and a-ANP (3K), the active circulating form. Adapted from Itoh et al., 1988. - 3 -ANP,.^, of the pro-ANP is co-secreted with a-ANP (Imada et al.,1987). Although it has a longer half-life (Thibault et al., 1988), it has no clear biological activity. A unique antiparallel dimer of ANP^.^ , comprising 56 amino acids (p-ANP) has also been identified in human plasma (Miyata et al.,1987). Though it has a slower and longer natriuretic-diuretic action than a-ANP (Miyata et al.,85; Kangawa et al., 1985), the physiological significance of this dimer is not clear at this time. 1.3 Release of ANP The release of ANP into the circulation seems to be stimulated by direct atrial stretch caused by increases in central blood volume and/or right or left atrial pressure (de Bold et al.. 1986; Weidman et al., 1989) (Fig. 2). Increased secretion of ANP is observed in response to intravenous loading with saline or water (Haller et al., 1987; Yamaji et al., 1986), an acute increase in sodium intake (Weidman et al., 1988, Hollister et al., 1986), dynamic exercise (Tanaka et al., 1986) or increased heart rate induced by pacing (Espiner et al., 1986). In addition, a central shift of volume produced by the change from the upright to the supine position (Haller et al., 1987; Hollister et al, 1986) or by water immersion (Epstein et al, 1986, Tajima et al., 1988) also caused an increase in ANP secretion. There is some evidence that ANP release may also be influenced by humoral agents. Sonnenberg and Veress (1984) reported that incubation of isolated rat atria in vitro with arginine vasopressin (AVP) and adrenaline but not with the non-pressor analogue of vasopressin (dVAVP), results in an increased release of ANP. AVP, phenylephrine, angiotensin II, isoprenaline and carbacholine infusion in vivo produced a - 4 -volume expansion vasoconstrictors TRAP TUNaV' TGFR Fluid Electrolyte Blood Pressure Homeostasis ^Aldosterone GTP cGMP uncoupled Clearance a-ANP Storage Form Y - A N P ^ T Release/Cleavage 1 selective enzyme Circulating Forms C-Terminus N-Terminus A N P V 9 8 l a 1/2 300 sec. Renal degradation Fig. 2 Schematic overview of ANP synthesis, storage, release, response, and manipulation. Abbreviations: ANP, atrial natriuretic peptide; C-terminus, carbosy terminus of the prohormone; N-terminus, amino terminus of prohormone; All, angiotensin II; AVP, arginine vasopressin; PVH, paraventricular nucleus of the hypothalamus; RAP, right atrial pressure. Adapted from Needleman P, ed., 1988. marked increase in immunoreactive ANP levels (Manning et al., 1985). Using an isolated heart model, Ruskoaho et al. (1985, 1986) have demonstrated a role of phosphoinositide system, cytosolic calcium and the cyclic AMP pathway in the regulation of ANP release. This suggests that factors which alter the concentration of calcium in heart muscle cells may influence ANP secretion. In the heart muscle cells, both adrenaline and acetylcholine alter ANP secretion rate by exerting inotropic and chronotropic effects on the heart, thereby suggesting the participation of autonomic nerves in the regulation of ANP release (Toth et al., 1986). 1.4 ANP Receptors Once released, a-ANP binds and interacts with specific receptors located in numerous target organ tissues (Fig. 2). ANP receptors have been found in rat renal glomeruli, medulla and papillary vasa recta (Bianchi et al., 1985), rat adrenal glomerulosa (Schiffrin et al., 1985), rat mesenteric artery (Schiffrin et al., 1985), rabbit aorta (Napier et al., 1984), cultured established epithelial (LLC PK,) cells derived from pig kidney (Napier et al., 1984), and cultured rat aortic smooth muscle (Hirata et al., 1984). In the central nervous system, specific high-density binding sites were found in the subfornical organ, median eminence, area postrema, nucleus tractus solitarius, and hypothalamic regions, including the anteroventral region of the third ventricle (Quirion et al., 1984). Two distinct ANP receptor subtypes have been identified and characterized in various tissues and cell types. The ANP B-receptor is a 120,000-dalton subunit protein that recognize only biologically active ANP fragments (Leitman and Murad, 1986). It has been linked with the generation of - 6 -guanosine 3', 5'-cyclic monophosphate (cGMP) In response to ANP binding (Waldman et al., 1984). The intracellular region of the receptor features homology with protein kinase catalytic domains, soluble guanylate cyclase and a brain adenylate cyclase (Krupinski et al., 1989). It is suggested that binding of ANP to the extracellular domain of its receptor initiates a conformational change in the protein kinase-like domain, resulting in derepression of guanylate cyclase activity (Clunkers et al., 1989). A second receptor, termed the ANP C-receptor, is distinct from the B-receptor in structure, physiological role, and ligand-binding specificity (16, Maack et al., 1987). The receptor is consisted of a single 60,000-dalton subunit that appears to exist predominantly as a homodimer on the cell surface (Pandey et al., 1987). Its structure is proposed to consist of a large amino-terminal extracellular ligand binding domain adjacent to a single transmembrane anchor and a short hydrophilic cytoplasmic tail (Fuller et al., 1988). It is believed that the extracelluar domain of this receptor is alone sufficient for specific high affinity ligand binding; no contribution is necessary from other membrane components or from the membrane or cytoplasmic domains (Porter et al., 1989). The C-receptor binds native ANP as well as various truncated and internal ring-contracted analogs of ANP with high affinity (Leitman and Murad, 1986). This receptor is not involved in the stimulation of particulate guanylate cyclase and does not appear to mediate directly any of the known biological effects of ANP. However, it has been suggested that the receptor is a clearance site that mediates the metabolic clearance and degradation of this hormone (Maack et al., 1987). - 7 -1.5 Intracellular Signals Mediating ANP Action Binding of ANP to its specific cell-surface receptors activate guanylate cyclase, resulting in the accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP) levels fWaldman et al., 1984) (Fig. 2). It has been suggested that this cyclic nucleotide is the intracellular mediator of many, if not all, of the physiological actions of ANP (Waldman et al., 1984). Injection of atrial extracts into rats resulted in a rise of plasma and urinary cGMP levels (Hamet et al., 1984). ANP-induced cGMP accumulation in the kidney was found to correlate well with the distribution of particulate guanylate cyclase (Garcia et al., 1985). The specific action of ANP on particulate guanylate cyclase activity has also been reported in rat kidney, aorta, lung, liver, intestine, and testes (Waldman et al., 1984). In cultured vascular smooth muscle cells and isolated rat renal glomeruli, a strong correlation between ANP binding to specific receptors and cGMP accumulation has been demonstrated (Ballermann et al., 1985). There are, however, major discrepancies between the kinetics of ANP binding and the dose-response curve of ANP induced cGMP accumulation fWaldman et al., 1984; Ballermann et al., 1985). Moreover, in vascular smooth muscle and cultured endothelial cells, some analogs of ANP that effectively compete for specific binding sites do not antagonize the ANP-induced increase in cGMP (Leitman and Murad, 1986). These data raise the possibility that cGMP is not the sole mediator of ANP actions. Inhibition of adenylate cyclase activity by ANP has also been reported. In aorta, mesenteric and renal artery, the basal and hormone-stimulated - 8 -adenylate cyclase activity was inhibited by ANP in a dose-dependent fashion (Anand-Srivastava et al., 1984). ANP inhibition of adenylate cyclase activity in anterior and posterior pituitary has also been reported (Anand-Srivastava et al., 1985). Others, however, did not find effect of ANP on adenylate cyclase activity in kidney (Waldman et al., 1984) or on urinary cAMP excretion (Hamet et al., 1984). This suggests that inhibition of adenylate cyclase may not be a universal phenomenon. 1.6 Physiology of ANP ANP has powerful natriuretic and vasorelaxant effects that can be divided into four categories: effects on vascular smooth muscle; alterations of renal hemodynamics and sodium excretion; changes of blood pressure and volume; and effects on renin-angiotensin-aldosterone system (Fig. 2). 1.6.1 Effects on Vascular Smooth Muscle A prominent physiological effect of ANP is the inhibition of smooth muscle contraction. In a variety of preconstricted tissues as well as in isolated arteries and arterioles from several vascular beds, application of atrial extracts caused a reduction in contractile tone (Kleinert et al., 1984). The aorta, renal vasculature, and facial vein of the rabbit are very sensitive to ANP's vasorelaxant action (Currie et al., 1983). In human subjects, infusions of the ANP have been associated with significant and dose-dependent increases in blood flow to the skin of the forearm (Bussien et al., 1986). ANP has also been shown to be particularly effective in antagonizing angiotensin II-induced contractions of the rabbit aorta (Kleinert et al., 1984). Maack el al. (1984) have reported that the vasorelaxant effect of ANP can not be blocked even with a high - 9 -concentrations of angiotensin II (9.7 nM) given to anaesthetized dogs. 1.6.2 Effects on Renal Hemodynamics and Sodium Excretion ANP has been demonstrated to produce a marked and sustained increase in glomerular filtration rate (GFR) in isolated perfused rat kidneys (Camargo et al., 1984), kidneys of intact animals (Maack et al., 1984) and human subjects (Weidman et al., 1986). The marked increase in filtration fraction following ANP suggests that it either selectively constricts the efferent or dilates the afferent arterioles of the glomerulus, thereby enhances the filtration rate (Maack, 1985). Alternatively, the hormone may act on glomerular membrane to directly increase its permeability (Maack, 1985). Consistent with the latter possibility is the presence of high-affinity ANP receptors in the rat glomeruli (Ballermann et al., 1985). An impressive physiological effect of ANP is its ability to enhance renal sodium and fluid excretion in anaesthetized dogs (Maack et al., 1984). However, since the natriuretic effect of the hormone can be abolished by partial occlusion of the renal artery (Sosa et al., 1986), it suggests that the increased glomerular filtration rate produced by ANP may be a necessary component of the natriuresis. In addition, the hormone causes a washout of the medullary osmotic gradient by altering intrarenal distribution of blood flow (Maack et al., 1985), thus increasing medullary flow (Fried et al., 1985). As a result of an increased distal delivery of filtrate (Maack et al., 1984), renal excretion of sodium is enhanced. The observation of reduced urine osmolality without a change in free water clearance during ANP-induced natriuresis supports such a view (Maack et al., 1984). - 10 -Although the renal hemodynamic actions of ANP appear to be essential for its ability to induce natriuresis, other tubular mechanisms cannot be excluded. Tubular effects of ANP that have been proposed include a specific inhibition of normal sodium reabsorption from the medullary collecting ducts (Sonnenberg and Cupples, 1982). This may involve altering the passive permeability of the papillary collecting ducts (Sonnenberg, 1986), or inhibiting apical sodium permeability (Cantiello and Ausiello, 1986). 1.6.3 Effects on Blood Pressure and Blood Volume Administration of ANP not only induces an increase in glomerular filtration rate and sodium excretion, but also consistently produces a dose-dependent fall in arterial pressure in several animal species (Maack et al., 1984, Garcia et al., 1985) and In men (Bussien et al., 1986). The blood pressure declines immediately after the beginning of infusion and returns to basal levels soon after termination of infusion (Maack et al., 1984). This may suggest that ANP acts directly upon the systemic vascular system to decrease blood pressure rather than indirectly through volume depletion. The fall in blood pressure has been ascribed to a decrease in cardiac output (Breuhaus et al., 1985; Maack et al., 1984). This is thought to bring about by ANP-induced relaxation of venous smooth muscles, leading to an augmentation of venous capacitance and a reduction of venous return (Breuhaus et al., 1985). Since ANP infusion also produces a rapid rise in haematocrit (Maack et al., 1984), the effect of ANP on venous return may reflect an alteration of capillary permeability. This causes - 11 -Intravascular fluid to shift into the interstitium. Regulation of intravascular volumes and pressures through changes of capillary permeability could be one of the major functions of ANP (Laragh, 1985). 1.6.4 Effects on Renin-Angiotensin-Aldosterone System ANP has been reported to produce a marked and sustained suppression of both renal renin secretion and plasma renin activity in anaesthetized and conscious dogs with inferior vena cava ligation (Maack et al., 1984, Freeman et al., 1985). These actions may be due to ANP-induced vasodilation of renal vasculature which blocks renin release (Maack et al., 1984). Increased glomerular filtration that exerts a negative feedback signal to reduce renin secretion (Maack et al., 1984), or a direct action of ANP on juxtaglomerular cells to suppress renin secretion (Krutz et al., 1986) may also be important. ANP directly and selectively lowers basal secretion of aldosterone by a direct action on adrenal glomerulosa cells. This has been shown to block angiotensin's stimulation of aldosterone release both in vitro (Goodfriend et al., 1984) and in vivo (Maack et al., 1984) studies. Suppression of aldosterone in vivo is amplified by the concurrent suppression of renin secretion (Maack et al., 1984). In this manner, ANP has four distinct presumed actions on the renin system (Laragh, 1985b): it reduces renin secretion; it blocks aldosterone secretion; it opposes angiotensin IPs vasoconstrictive effect; and it decreases aldosterone's sodium-retaining action. Although not every one of these actions is important in acutely induced natriuresis, all may play some role in the long-term regulation of sodium balance and blood pressure. - 12 -1.7 Pathophysiological Implications of ANP The available data briefly summarized above strongly implicate that ANP is involved in the regulation of blood pressure and sodium balance in healthy men and in patients with diseases characterized by volume overload. Administration of ANP produces a dose-dependent increase in diuresis and natriuresis in patients with chronic renal failure or nephrotic syndrome (Suda et al., 1988, Windus et al., 1989). In congestive heart failure patients, ANP infusion caused an acute decrease in preload and peripheral vascular resistance, an increase in cardiac output, a mild reduction in blood pressure, a lowered plasma aldosterone, and an enhanced diuresis and natriuresis (Riegger et al., 1986). In patients with essential hypertension, infusions of ANP produce an exaggerated diuresis and electrolyte excretion compared to normotensive subjects (Cody et al., 1987). Collectively, these data suggest that an excess or absence of the hormone may be involved in the pathophysiology of several clinical disorders. Inadequate ANP secretion, for example, might be implicated in low-renin essential hypertension and in other states that are characterized by excessive sodium-volume retention (Laragh, 1985). High levels of circulating ANP have been reported in patients with congestive heart failure (Ding et al., 1987), cirrhosis (Gerbes et al., 1985), hypertension (Cantin et al., 1988), renal failure (Ogawa et al., 1987), and atrial arrhythmias (Yamagi et al., 1985b). Whether this alteration in plasma ANP levels reflects an involvement of ANP in the pathogenesis of these disorders or a consequence of such disorders remains unclear. - 13 -1.8 Aging The physiological changes that occur with aging have a significant impact on the health of elderly individuals. Changes in renal function are particularly important, because the kidney plays a central role in drug excretion, fluid regulation and electrolyte balance. There are a host of renal structural and functional abnormalities associated with advancing age. The kidney as a whole progressively loses mass with age. Studies have shown that the normal kidney loses about 20 percent of its mass between the ages of 40 and 80 years (Tauchi et al., 1971) . This loss is much greater in the cortex than in the medulla, with vascular changes accompanying the loss of tissue (Takazakura et al., 1972) . Histologic examinations of the aging kidney frequently show complete destruction of the preglomerular arterioles in the cortex, and the formation of shunts between the afferent and efferent arterioles of the juxtamedullary area (Ljungqvist and Lagergren, 1962). There is also age-related chronic glomerular lesions observed in rats, hamster, and mice. With age, there is a thickening of the glomerular basement membrane due to accumulation of basement membrane-like material on the epithelial side of the lamina densa (Yagihashi, 1978). There is a thinning of the lamina rarae interna and externa, and an increasing of mesangial matrix with age. Finally, there is an increase in the collagen content of whole glomeruli and basement membrane with age (Kalant et al., 1977). In addition to these structural abnormalities, there is a functional loss of glomeruli with preservation of blood flow to the renal medulla (Luft et al., 1985). The human kidney contains 800,000 to 1,000,000 glomeruli from birth to age 40, following which this number may be reduced by one-third - 14 -to one-half of its original number by the time the subject reaches 70 (Moore, 1943). These pathological alterations in the glomeruli may contribute, in part, to the observed age-related decline in renal function after the age of 30 (Davies and Shock, 1950). Bricker (1969) was the first investigator to speculate that decreases in specific renal functions with age parallel the decrease in glomerular filtration rate. In 70 healthy male subjects, Davies and Shock (1950) reported that renal blood flow decreases from 600 ml/min at age 40 to 300 ml/min at age 85. As a result, glomerular filtration rate falls from 120 ml/min to 60 - 70 ml/min at age 85. Creatinine clearance, which closely parallels glomerular filtration rate, also decreases with age. This decrease begins at age 40 and continues in a more or less linear fashion at 0.8 ml/min per year per 1.73 m 2 of body surface area (Rowe et al., 1976). Since virtually all the ingested NaCl are eliminated by the kidneys (Luft and Weinberger, 1982), alterations in renal functions due to aging may have major influences on sodium homeostasis. When subjected to salt deprivation, older individuals are unable to conserve sodium as rapidly and efficiently as younger individuals (Epstein and Hollenberg, 1976). Although the exact cause for this is not clear, it may be related to alterations in the renin-aldosterone system. In addition, elderly subjects have lower plasma renin activities and urinary aldosterone excretion when placed on an unrestricted diet or immediately following sodium restriction Weidman et al., 1975). On the other hand, elderly subjects are more likely to develop an exaggerated natriuresis after adminstration of a water - 15 -or saline load than younger subjects (Lindeman et al., 1970; Schalekamp et al., 1971). Collectively, the data indicates that the ability of the aging kidney to regulate sodium excretion in response to volume contraction and expansion is compromised. In addition to the inappropriate responses in maintenance of sodium homeostasis, the aging kidney can neither dilute nor concentrate urine as well as a younger kidney (Rowe et al., 1976). One explanation attributes such a change to the age-related decrease in the number of functional nephrons thus causing an increase in solute load per nephron. Hence, an obligate diuresis limits the capacity of the kidney to concentrate urine maximally (Rowe et al., 1976b). Another possible explanation for the reduction in concentrating ability relates to changes in distribution of renal blood flow. An increase in the medullary blood flow causes a washout of the normally hypertonic fluid in the renal medulla and thereby reduce the concentrating capacity of the countercurrent system (Takazakura et al., 1972). The ability to concentrate urine also depends on the response to vasopressin. The aging kidney is less sensitive to the effects of vasopressin (Philips et al., 1984). In vitro experiments have shown that with aging the cyclic AMP generation is reduced in response to vasopressin, and this might also play a role in the age-related decline in urine concentrating ability (Goddard et al., 1984). Further studies have demonstrated that serum vasopressin levels are actually higher in older people than in younger subjects with similar levels of dehydration (Miller et al., 1953). These findings indicate that although the elderly are able to produce an adequate amount of vasopressin, their kidneys do not respond to vasopressin as well as those of younger people. - 16 -Recently, there have been several reports that document an elevated ANP concentration In the plasma of the aged individual, and have established a significant positive correlation between plasma ANP levels and age (Haller et al., 1987; Ohashi et al., 1987; Richards et al., 1986; Sagnella et al., 1986; Larochelle et al., 1987; Ezaki et al., 1988). In addition, it was demonstrated that infusion of 500 ml of 0.15M NaCl produces a more elaborate increase in ANP concentration in elderly men relative to young men (Ohashi et al., 1987). Haller et al. (1987) reported that the increase in circulating ANP levels following saline loading is correlated with age. Yamasaki et al. (1988) showed that infusion of hypertonic saline (2.5% NaCl) caused a 119% increase in plasma ANP concentration in elderly men (51 - 61 years) compared to 37% in young men (20 - 22 years). Moreover, Heim et al. (1989) demonstrated that a small (33 mg) intravenous administration of a-ANP produced an exaggerated hypotensive, diuretic, natriuretic, and calciuretic responses in the elderly men. In another study, Tajima et al. (1988) reported that head-out water immersion induced a pronounced release of ANP in the elderly relative to the young. This immersion-induced increase in plasma ANP levels was related to exaggerated natriuretic and diuretic responses in the elderly (Epstein et al., 1987). Collectively, these findings suggest that the homeostatic balance of ANP and the sensitivity to ANP in aged individuals may somehow be altered. Since aging is associated with a compromised ability in maintaining sodium balance, it is of interest to investigate if this is related to the alteration in ANP homeostasis. Thus, the present experiments were designed to examine some of the putative age-related disturbances - 17 -in ANP homeostasis. Specifically, the intracellular storage of ANP, the processing of pro-ANP to active a-ANP, the secretion of ANP from the atrium, circulating ANP concentrations, and ANP receptor binding kinetics were studied. Atria from rats of different rats were isolated and superfused in a modified Langendorff apparatus to measure the rate of spontaneous ANP secretion. The atrial tissue and plasma was quantitatively evaluated for ANP content. Reverse-phase high performance liquid chromatography (RP-HPLC) was employed to analyze the various molecular species of ANP in the atrial tissue, perfusing medium and circulation, in order to assess age-related modifications. ANP receptors from glomerular membranes were isolated and radioligand binding studies performed to appraise if receptor affinity and density are affected by age. - 18 -2.0 MATERIALS AND METHODS 2.1 Materials 2.1.1 Rats Male Wistar rats were purchased from Charles River Breeding Laboratories, Wilmington, MA, and housed in climate-controlled rooms. They were fed with standard rat chow (Ralston Purina Co., St. Louis, MO) and had free access to tap water. They were studied at the desired ages. The rats were divided into four age groups: group 1 rats, 1-month-old (aged 33+1 days); group 2 rats, 3-month-old (84 + 3 days) and is used as the control group; group 3 rats, 10-month-old (289 ± 12 days); and group 4 rats, 20-month-old (596 ± 4 days). In some instance, rats whose age fall outside of the four specified age groups were also used for the determination of plasma ANP levels. 2.1.2 Chemicals Unless otherwise stated, all chemicals were purchased from Sigma Chemicals, St. Louis, MO. 2.2 Methods 2.2.1 24-Hour Clearance Studies Rats from each age group were housed in metabolic cages and had free access to tap water and rat chow. After readjustment to the new environment in the metabolic cages for 7 days, the first baseline clearances were determined. Twenty-four hour urine and free-flowing tail vein blood were collected from each rat. Sodium was analyzed with a flame photometer (Instrumentation Laboratory 943, Milan, Italy). - 19 -Plasma and urinary creatinine were determined with a VP Abbott spectrophotometer (Abbott Laboratory, Dallas, Tex). The daily sodium intake for the rats varied from 7.2 to 14.8 g. 2.2.2 Measurement of Plasma ANP Levels The rats were anaesthetized with sodium pentobarbital (65 mg/kg) (M.T.C. Pharmaceutical, Cambridge, Ontario) administered intraperitoneally. Blood ( 2 - 3 ml) was withdrawn from the abdominal aorta after the rats were anaesthetized for at least 45 minutes. The samples were transferred to chilled siliconized Vacutainer tubes containing 50 ul aprotinin (1,000 kallikrein inactivator units/ml) and EDTA (ethylenediaminetetraacetic acid) (1 mg/ml), then immediately placed on ice and promptly centrifuged at 4°C at 3,000 rpm. Plasma samples were first subjected to ANP extraction before analyzed by radioimmunoassay (RIA) methods . Extraction was carried out using Sep-Pak C-18 cartridges (Waters Association Inc. Milford, MA) activated with 100% methanol (5 ml) and washed with distilled water (12 ml). Plasma samples were diluted in equal amounts of 0.1 N Hcl, and then allowed to pass through the cartridge twice by gravity. The cartridge was then washed with water (12 ml) and the wash was discarded. Elution of the absorbed ANP was carried using 2 ml of 80% methanol. The effluent was collected and air dried. The dry sample was reconstituted in RIA phosphate buffer containing 0.1% BSA, 0.01% triton X-100, and 0.1% NaN3, and analyzed for ANP concentration. The remaining sample was retained for HPLC analysis. - 20 -2.2.3 Spontaneous Release of ANP After the blood samples were taken, the heart was removed. Both atrial appendages were quickly excised, and the right atrium was incubated at 37°C in 10 ml of Tyrode's solution (in Mm: CaC1.2H20, 1.8; Kcl ,2.7; MgC1.6H20, 1.1; NaCl, 13.7; Na^PO^ 0.4; D-Glucose, 5.6). The left atrium was save for later determination of ANP content. Both kidneys were also removed for receptor binding studies. The perfusing medium was gassed continuously with 95% 0 2 and 5% C 0 2 . Atrial appendages from the 4 different age groups of rats were perfused simultaneously in separate chambers in a modified Langendorff apparatus schematically represented in Fig. 3. The reservoir chamber consisted of a 3-ml syringe with an inflow tube (PE-100) inserted into the top of the syringe for the addition of perfusate and held in place with a rubber stopper. A similar tube was connected to the tip of the syringe to allow outflow of perfusate. The entire chamber was secured to a stand by a clamp. The perfusion rate was set at a constant flow rate of 0.5 ml/min by a peristaltic pump (model PP03, Scientific Instruments, Inc., New York). The atria were perfused for 40 minutes to allow the hormone secretion rates to stabilize. After the stabilization period, the perfusate was collected at 5-rninutes intervals for 100 minutes. The perfusate was collected by LKB fraction collectors (Bromma, Sweden) and immediately placed on ice until assay the same day. 2.2.4 Atrial ANP Content Determination To determine atrial ANP content, both the right and left atria obtained from the perfusion studies were placed in 2 ml of 0.1 M acetic acid and kept at -20°C until assay. On the day before assay, the atria were - 21 -Incubator (37 c 5% C 0 2 Air) Fig. 3 Schematic representation of the modified Langendorff apparatus used for perfusing atria. - 22 -homogenized by VisTishear tissue grinder (VisTis Co., Gardiner, N.Y.) separately for 2 minutes in acetic acid and centrifuged for 30 minutes at 30,000 rpm. The pellet was discarded and the supernatant was stored at -20 °C overnight. On the day of assay, the supernatant was thawed and centrifuged again at 30,000 rpm for 20 minutes to further disrupt the membranes. The supernatant was then collected for radioimmunoassay. 2.2.5 HPLC Analysis of ANP Molecular Species Plasma, perfusate, and tissue samples were subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) using a Whatman Partisil ODS-3 C i 8 column (Waters Association Inc. Milford, MA). The column was first equilibrated with 10% acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). The gradient elution was carried out over 55 minutes using a linear gradient (slope 0.4%/min) of 19% - 40% CH 3 CN in 0.1% TFA for the first 24 minutes and then at a constant 60% CH 3 CN in 0.1% TFA for the remaining period . The flow rate was maintained at 1 ml/min (Shimadzu model LC-6A pumps, Kyoto, Japan). The column effluent was monitored at 215 nm (Shimadzu model SPD-6AV spectrophotometric detector, Kyoto, Japan) and collected at 1-minute intervals (LKB fraction collector, Bromma, Sweden). The column was maintained at a constant temperature of 30 °C (Shimadzu model CTO-6A oven, Kyoto, Japan), and the column pressure ranged from 90 to 120 kgf/cm2. Fifty pi of bovine serum albumin (0.2 g/1) was added to each collected fraction (50 ,ul) before freezing. After freeze-drying, the fractions were resuspended in RIA buffer, and the ANP level in each fraction was measured by radioimmunoassay. Calibration was -23 -performed against synthetic a-ANP (#9103, Peninsula Laboratories, Belmont, CA), synthetic |3-ANP (#9105, Peninsula Laboratories, Belmont, CA) and y-ANP purified from rat atrium using the method of Kangawa et al. (1985). 2.2.5.1 Plasma Samples The extracted plasma samples were frozen and lyophilized overnight. The dry sample was reconstituted in 50 ul of HPLC solvent (60% CH 3 CN in 0.1% TEA) and injected into the HPLC system by autoinjector (Shimadzu model SIL-6A, Shimadzu Corp. Kyoto, Japan). 2.2.5.2 Perfusate Effluents from an isolated rat atrium collected over the 100-minute perfusion period were pooled together, and lyophilized. The dry powder was then reconstituted in 200 ul of HPLC solvent, centrifuged, and 25 ul sample was injected into the HPLC system. 2.2.5.3 Atrial ANP The homogenized atrial suspension was diluted 20 times in HPLC solvent, centrifuged, and a 20-ul aliquot was analyzed by the HPLC system. 2.2.6 Radioimmunoassay for ANP 2.2.6.1 Preparation of Radiolabeled ANP Radiolabelling of ANP was achieved by the Chloramine-T method (Greenwood and Hunter, 1963). A small amount of rat a - A N P ^ (1-2 ug) was pipetted into a small plastic vial, together with 20 pi of 0.05 M - 24 -phosphate and 0.15 M NaCl buffer, Ph 7.4. Fifty ul of 0.5 M phosphate buffer, pH 7.0, was then added to the mixture. One mCi of carrier-free, high-specific activity Na 1 2 5I (about 2 - 3 ul) (Amersham, Oakville, Ont) was added and thoroughly mixed by finger-flicking the tube. Freshly prepared Chloramine-T (2.5 ug in 5 ul of 0.15 M NaCl, 0.05 M phosphate buffer, pH 7.4) was added to the mixture to start the reaction. The reaction was allowed to continue for 10 - 20 seconds while the vial was generously mixed by finger-flicking the tube. The reaction was stopped by adding 300 ul of bovine serum albumin (BSA) (10 g/1) and the contents of the reaction vial was then quickly transferred to a gel-filtration chromatography column (0.5 x 15 cm) packed with Sephadex G-10 beads, to separate 1 2 5I-ANP from the free 1 2 5I. The eluted fractions were collected at 5 minutes intervals. A small aliquot (2 ul) from each fraction was counted in LKB 1275 minigamma counter (Wallac, Finland) to determine the positions of the 1 2 5I-ANP and the 1 2 5I peaks (Fig. 4). The 1 2 5I-ANP fraction was stored in 25 ul aliquots at -70°C. 2.2.6.2 Radioimmunoassay (RIA) of ANP Measurement of plasma, perfusate, and tissue ANP levels were performed using RIA method. ANP antibodies were purchased from Peninsula Laboratory Co. (Belmont, CA). The antibodies bind to the C-terminal end of the peptide and has a 100% cross-reactivity with human a-ANP, rat a-ANP, and atriopeptin III (Peninsula Laboratory catalog). The radioimmunoassay was performed in RIA phosphate buffer containing 0.1% BSA, 0.01% triton X-100, and 0.1% NaN 3. Synthetic rat a-ANP at a concentration of 1000, 500, 250, 125, 62.5, 31.25, 15.6, -25 -Radioact iv i ty (cpm x 5 i million) [125-ll-ANP Fract ion No. Fig. 4 Chromatographic separation of the 125I-ANP and 1 2 5l peaks in the labelling of ANP with fodine-125. - 26 -7.8, 3.9, 1.95 pg/tube was used to construct standard curves. The incubation mixture consisted of 100 ul of standard or sample and 100 )il of antibody, and was incubated overnight at 4°C. Iodinated ANP (100 jil) (approximately 24,000 cpm) was added, mixed, and incubated overnight at 4°C. Bound and free ligands were separated by the double-antibody method utilizing goat anti-rabbit gamma-globulin (100;ul, 1:50) in the presence of 1% pooled normal rabbit serum. After the addition of 750 pi of 5% polyethylene glycol 8000 to facilitate precipitation, the tubes were centrifuged at 3,000 rpm at 4°C for 30 minutes. The supernatant was aspirated using a vacuum suction and the radioactivity in the precipitate was measured in LKB 1275 minigamma counter (Bromma, Sweden). The radioactivity in the sample was converted to picogram of ANP by fitting the counts in the unknown samples to the standard curve by using Least Squares Fit computer program. 2.2.7 Receptor Studies 2.2.7.1 Preparation of Glomerular Membranes Kidneys obtained previously from the perfusion experiments were used to prepare glomerular membrane. The kidneys were dissected longitudinally, and the medulla and papilla discarded. The resulting cortical rims were finely minced with a razor blade and gently pressed with the blunt end of a syringe inner piston. The tissue was pressed through a stainless steel sieve (Bellco Biotechnology, Vineland, NJ) with a pore size of 150;um, and then rinsed from the undersurface through stacked sieves with pore sizes of 100 jum (to remove large debris) and 75 jum (to retain glomeruli), respectively. Glomeruli retained on the 75-pm sieve were thoroughly washed with ice-cold Dulbecco's Phosphate-- 27 -Buffered Saline (PBS) (Gibco Laboratories, Grand Island, NY) and collected into a 50-ml centrifuge tube. A small suspension was placed under light microscope to check for purity. Usually the glomerular preparation was >95% pure. The glomeruli were washed twice by centrifugation at 1,000 rpm at 4 °C and resuspended in 50 mM Tris HC1 buffer, pH 7.4. The glomeruli were then kept on ice and homogenized with a Caframo stirrer (Wiatron, Ont) at setting 8 for 90 seconds. The homogenate was sedimented at 1,000 rpm for 10 minutes to remove unbroken cells, nuclei, and debris. The resulting supernatant was centrifuged at 15,000 rpm for 30 minutes. The pellet was suspended in Tris buffer with 1% Tyrode salt and vortexed to disperse the pellet. The glomerular membrane obtained from the kidneys of one rat were suspended in 2.0 ml of Tris-Tyrode buffer. Two 50 vol aliquots were taken for protein determination by the Lowry method (Lowry et al., 1951). The glomerular membrane suspension was kept frozen in 1 ml aliquots at -70 °C. 2.2.7.2 Receptor Binding Assay a) Dissociation Curves The glomerular membrane fractions were thawed and diluted in assay buffer to yield a final concentration of 0.5 mg/ml membrane protein. The assay buffer contains 50 mM Tris HC1 (pH 7.4), 1% Tyrode salt, 1 pM aprotinin, 0.4% phenylmethylsulfonyl fluoride (PMSF), and 0.2% bovine serum albumin (BSA). To determine the effect of incubation time, glomerular membranes were incubated with 120 fmol 1 2 5I-ANP (70,000 cpm) for varying periods up to 120 minutes. Dissociation - 28 -rates were determined by adding 100 nM unlabelled ANP at binding equilibrium and determining the remaining radioactivity as a function of time up to 120 minutes. To determine the optimal amount of receptor protein on the specific binding of 1 2 5I-ANP, increasing amount of glomerular membrane fraction (0.01 to 1.0 mg/tube) were incubated with 120 fmol (70,000 cpm) for 10 minutes at 20°C in the presence or absence of 100 nM of unlabelled ANP. Competitive Binding Study Synthetic unlabelled rat a-ANP was prepared by diluting in assay buffer to concentrations of 100,000, 4,000, 2,000. 1,000, 750, 500, 250, 62.5, 15.6, and 3.9 pg/20 ;ul to determine glomerular receptor density and equilibrium dissociation constant using competitive binding techniques, . The experiment was started by adding 20 pi of unlabelled ANP to each assay tube to saturate the binding sites on the membrane. Each tube was extensively vortexed and incubated in water bath at 20 °C. After 5 minutes, 100 ^ll of radiolabelled ANP (60,000 - 80,000 cpm) was added to each tube and vortexed. The incubation was continued in the water bath at 20 °C for another 10 minutes to allow competition of labelled and unlabelled ANP for the receptor binding sites. Glass microfiber filters (Boehringer Mannheim, Indianapolis, IN) were placed into 1% polyethylenimine and soaked for 2 to 4 hours at room temperature. At the end of the incubation period, the pre-soaked filters were placed on a filtration apparatus, and - 29 -excess polyethylenlmine was removed by briefly applying suction. Twelve ml of ice-cold wash-buffer (PBS containing 0.2% BSA) were added to the first tube, vortexed, and poured onto the first filter, followed by a 12-ml wash of the first tube. Suction was then applied to separate bound radioactivity from free, and the filter was washed with an additional 12 ml of wash buffer. In the same fashion, one duplicate set of standards was separated first, followed by all samples, and then by the second set of standard tubes, also in duplicate. Filter-associated radioactivity was counted in a LKB 1275 minigamma counter (Wallac, Finland) with 75% counting efficiency. Averages of duplicate determinations of bound 1 2 5I-ANP were used for data analysis. 2.3 Data Analysis Results are expressed as mean + standard error of the mean (SEM) for separate experiments. Glomerular ANP receptor density (B^) and equilibrium dissociation constant (KJ were determined from the binding data using a computer-based LIGAND program (Munson and Rodbard, 1980). Linear regression analysis was used to determine correlations between variables. Statistical analysis was performed using unpaired Student's t test or analysis of variance when appropriate, to evaluate the significance of the difference between means. P value of less than 0.05 was considered to be significant. - 30 -3.0 RESULTS 3.1 Physical Characteristics of the Rats The rate of growth of the Wistar rats was carefully observed over the duration of the present study. This is illustrated in Fig. 5. The rats exhibited two growth phases: an initial rapid-growth phase, as the rat rapidly gained weight from birth to approximately 100 days of age; and a plateau phase, in which a slower growth rate was observed. Physical characteristics of the four age groups of rats: whole body weights, whole heart, right atrium, left atrium, and kidneys, are summarized in Table 1. 3.2 Effect of Age on Renal Excretory Function Table 2 summarizes glomerular nitration rate (GFR), 24-h urinary sodium excretion (UNaV), fractional sodium excretion (FEpjJ, plasma sodium (PNa), and fractional excretion of water (FE^o) from the 24-hour clearance study. GFR steadily increased from 1 to 10 months (both p < 0.005 vs 3-month-old), and declined with advancing age to 20 months (p < 0.05 vs 3-month-old, p < 0.0005 vs 10-month-old). The changes in U N a V seemed to parallel the changes in G F R U N a V increased from 1 to 10 months (both p < 0.05 vs 3-month-old), and decreased at 20 months (p < 0.05 vs 3-month-old). The difference between the 10- and 20-month-old groups was statistically significant (p < 0.05). -31 -800 Body weight (g) 600 400 200 0 m 0 OB m a W 0 100 200 300 400 Age (day) 500 600 700 Fig. 5 Growth curve of male Wistar rats. Rats were purchased at 1 or 3 months old and allow to grow to the desire age. Each value represents the mean of 2 to 5 rats. TABLE I AGE. BODY AND ORGAN WEIGHTS IN THE FOUR AGE GROUPS OF RATS AGE GROUP (month) 1-Month 3-Month 10-Month 20-Month No. of Rats 36 48 29 21 Age (days) - Range - mean ± SEM 30-36 33 ± 1 70-95 84 ± 3 200 - 350 289 ± 12 580 - 620 596 ± 4 Weight (g) - Range - mean ± SEM 126 - 165 150 ± 3 280 - 360 334 ± 7 550-710 600 ± 15 520 - 655 576 ± 19 Heart Weight (g) 0.65 ± 0.05 0.94 ± 0.02 1.66 ± 0.04 1.55 ± 0.04 Right Atrium Weight (mg) 23.2 ± 1.1 33.7 ± 0.8 54.7 ± 2.3 59.4 ± 2.2 Left Atrium Weight (mg) 17.4 ± 0 . 9 22.7 ± 1.0 42.1 ± 2.7 41.0 ± 3 . 9 2 Kidneys Weight (g) 1.53 ±0 .05 2.17 ±0 .05 3.80 ±0 .14 3.40 ± 0.21 Values are expressed as mean ± SEM -33 -TABLE II SUMMARY OF 24-HOUR RENAL CLEARANCE DATA AGE GROUP (month) 1-Month 3-Month 10-Month 20-Month No. of Rats 15 22 14 10 P N a (mEq) 143 + 0.3 a 142 ±0 .3 144 ± 0 . 2 " 1 4 5 ± 0 . 3 b t GFR (ml/min) 1.90 ± 0.07 b 3.21 ± 0.07 4.50 ± 0.10 b 2.43 ± 0.06 a f UN aV (mEq/24h) 1.79 ± 0.04 b 2.32 ± 0.06 2.72 ± 0.07 a 2.01 ± 0.06 a FE N a (%) 0.66 ± 0.07 b 0.51 ± 0.03 0.42 ± 0.03 b 0.57 ± 0.05 b F E ^ (%) 0.71 ± 0.05 b 0.50 ± 0.04 0.44 ± 0.05 b 0.55 ± 0.04 b Values are expressed as mean + SEM Abbreviations: P N a - plasma sodium GFR - glomerular filtration rate UN aV - 24-h urinary sodium excretion FE N a - fractional sodium excretion FEH ; > r i - fractional excretion of water a - p < 0.05 as compared to 3-month-old b - p < 0.005 as compared to 3-month-old t - p < 0.05 as compared to 10-month-old - 34 -Similarly, F E ^ and F E j ^ q values were altered due to the changes in GFR. Both FElia and F E ^ q was increased in a stepwise fashion from 1 to 10 months of age (both p < 0.005 vs 3-month-old), and decreased with advancing age to 20 months (p < 0.005 vs 3-month-old). Plasma sodium concentration (PNa) decreased from 1 to 3 months (p < 0.05), and increased at 10 months and 20 months of age (both p < 0.001). Between 10- and 20-month-old, P N a was statistical different (p < 0.05). 3.3 Radioimmunoassay As shown in Fig. 6, binding of 1 2 5I-a-ANP to the anti-ANP serum was inhibited by unlabelled a-ANP. The 50% binding intercept of the standard curve was 45 ± 12 pg/tube for 22 determinations. The minimum detectable quantity was 5 pg/tube. The inter- and intra-assay coefficients of variation of IR-ANP measurements were less than 12%. The recoveries of various quantities (1.25 to 900 pg) of a-ANP added to a pool of rat plasma or buffer are shown in Fig. 7. The regression line calculated from the amount of a-ANP added vs. the amount recovered was almost superimposable on the theoretical line (100% recovery). 3.4 Effect of Age on Plasma ANP Levels Sodium pentobarbital was chosen in the present study to anaesthetize the animals because it is an anaesthetic agent widely used and has the least effect on the circulating ANP levels (Horky et al., 1985). Only a -35 -B / B o {%) Fig. 6 Representative standard binding curve of a-ANP. B/Bo, Bound to free ratio. - 36 -ANP found (pg/tube) 1200 i 0 200 400 600 800 1000 A N P added (pg/tube) 100% recovery Found Fig. 7 Recovery of a-ANP added to a pool of rat plasma. The dotted line represent 100% recovery. The results shown are the mean+SEM of triplicate determinations. - 37 -small amount of blood ( 2 - 3 ml) was taken from each animal to minimize any effect of volume depletion on the release of ANP. During the anaesthetization and blood withdrawal process, extreme care was exercised to avoid any stimulation to the rats. The mean plasma irnrnunoreactive ANP (IR-ANP) concentration in 1-month-old rats was 75 + 4 pg/ml (N = 31, range: 46 to 124 pg/ml), and this was increased to a mean of 91 ± 4 pg/ml (N = 48, range: 51 to 132 pg/ml) in 3-month-old rats (p < 0.005). The 10-month-old rats had a mean level of 115 ± 8 pg/ml (N = 27, range: 52 to 210 pg/ml, p < 0.005 vs 3-month-old). The highest level of ANP was found in the 20-month-old, at 160 ± 17 pg/ml (N = 18, range: 91 to 328 pg/ml) and it was significantly higher than the other three younger age groups (p < 0.001) (Fig 8). Together with additional data obtained from rats with various ages, there was a significant positive correlation between plasma ANP levels and age (N = 147, r = 0.59, p < 0.0005) (Fig 9). 3.5 Effect of Age on ANP Secretion Rate The spontaneous release of ANP from the isolated right atrium in each age group is illustrated in Fig. 10. The release of ANP increased from 5.74 + 0.42 pg/min/mg to 10.0 ± 0.65 pg/min/mg as the rat matured from 1 to 3 months (p < 0.005). However, as the rat aged, the release of ANP was significantly decreased to 7.30 ± 0.43 pg/min/mg in the 10-month-old (p < 0.005 vs 3-month-old) and 4.70 ± 0.25 pg/min/mg in the 20-month-old (p < 0.005 vs 3-month-old). Fig 11 illustrates the mean secretion rate in the different groups of rats. There was no apparent correlations between the rate of ANP release and the plasma - 38 -Plasma IR-ANP (pg/ml) 200 i 1-month 3-month 10-month 20-month Age Fig. 8 Effect of age on circulating ANP levels. The results shown are mean+SEM. N = 31, 48, 27, and 18, for 1-, 3-. 10-, and 20-month-old rats, respectively. * p < 0.005 as compared to 3-month-old - 39 -Plasma IR-ANP level(pg/ml) N - 147 r * 0.59 p < 0.0005 Y ' 0.15 X * 78.2 a » 8 \f 0 100 200 300 400 Age (day) 500 600 700 Fig. 9 Relationship between plasma ANP levels and age. (N • 147, r = 0.59, p < 0.0005). Rate of IR-ANP secret ion (pg/min/mg) 14 -12 -2 -0 H 1 1 1 1 1 r— 1 1 1 r~ 0 10 20 30 40 50 60 70 80 90 100 Time (minute) A " - 1-month 3-month - Q - 1 0 - m o n t h - i ^ - 2 0 - m o n t h Fig. 10 Effect of age on the rate of ANP secretion from isolated right atrium. The results shown are mean + SEM. N - 15, 24,16, and 12, for 1-, 3-, 10-, and 20-month-old rats, respectively. Rate of IR-ANP secret ion (pg/min/mg) 1-month 3-month 10-month 20-month Age Fig. 11 Summary of ANP released from each age group. The results shown are mean+SEM. N = 15, 24, 16, and 12, for 1-, 3-. 10-, and 20-month-old rats, respectively. * p < 0.005 as compared to 3-month-old. - 42 -ANP concentration (N = 44, r = 0.057, p > 0.5), suggesting that high plasma ANP concentrations found in the older rats were not due to inappropriate secretion. 3.6 Effect of Age on Atrial ANP Content The concentration of ANP in right and left atrium of the four age groups of rats is illustrated in Fig. 12. The average right and left atrium of the 1-month-old rats contained 230 ± 1 4 and 182 ± 6 ng ANP/mg tissue, respectively; this was increased to 360 ± 1 5 and 315 ± 9 ng ANP/mg tissue, respectively, in the 3-month-old rats (p < 0.005 in both atria). On the other hand, ANP content in right and left atrium were slightly lower but did not reach statistical significance in the 10-month-old (329 ± 1 7 and 286 ± 1 2 ng/mg tissue, respectively, p > 0.05 in both atria vs 3-month-old). In 20-month-old, the ANP content were further reduced to 175 ± 9 and 140 ± 8 ng/mg tissue, respectively, (p < 0.005 in both atria vs 3-month-old). ANP content in both atria was lower in the 20-month-old group than the 10-month-old group (p < 0.005). In all age groups, the right atrial ANP concentration was significantly greater than the left (p < 0.05). Interestingly, there was a positive correlation between the rate of ANP release and the right atrial ANP content (N = 42, r = 0.50, p < 0.01) (Fig. 13). This suggests that the release of ANP from the right atrium may depend on the atrial ANP content. On the other hand, there was no correlation between plasma ANP levels and atria ANP concentrations (N = 41, r = 0.034, p > 0.5), again confirming the previous results that elevated plasma ANP levels found in the older rats may not be due to - 43 -Atrial IR-ANP concentrat ion (ng/mg) 400 i Fig. 12 Effect of age on atrial ANP content. The results shown are mean+SEM. N = 15, 24,16, and 12, for 1-, 3-, 10-, and 20-month-old rats, respectively. * p < 0.005 as compared to 3-month-old; " p < 0.005 as compared to 10-month-old. - 44 -Fig. 13 Relationship between amount of ANP released and the atrial ANP content. (N p < 0.01). = 42, r = 0.50, altered secretion. 3.7 Effect of Age on Molecular Species of Circulating ANP i To characterize the molecular species of plasma ANP in the different age groups of rats, plasma extracts were analyzed by RP-HPLC coupled with RIA. The elution positions of standard a-, B-, and purified y-ANP are depicted in (Fig. 14). Representative RP-HPLC profiles of plasma extracts from each age group of rats are illustrated in Fig 15. The plasma IR-ANP in all age groups consisted of two major components with different molecular weights. The first peak eluted at the position of synthetic a-ANP, and the second emerged with an apparent molecular weight (13 K daltons) of y-ANP. In 5 plasma extracts from the 1- and 3-month-old rats, about 70 - 100% of the IR-ANP in the plasma had a molecular size similar to that of a-ANP (Fig 16). A smaller but distinctive peak was eluted at an apparent molecular weight of y-ANP in all but two of the samples, and represented between 8% to 30% of the total IR-ANP. With increasing age, the proportion of the two molecular species of ANP was changed. Fig. 17 summarizes the mean percentage of a- and y-ANP, The result represents the average percentage from 5 plasma extracts analyzed separately. The mean percentage of y-ANP in the total IR-ANP was 13 ± 4%, 16 ± 7%, 26 ± 7%, and 39 ± 4% for 1-, 3-, 10- and 20-month-old, respectively. Thus, the y-ANP concentration and the percentage of y-ANP in IR-ANP were markedly Increased in 20-month-old rats (p < 0.01 vs 3-month-old). - 46 -IR-ANP (fmol/fraction) C H 3 C N gradient {%) 300 | 1 70 250 200 150 100 Alpha-ANP Gamma-ANP 60 6 11 16 21 26 31 36 Fract ion No. 41 IR-ANP C H 3 C N gradient Fig. 14 Elution positions of synthetic a-ANP, synthetic p-ANP, and purified y-ANP on a C 1 8 Partisil column eluted with a linear gradient of 0.1% TFA-acetonitrile. The a-IR-ANP level in each fraction was assayed by the RIA for a-ANP. - 47 -A) 1-month-old IR-ANP (fmol/fraction) CH3CN gradient (%) 50 i 1 100 Alpha-ANP Gamma-ANP 80 60 40 1 6 11 16 21 26 31 36 41 Fraction No. • IR-ANP CH3CN gradient 35 30 25 20 -B) 3-month-old IR-ANP (fmol/fraction) CH3CN gradient (%) 15 Alpha-ANP Gamma-ANP 100 80 60 - 40 Q j I 1 I I I I | I I I 1 | I I I I | I I I I | I V I I | I I I I | I I I I | I I I l Q 1 6 11 16 21 26 31 36 41 Fraction No. IR-ANP CH3CN gradient 30 25 20 15 -c) 10-month-old IR-ANP (fmol/fraction) CH3CN gradient (%) Alpha-ANP Gamma-ANP 100 80 60 40 0 | I I I I | • • I I | I I I I | I • I • | n I V J C P I I I | I t I I | I I i I | I I I I Q 1 6 11 16 21 26 31 36 41 Fraction No. 25 D) 20-month-old IR-ANP (fmol/fraction) CH3CN gradient (%) Alpha-ANP Gamma-ANP 100 80 Q I I I I | I I I I | I I I I |Tl I I | I I I I | I I I I | I I I I | I I I I | I I I I Q 1 6 11 16 21 26 31 36 41 Fraction No. • IR-ANP • CH3CN gradient • IR-ANP CH3CN gradient Fig. 15 Representative RP-HPLC profiles of the plasma IR-ANP. A) 1-month-old B) 3-month-old C) 10-month-old D) 20-month-old. The analysis was carried out in C 1 8 Partisil column eluted with a linear gradient of 0.1 % TFA-acetonitrile. The cc-IR-ANP level in each fraction was assayed by the RIA for a-ANP.The elution positions of synthetic a-ANP and purified y-ANP are indicated. - 48 -Fig. 16 Schematic illustrations of individual plasma concentrations of a-ANP and y-ANP in the total plasma IR-ANP concentration. * p < 0.01 as compared to 3-month-old. % in plasma IR-ANP Fig. 17 Schematic illustrations of average percentages of a-ANP and y-ANP in the total plasma IR-ANP concentration from 5 RP-HPLC runs in each group. * p < 0.01 vs 3-month-old - 50 -3.8 Effect of Age on Molecular Species of Secreted ANP In all four age groups, virtually all of the immunoreactlve ANP secreted by the isolated right atrium had a molecular size similar to that of a-ANP (Fig. 18). A small percentage of perfusate (1% - 8%) was chromatographically identical to y-ANP. Fig 19 illustrates the relative percentage of a- and y-ANP from each RP-HPLC and RIA analysis. The mean of 3 analyses in each age group is summarized in Fig 20. The y-ANP represented approximately 1% of the total perfusate ANP in the 1-, 3- and 10-month-old groups, but close to 8% of total ANP in the 20-month-old was y-ANP (p < 0.05 vs 3-month-old). It indicates that the cosecretional processing mechanism of y-ANP into a-ANP is influenced by age. 3.9 Effect of Age on Molecular Species of Atrial ANP Representative RP-HPLC profiles of atrial extracts from the right or left atrium in each age group are shown in Figs. 21 and 22, respectively. Evidently, the only component of IR-ANP was y-ANP like material in all 4 age groups, and neither a- nor p-ANP were present in either atrial tissues. There was no significant differences in elution pattern between the right and left atrium. No major difference was seen between the four groups of animals. Previous reports demonstrated that y-ANP may undergo partial degradation during the extraction procedure to form a-ANP or (3-ANP (Sugawara et al., 1988). Synthetic a-ANP may also dimerize to form p-ANP during its exposure to the conditions used for the extraction (Kangawa et al., 1985). In the present study, the atrial form of ANP was - 51 -A) 1-month-old IR-ANP (Imol/fraction) CH3CN gradient {%) 1000 800 600 400 200 -Alpha-ANP " 111111 Gamma-ANP 100 80 60 40 20 11 16 21 26 31 36 41 Fraction No. IR-ANP CH3CN gradient 1400 1200 -1000 800 600 400 -200 -B) 3-month-old IR-ANP (tmol/fraction) CH3GN gradient (*) 100 1 6 11 16 21 26 31 36 . 41 Fract ion No. - e - IR-ANP CH3CN gradient C) 10-month-old IR-ANP (fmol/fraction) CH3CN gradient («) 1600 1200 9 0 0 600 300 Otf Alpha-ANP Gamma-ANP 100 80 60 - 40 20 11 16 21 26 31 36 41 Fraction No. • IR-ANP - CH3CN gradient D) 20-month-old IR-ANP (fmol/fraction) CH3CN gradient (« ) 3501 1100 300 250 200 150 100 50 Alpha-ANP Gamma-ANP 8 0 6 0 40 2 0 1 6 11 16 21 26 31 36 41 Fract ion No. • IR -ANP - CH3CN gradient Fig. 18 Representative RP-HPLC profiles of the perfusate IR-ANP. A) perfusate from 1-month-old rats. B) perfusate from 3-month-old rats. C) perfusate from 10-month-old rats. D) perfusate from 20-month-old rats. The elution positions of synthetic a-ANP and purified y-ANP are indicated. - 52 -CO R-ANP Concentrat ion (pmol/mg tissue) 10 8 6 -4 -0 \/wmA 7\ \A \A V 1 - m o n t h 3 - m o n t h 1 0 - m o n t h 2 0 - m o n t h Age CZH A lpha-ANP H i G a m m a - A N P Fig. 19 Schematic illustrations of individual perfusate concentrations of a-ANP and yANP in the total perfusate IR-ANP concentration. A lpha -ANP {%) Gamma-ANP {%) 100 -8 0 -60 40 -20 0 10 8 6 - 4 - 2 0 1-month 3-month 10-month 20-month Age A l p h a - A N P H Gamma-ANP Fig. 20 Schematic illustrations of average percentages of a-ANP and "y-ANP in the total perfusate IR-ANP concentration from 3 RP-HPLC runs in each group. * p < 0.05 as compared to 3-month-old. - 54 -300 250 200 150 A) 1-month-oId IR-ANP (Imol/lraction) CH3CN gradient («) 100 1 6 11 16 21 26 31 36 41 Fraction No. B) 3-month-old IR -ANP <(mol/1raction) CH3CN gradient («) ICO 350 300 " 250 200 150 lOO 1 6 11 16 21 26 31 36 41 Fraction No. • IR-ANP - CH3CN gradient •IR-ANP • CH3CN gradient 300 250 200 150 C) 10-month-old IR-ANP (fmol/lractloo) CH3CN gradient (%) D) 20-month-old 100 100 1 6 11 16 21 26 31 36 41 Fraction No. • IR-ANP CH3CN gradient IR-ANP (fmol/fraction) CH3CN gradient (*) 3001 1100 260 200 150 100 Gamma-ANP o 80 60 40 20 U 1 1 1 imprwTmjffflTrTTrTrfnnpnm 111 t*wprmfni 11111q 1 6 11 16 21 26 31 36 41 Fraction No. • IR-ANP - OH3CN gradient Fig. 21 Representative RP-HPLC profiles of right auricular extracts of the four age groups of rats. A) extract from a 1-month-old rat. B) extract from a 3-month-old rat. C) extract from a 10-month-old rat. D) extract from a 20-month-old rat. The elution position of purified y-ANP is indicated. - 55 -A) 1-month-old B) 3-month-old 350 300 250 200 IR-ANP ((mol/fraction) CH3CN gradient (*) 150 -1O0 1 6 11 16 21 26 31 36 41 Fraction No. IR-ANP (fmoi/fraction) OH3CN gradient (*) 4001 1100 3O0 -200 -100 -•IR-ANP • CH3CN gradient 1 6 11 16 21 26 31 36 41 Fraction No. -e- IR-ANP CH3CN gradient C) 10-month-old „ IR-ANP (f mol/fraction) OH3CN gradient (%) 3501 2 l_i 1 0 0 300 -250 -200 -150 -100 -6 11 16 21 26 31 36 41 Fraction No. • IR-ANP ' CH3CN gradient D) 20-month-old IR-ANP (fmol/fraction) CH3CN gradient {%) 100 250 -200 150 -100 1 6 11 16 21 26 31 36 41 Fraction No. - e - IR-ANP CH3CN gradient Fig. 22 Representative RP-HPLC profiles ot left auricular extracts of the four age groups of rats. A) extract from a 1-month-old rat. B) extract from a 3-month-old rat. C) extract from a 10-month-old rat. D) extract from a 20-month-old rat. The elution position of purified y-ANP is indicated. - 5 6 -found to be predominant y-ANP with little or no a-ANP present. No p-ANP was detected in plasma, perfusate or atrial extract samples. This suggests the extraction condition used in this study do not alter the natural molecular form of ANP. 3.10 Effect of Age on Glomerular Receptor Binding Characteristics 3.10.1 Optimal Conditions for Radiolabelled Receptor Assay Specific binding of 1 2 5I-ANP to the glomerular membrane fraction was a time-dependent process in all four groups of the rats (Fig 23). When determined at 20 °C in the presence of 0.12 nM labelled ligand (70,000 cpm), the specific binding increased rapidly and reached a peak stable equilibrium value within 5 minutes. This was stable for at least another 15 minutes before a gradual decline in binding was observed. After 120 minutes of incubation, the specific binding had decreased to 60% of the maximum binding. At 20°C, the specific binding of 1 2 5I-ANP to the glomerular membrane fraction was proportional to the membrane protein concentration from 0.05 to 0.20 mg/tube (Fig. 24). 3.10.2 Competition and Scatchard Plots A representative plot of the competitive inhibition binding study using glomerular membranes from 1-, 3-, 10-, and 20-month-old rats is depicted in Fig. 25. Nonspecific binding was determined by the addition of 100 nM of unlabelled ANP. In this series of competitive binding inhibition experiments, nonspecific binding was linear and accounted for 2 - 3% of total radioactivity. Binding was not further reduced by - 57 -CO 500 125-1 ANP bound (fmol/mg protein) 400 -300 200 100 0 20 i r 40 60 80 Incubation Time (min) 100 120 1-month - ^ 3 - m o n t h - B - 10-month 20-month Fig. 23 Line graph showing the effect of incubation time oh the binding of 12SI-ANP to the rat renal membrane fractions from the four age groups of rats. Membrane suspension (0.1 mg/tube) was incubated with 70,000 cpm (0.12 nmol) 125I-ANP at 20°C. Values are mean of triplicate determinations. Spec i f i c Binding of 1251-ANP (cpmxIOOO) 16 i :  0 0.2 0.4 0.6 0.8 1 Glomerular membrane (mg protein/tube) Rg. 24 Line graph showing effect of the amount of receptor proteins on the specific binding of ^l-ANP. Increasing amount of the renal membrane fraction (0.01 to 1.0 mg/tube) were incubated with 70,000 com (120 fmol) for 10 minutes at 20°C in the presence or absence of 100 nM of unlabelled ANP. Values are means of triplicate determinations. - 59 -0.09 0.08 -0.07 -0.06 0.05 -0.04 -0.03 Bound/Total 0.02 •10 -9.5 -9 -8.5 -8 -7.5 Log Total A N P (M) ^ - 1 - m o n t h - 0 - 3 - m o n t h Q - 1 0 - m o n t h - ^ f - 2 0 - m o n t h -7 -6.5 Fig. 25 Representative competitive inhibition plot of 12SI-ANP binding to membrane fractions from rat kidneys in each age group. Membranes (0.1 mg/tube) were incubated with increasing amount of unlabelled ANP (0 to 100,000 pg/tube). Each value represents the mean of duplicate determinations. - 60 -higher (up to 100 uM) concentrations of unlabelled ANP. In the absence of unlabelled ANP, 6 - 9% of total added radioactivity was specifically bound to the glomerular membrane. Specific binding of 1 2 5I-ANP was saturable with increasing concentrations of 1 2 5I-ANP. Glomerular membranes isolated from all four groups of rats exhibit a similar pattern. Scatchard analysis of the above data revealed that all 4 groups of rats has a single high class of binding sites with an apparent equilibrium dissociation constant (KJ (Fig. 26). The result from all competitive binding inhibition experiments is summarized in Fig. 27. Glomerular ANP receptor density increased as the rats matured from 1 to 3 months of age and consistently decreased with advancing age. On the other hand, receptor binding affinity declined (higher k,,) from 1 to 3 months of age and enhanced at 20 months of age. - 61 -Bound/Free 0.1 i 0 2 4 6 8 10 12 14 16 Bound (pmol/L) •A- 1-month 3-month • S - 10-month 20-month Fig. 26 Representative Scatchard plot of ,25I-ANP binding to membrane fractions from rat kidneys in each age group. Membranes (0.1 mg/tube) were incubated with increasing amounts of unlabelled ANP (0 to 100,000 pg/tube). Each value represents the means of duplicate determinations. In this example, k,, and B m j i x are 0.14 nM and 162 fmol/mg for 1-month-old (triangle), 0.30 nM and 485 fmol/mg for 3-month-old (diamond), 0.29 nM and 353 fmol/mg for 10-month-old (square), and 0.078 nM and 221 fmol/mg for 20-month-old (star), respectively. - 6 2 -Bmax (fmol/mg) 5 0 0 f— Kd (nM) Fig. 27 Summary of the receptor binding results - equilibrium dissociation constants (K„) and maximum binding capacities (B^). The results shown are means+SEM of 11 experiments. * p < 0.001 as compared to 3-month-old. ** p < 0.05 as compared to 3-month-old. - 6 3 -4.0 DISCUSSION Using senescent (the period around or beyond the median life span) laboratory animals to assess the potential age-related changes in ANP physiology may clearify any understanding of this hormone in both normal and pathological states. The results obtained in this aging model may also provide a natural extension to the current understanding in young adult rats. The life span of laboratory rats is relatively short, approximately 2 - 3 years, allowing gerontological studies of this species to be conducted within a reasonable time-frame. The male rat reaches puberty at around 50 ± 10 days, and the body weight of the young adult is around 300 - 400 grams (Baker et al., 1979). It is generally accepted that senescence is about 18 months or older (Dax, 1987). The selection of age groups in the present study (1-, 3-, 10-, and 20-month-old) is based on this information. 4.1 Effect of Age on Renal Functions Results from the 24-hour clearance studies clearly indicate the kidney's ability to excrete salt and water is affected by age. Maturation of rats, from 1 to 3 months, was associated with an increase in GFR and U N a V, and a decrease in P N a , FE,^, and F E ^ Q (Table 2). GFR was at the highest value when the rat reached about 10 months of age; at which time both U N a V and P N a were increased while FE,^ and F E ^ Q were further reduced. With the aging of rats to 20 months of age, GFR and U N a V were significantly reduced, resulting in elevation of F E ^ , FE^o and P N a . Values obtained in these clearance study are comparable to other published results (Peiico et al., 1989; Soejima et al., 1988; Hirata et al., 1985). - 64 -These observations are consistent with the fact that newborn and young animal of many species is characterized by an attenuated natriuretic response to sodium loading (Kleinman, 1982). Saline expansion of the newborn animal is often associated with the development of edema, weight gain, and increase in the serum concentration of sodium (McCance and Widdowson, 1957). These are thought to be caused by inappropriate reabsorption of sodium in the distal nephron of the kidney (Kleinman and Bank, 1983). On the other hand, a host of abnormalities in renal structure and function accompany advancing age (Papper, 1973). Senescence, for example, results in renal atrophy with a reduction in renal cortical mass (Darmady et al., 1973) and a reduction in renal blood flow and filtration rates (Davies and Shock, 1950). Furthermore, a reduction in the tubular maximum capacity (Tm) for glucose (Miller et al., 1952), and an impairment in the kidney's capacity to concentrate urine (Miller et al., 1952) and handle an acid load (Adler et al., 1968) have been associated with aging. These alterations in kidney function have been implicated to alter the kidney's ability in maintaining sodium homeostasis. 4.2 Effect of Age on Plasma ANP Levels Plasma levels of ANP measured by radioimmunoassay vary widely from approximately 60 to 1650 pg/ml in rats and from 25 to 100 pg/ml in humans (Lang et al., 1985; Gutkowska et al., 1984; de Bold, 1985). The discrepancies may be accounted for by differences in anaesthetic agents used (Horky et al., 1985), procedures for collecting and storing samples, varying recoveries of extraction methods (Rankin et al., 1987), or specificity of the antibody employed. The plasma ANP concentrations - 65 -measured in this study ranged from 50 to 350 pg/ml, which are comparable to most of the published reports. Although a few studies noted that circulating ANP levels in healthy men do not vary with age (Weil et al., 1986; Rascher et al., 1987). the majority of reports described a tendency for ANP levels to increase with age. Ohashi et al. (1987) demonstrated an eight-fold increase in plasma ANP levels in elderly men (64 - 91 years old) compared to young men (24 -28 years old) (120 ± 22 vs 25 ± 5 pg/ml). Haller et al. (1987) observed a 7 times higher ANP concentration in older subjects than in younger subjects (167 + 31 vs 24 + 3 pg/ml). Furthermore, plasma levels of ANP were positively and significantly correlated with age in healthy volunteers (r = 0.71, p < 0.001 by Sagnella et al., 1986; r = 0.35, p < 0.02 by Larochelle et al., 1987; r = 0.82, p < 0.001 by Haller et al., 1987; r = 0.61, p < 0.001 by Richards et al, 1986; and r = 0.46, p < 0.01 by McKnight et al., 1989). However, whether this relationship also exists in rats has not been previously investigated. The present study demonstrated that plasma ANP levels in rats is correlated with age. The aged rats (20-month-old) have significantly higher levels of plasma ANP than the other three younger groups, 1-, 3-, or 10-month-old (160 ± 17 vs 75 ± 4, 91 ± 4, and 115 ± 8 pg/ml, respectively). The mean plasma ANP levels in the 20-month-old was 76% greater than in the 3-month-old rats. In addition, a close relationship between plasma ANP levels and age was found (Fig. 9), indicating to the importance of age as a determinant of plasma ANP levels. - 66 -The reasons for the elevation in plasma ANP concentration in the aged are not clear. It may be speculated that the elevation is due to a secondary effect to hyperaldosteronism (Wong et al., 1989; Lendenson et al., 1987), chronic heart failure (Ezaki et al., 1988), chronic renal failure (Suda et al., 1988), or hypertension (Sagnella et al., 1986; Cantin et al., 1988), which are conditions often associated with old age (Rowe and Minaker, 1985). Alternatively, aging can affect the sensitivity of physiological regulatory centres thereby causing a raise in basal levels of circulating hormones (Helderman et al., 1978; Kirkland et al., 1984; Rondeau et al., 1982). It is also possible that the set-point of ANP basal levels in the plasma is raised in the aged subjects. Finally, the raised plasma ANP level may reflect alterations in metabolic clearance of the circulating peptides. This aspect will be discussed further in the following section. 4.3 Effect of Age on ANP Secretion The elevated plasma ANP levels detected in the aged rats may be a result of increased release of ANP from the atria or reduced clearance of ANP from the circulation. To determine which mechanism is responsible, isolated atria were perfused in a modified Langendorff apparatus to measure the in vitro secretion of ANP. The results suggested that ANP secretion increased with maturation (from 1 to 3 months old) and progressively decreased with age from 3 to 20 months (Fig. 11). ANP secretion was reduced by 27% in the 10-month-old and 53% in the 20-month-old when compared to the 3-month-old. These data clearly indicate that the spontaneous release of ANP increases with maturation and decreases with aging. - 67 -Although the spontaneous ANP secretion in the aged rats were decreased, their plasma ANP levels remained elevated; this suggests that the metabolic clearance of ANP was altered. Since the kidney has been demonstrated to be an important site for the catabolism of atrial peptides (Berg et al., 1988; Tang et al., 1984; Sonnenberg et al., 1988) and that aging has been shown to be accompanied by a variety of abnormalities in the structure and function of the kidney (Papper, 1973), it is conceivable that the renal degradation of ANP is compromised by age. Studies have indirectly demonstrated that when the renal clearance function is impaired, ANP concentration in the plasma quickly rise. For example, in nephrectomized rats (Petersen et al., 1988; Smith et al., 1986) and chronic renal failure patients (Shenker et al., 1987), the plasma ANP concentration was found to be significantly raised. 4.4 Effect of Age on Atrial ANP Content The result of the current study clearly indicated there was age-related changes in atrial ANP content. Atrial ANP contents in both right and left atrium of the rats were elevated from 1 to 3 months of age, and declined slightly but not reaching statistical significance at 10 months. At 20 months, a dramatic reduction was seen; the atrial ANP concentration was only 49% of the 3-month-old (Fig. 12). This data is consistent with previous reports that described a lower atrial ANP content in the newborn (19 - 21 days old) compared to adults (8 -16 weeks old) (Pollock and Banks, 1985; Imada et al., 1985). However, the changes between adult and older animals have not been previously examined. This study also revealed that within the same age group the - 68 -concentration of ANP was greater in the right atrium than in the left atrium (Fig. 12), agreeable with many published reports (Imada et al., 1985; Cantin and Genest, 1985; Katsube et al., 1985; Tsunoda et al., 1986). On the other hand, only a few reports shown that ANP concentration between the right and left atrium is similar (Dolan et al., 1989; Wei et al., 1987). Since tissue ANP concentration is determined by synthesis, storage and secretion, the age-associated decrease in ANP concentration in the atrium per se does not imply a decrease in synthesis. A depletion of atrial tissue due to excessive secretion could have been the cause. However, results obtained from the present study indicate that diminish synthesis is the cause. The aged atrium (20-month-old) not only contained the least amount of ANP in the atrial tissue, but also had the lowest secretion rate compared to the other three groups (Figs. 11, 12). A significant correlation was also found between the rate of ANP release and the atrial ANP concentration regardless of age group (Fig. 13), suggesting that the aging atrium secretes the same proportion of ANP with respect to its ANP content as do atrium from the other three younger groups. Therefore, it is reasonable to conclude that the reduction in ANP secretion by the aging heart is a consequence of decreased synthesis. In an earlier study. Pollock and Banks (1985) reported a significantly lower level of ANP content in atrial extract prepared from newborn rats (19 days of age) than from adult animals (56 days of age). When comparing the natriuretic activities of the extracts, they found that the - 69 -newborn atrial extracts have lower natriuretic activity, and speculated that the compromised ability of newborn animals to excrete a sodium load is related to a deficiency of ANP in the atrium. The data obtained in the present study is consistent with their results. Comparing to the adults (3-month-old), the young rats (1-month-old) were found to contain a lower concentration of ANP in the atrium concurrently with a low sodium excretion (low UN aV) by the kidney (Table 2). In another study, Inscho et al. (1987) noted a significantly lower hypotensive and natriuretic activities of atrial extracts prepared from 290-day-old than from 15-, 39-, or 56-day-old animals. They also observed that the atrial extracts from the old rats (290 days old) was found to contain more ANP per tissue DNA level but had reduced biological activities. This indicates that the composition of individual extracts may be changed with age. Accordingly, this may provide yet another reason as to why the ability of the aging kidney to excrete sodium is curtailed. 4.5 Effect of Age on Molecular Species of Circulating ANP Chromatographic analyses of healthy human plasma samples consistently showed single ANP peaks that correspond to the active a-ANP molecules (Yamaji et al., 1985; Sugawara et al., 1985; Theiss et al., 1987). No other forms of ANP were detected. In normal rat plasma, one major peak that corresponds to a-ANP was detected, along with smaller quantity of the large molecular-weight y-ANP (Kangawa et al., 1984; Misono et al., 1984). In pathological conditions such as chronic renal failure or congestive heart failure, y-ANP was found to increase significantly in proportion to the total ANP concentration (Ogawa et al., 1987; Ding et al., 1987). - 70 -The result from the present study clearly indicates that both a- and y-molecular forms of ANP are present in rat plasma extracts from all four age groups (Figs. 15, 16). In a total of 20 analyses, no {3-ANP was detected (Fig. 15). This is in accordance with other's findings (Kangawa et al., 1984; Misono et al., 1984). However, y-ANP increases significantly in proportion to the total ANP concentration with age (Fig. 17). Close examination of Fig. 16 revealed that the increase in total plasma ANP concentration with age consisted of a smaller increase in the a component and a larger increase in the y component. This may have pertinent significance; as y-ANP is not biologically active, it may be one of the reasons why age-related reduction in renal sodium excretion occurs in the presence of elevated plasma ANP levels. In contrast to most studies where y-ANP was found to account for only a small percentage of total ANP in the plasma, the present study showed y-ANP represented as much as 39% of the total ANP (Fig. 17). This discrepancy may be explained by the fact that the rats used in most studies were relatively young, ranging from 150 to 350 g in body weight. According to the growth curve of the rats used in the present experiment, this can be translated to an age of 1 to 3 months (Fig. 5). Hence, it is only logical that results obtained from these young rats are compared to 1- and 3-month-old groups in the present study. In these two groups, the average percentage of y-ANP in the plasma were about 14% (Fig. 17), agreeing well with the published results. Larger quantities of y-ANP were found only in the older groups (10- and 20-month-old), which had not been previously examined. - 71 -In a human study done by Ohashi and his colleagues (1987), it was demonstrated that the increase in plasma ANP in the elderly was due to an increase in a-ANP and not the y-ANP molecule. Species differences could have accounted for the discrepancy. As mentioned before, y-ANP is usually not found in normal human plasma (Yamaji et al., 1985; Sugawara et al., 1985; Theiss et al., 1987), while it is known to exist in normal rat plasma (Kangawa et al., 1984b; Misono et al., 1984). Increase in circulating y-ANP may be due to the fact that a-ANP and y-ANP have different half-lifes (T, /2) in the circulation. T 1 / 2 of a-ANP was calculated to be around 30 seconds (Katsube et al., 1986; Luft el al., 1986), while y-ANP, in contrast, is thought to have a much longer T 1 / 2 . Because a complete molecule of y-ANP has not been synthesized in vitro, the precise T 1 / 2 of y-ANP is difficult to determine. However, T 1 / 2 of other large fragments of ANP molecules derived from the y-ANP, such as ANPi 3 . 1 0 4 and N-terminal peptide (ANPj.gg), have been evaluated. ANPig.,^ lacks most of the biologically active C-terminal portion of the molecule (ANP 9 9. 1 2 6), but retains the majority of the remaining N-terminal region; its T , / 2 was estimated to be 9 times longer than that of a-ANP (Katsube et al., 1986). N-terminal peptide (ANP g^g) has the intact N terminal of the y-ANP molecule but lacks the active C terminal; its T 1 / 2 was 150 seconds, which was 5 times longer than that of a-ANP (Thibault et al., 1988). Since y-ANP (ANPi.126) has a molecular size larger than both A N P 1 3 . 1 0 4 or ANP,.^, it is appropriate to assume that its T 1 / 2 is at least the same, if not greater, than that of ANP 1 3 . , 0 4 or ANP,.^. - 72 -Thus, a diminished degradation due to its longer T 1 / 2 and/or increased y-ANP secretion, y-ANP's concentration in the plasma may quickly rise. It is possible that the significant increase in the y-ANP concentration in the present study is due to reduced renal clearance function as a result of age-related abnormalities in the structure and function of the kidney. Since ANP has been shown to be eliminated predominantly by the kidney (Tang et al., 1984; Berg et al., 1988; Sonnenberg et al., 1988), a decrease in the renal clearance would easily lead to an accumulation of y-ANP. Recently, a bound form of ANP have been detected both in men (Miyata et al., 1987; Richards et al., 1987) and in rats (Kato et al., 1988). Another explanation as to why plasma y-ANP may appear to accumulate in the 10- and 20-month-old rats In the present study is that a-ANP may exist as a bound form in the circulation. This bound form of ANP may resemble a large y-ANP molecule in the HPLC analysis, thus leading to the illusion of excess y-ANP concentration in the plasma. Also, it is conceivable that age may alter the ratio of free to bound form of plasma ANP in some unknown way, as it is reported in some pathological conditions (Kato et al., 1988). Other studies are required to elucidate the cause and ascribe a significance for the apparent accumulation of y-ANP in the circulation. 4.6 Effect of Age on Molecular Species of Secreted ANP Reverse phase-HPLC analyses of the perfusate coupled with radioimmunoassay demonstrated that the predominant form of ANP released was chromatographically identical to a-ANP in all four age - 73 -groups {Fig. 18). However, a minor peak that is chromatographically identical to y-ANP also appeared i n many perfusate analyses. The relative proportion of y-ANP to a -ANP was increased wi th age (Figs. 19, 20). In the 1-, 3-, and 10-month-old groups, y-ANP accounted for approximately 1% of the total A N P ; i n the 20-month-old group, close to 8% was y-ANP. Th is suggests a possible age-related defect i n the processing of y-ANP to the active a-ANP form prior to secretion. This finding is consistent wi th previous observations that the predominant molecular form of A N P found i n the perfusion med ium collected from isolated adult (Saito et a l . , 1986; Lang et al . , 1985; Curr ie et al . , 1984) or neonatal (Shields and Glembotski, 1987) rat hearts is a low molecular-weight material. The precursor protein represented only min imal amount, i f any, of total A N P concentration (Lang et al . , 1985; Curr ie et al . , 1984; Ruskoaho 86, Sugawara 1985 & 1986). Similar results were obtained i n studies on human, where the main molecular form of A N P i n the p lasma taken at the coronary sinus dur ing cardiac catheterization was a -ANP (Sugawara et a l . , 1985; Yandle et a l . , 1986b). Together wi th our results, these observations support the theory that proteolytic conversion of the prohormone to circulating A N P occurs mainly within cardiac myocytes, and that the low molecular-weight a-ANP found i n p lasma is released directly from the heart. There are evidences to support the speculation that the prohormone y-A N P is released into the circulat ion due to increased biosynthesis and /or defective conversion or storage. In renal juxtaglomerular apparatus, both active renin and inactive pro-renin have been shown to - 74 -be released (Hsueh et al., 1983), and their ratio in plasma is altered in certain physiological and pathological conditions (Delevia et al., 1976; Day and Luetscher, 1974). Similarly, both the precursor and the active circulating forms of insulin were detected in the plasma (Carroll et al., 1988; Cohen et al., 1988; Robbins et al., 1984). Therefore, the increasing concentration of y-ANP in the perfusate reflects a possible abnormality in the processing of the precursor hormone to the active a-ANP in the aging heart. 4.7 Effect of Age on Molecular Species of Atrial ANP Accumulated evidences have established that in both man and animal the predominant form of ANP in the heart is the high molecular-weight y-ANP (Nakao et al., 1984, 1987; Thibault et al., 1987; Kangawa et al., 1984b). In the present study, y-ANP is the predominant and the only form of ANP present in all four groups of atrial tissues (Figs. 23 and 24). Apparently, age has no effect on the molecular species of ANP in the atrial tissue. This is an intriguing observation; since data from the present isolated atrial perfusion study indicated that perfusing medium contains predominantly a-ANP, it is apparent that the post-translational cleavage to form a-ANP occur within cardiocytes and does not require protease from the circulation. With aging, the atrium secretes an increasing proportion of y-ANP, suggesting that this processing mechanism is somehow altered by age. This may imply the capacity of the putative processing enzymes is exceeded in the aged subjects. 4.8 Effect of Age on Glomerular ANP Receptor Binding Characteristics The actions of ANP, like those of other peptide hormones, are believed - 75 -to be mediated through binding to specific receptors on cell plasma membranes. It is commonly accepted that circulating peptide hormones may directly regulate the numbers of receptors at target cells, and changes in the ambient concentrations of many peptide hormones are commonly associated with reciprocal changes in the density of their specific receptors in target tissue (Lefkowitz and Michel, 1985). To explain the diminished GFR and reduced sodium excretion in the presence of chronic elevation in circulating ANP levels, the possibility of an abnormality at postglomerular level was investigated. Glomerular membranes were chosen because many investigators (Ballermann et al., 1985; Ogura et al., 1989; Garcia et al., 1989) have previously reported that high affinity binding sites of ANP existed in the rat kidney, especially in the renal cortex by radiolabeled receptor assay (Ogura et al., 1985) and autoradiography (Yamamoto et al., 1987). Furthermore, since this tissue contains high density of specific ANP receptors and it is easily accessible, the renal glomerular ANP receptor is ideal for studying alterations in receptor density and affinity that may be accompanied by age-related changes in circulating ANP levels. The specific binding of glomerular membrane to 1 2 5I-ANP was linear between 0.05 and 0.2 mg of tissue (Fig. 24), subsequently, a concentration of 0.1 mg of glomerular membrane per assay tube was used throughout the study. In addition, the ANP binding capacity was greatly influenced by incubation time (Fig. 23), as Napier et al. (1984) and Ogura et al. (1989) have previously reported. Since the maximum binding was achieved in 5 minutes and remained stable for 15 minutes. - 76 -the ANP radiolabelled receptor assay was conducted for 10 minutes at 20°C. When data from competitive binding inhibition studies (Fig. 25) were subjected to computerized Scatchard analysis, it was apparent that ANP interacted with one population of sites of a single affinity in glomeruli isolated from all four age groups of rats (Fig. 26). The receptor binding to ANP was saturable (Fig. 25) and with high affinity. Nonspecific binding was low, and contributed to less than 3% of the total count. The glomerular receptor density (Bmax) in the four age groups of rats was calculated to be ranged from 180 to 480 fmol/mg tissue protein (Fig. 27), values that are almost identical with those reported by Ballermann et al. (1985) (116 to 426 fmol/mg protein) and Gauquelin et al. (1988) (103 to 500 fmol/mg protein). The B , ^ in 1- and 3-month-old rats were calculated to be 182 and 446 fmol/mg protein, respectively, in excellent agreement with values reported by Garcia and his colleagues (1989) of 170 fmol/mg protein for 4-week-old and 503 fmol/mg protein for 12-week-old Wistar rats. Similarly, equilibrium dissociation constant (Kj) of the ANP binding sites in glomerular membrane ranged from 0.06 to 0.35 nM (Fig. 27), agree well with values reported by Ballermann et al. (1985) (0.02 - 0.55 nM), and many other groups (Gauquelin et al., 1988; Garcia et al., 1988; Ogura et al., 1989). Glomerular ANP receptor density was greater in the 3-month-old than in the 1-month-old (Fig. 27), which is probably due to the adaptive changes of the rat as a result of maturation. In agreement with this - 77 -observation is the recent study that documented a progressive increase in the density of the glomerular ANP binding sites with age, from 4- to 16-week-old rats (Garcia et al., 1989). The glomerular receptor affinity, on the other hand, is lower (higher k j in the 3-month-old than in the 1-month-old (Fig. 27). These observations suggest that even though the density of ANP receptors was lower in the 1-month-old, it is compensated by a more effective ANP binding (higher affinity, lower KJ. Consistent with this finding is the observation that extracts prepared from atria of adult rats (>2 months of age) contained significantly greater natriuretic activity than extracts from pups (19 days of age) (Pollock and Banks, 1985). This up-regulation of receptor density may be an indication of the still developing and "tuning-up" of many physiological processes in the young rats. With advancing age (from 3 to 20 months), the receptor density steadily declined. The oldest group of rats, 20-month-old, contained the least amount of receptor compared to the other 3 age groups (Fig. 27). This lower density of glomerular ANP receptors in the 20-month-old rats was accompanied by a higher binding affinity (lower k j . In the 10-month-old glomerulus, although the binding affinity was similar to the 3-month-old, the receptor density was significantly lower, thus reflecting regulation of receptors as a result of aging. With further reduction in the receptor density in the 20-month-old (to about 55% of the 3-month-old), the receptor affinity was dramatically increased, which indicates the body's attempt to compensate for the down-regulation in receptor density. - 78 -Down regulation of ANP receptors have been previous reported in various experimental, physiological and clinical conditions in which plasma ANP levels are elevated (Morton et al., 1987; Takayanagi et al., 1986; Roubert et al., 1987; Lynch et al. 1986; Ballermann et al. 1985; Hirata et al. 1985). For instance, Lynch et al. (1986) demonstrated that water deprivation, which depresses plasma ANP levels, augments binding receptor number, but salt loading, which increases plasma ANP level, decreases receptor density in the kidney of the rats. In spontaneous hypertensive rats (SHR), a marked (60%) down-regulation of ANP receptors in aortic smooth muscles and adrenal capsules was observed to correspond to an elevation in plasma ANP level (Takayanagi et al., 1986). In cultured rat aortic smooth muscle cells pretreated with ANP, a significantly lower binding capacity was observed when subsequently exposed to 1 2 5I-ANP (Roubert et al., 1987). This apparent decrease in efficiency or capacity of physiological processes is a common feature of aging. The changes in the number of receptors with age may be due to modulations in the rate of receptor synthesis and degradation, or alterations in receptor regulation (Scarpace and Abrass, 1988). Similarly, the down-regulation of the glomerular receptor in the aged rats may due to age-associated alterations in the structure, composition or function of the membrane (Naeim and Walford, 1985; Heron et al., 1980). On the other hand, the responsiveness of a variety of tissues to stimulation by hormones, neurotransmitters, antigens, and mitogens may decrease with age (Walford et al., 1981), and this may explains the blunted responses of the aged rats to a chronically elevated circulating ANP. In adrenergic - 79 -receptors (Scarpace and Abrass, 1988; Forn et al., 1970), corticosteroids and androgen receptors (Roth, 1976; Robinette and Mawhinney, 1977), there are many reports that document a reduction in biological responsiveness concurrent with a decline in receptor binding. Taken together, these observations support the notion that the number of ANP receptors changes reciprocally in response to changes in plasma ANP, and may provide an insight into the mechanism whereby binding sites of ANP in the kidney are reduced in old rats. There are several mechanisms that have been postulated to explain this receptor down-regulation phenomenon, but available evidence favours two hypotheses. On hypothesis postulated that the total number of receptors in the cell is normal, but hormone-mediated internalization has removed a sufficient fraction of receptor such that fewer receptors are on the surface of the cell available for binding (Krupp and Lane, 1981). The other suggested that the total number of receptors in the cell (as well as the number of receptors on the cell surface) is reduced as a result of increased degradation of the internalized receptors (Jacobs and Cuatrecasas, 1983). Which mechanism is responsible for down-regulation in the glomerular receptor of 10- and 20-month-old rats in the present study cannot be deduced from the data, and the exact mechanism remains to be elucidated. Recently, two subtypes of ANP receptors have been found: a B- or R,-receptor subtype, in which the receptor is coupled to guanylate cyclase, and is related to physiological effects in certain tissues (Takayanagi et al., 1987; Leitman and Murad, 1986); and a C- or Ra-receptor subtype, - 80 -which recognizes biological active ANP fragments as well as inactive truncated forms such as A N P 1 0 2 . 1 2 1 (so called C-ANP) (Maack et al., 1987; Leitman and Murad, 1986). Although the latter receptor type represented more than 90% of the total number of ANP binding sites, the receptor does not generate biological responses and seems to serve merely as a "clearance receptor" that helps to regulate ANP concentration in the circulation (Maack et al., 1987). Thus, modifications in the proportion of these receptors in target tissue could lead to profound physiological and pathophysiological effects. The present data of ANP receptor down-regulation caused by age-related elevation in plasma ANP levels do not allow one to differentiate between the two populations of receptors, since a-ANP binds to both subtypes of ANP receptors. However, Maack et al. (1987) have postulated that ANP receptor regulation largely represents the regulation of the ANP clearance receptor (C-receptor). This speculation is further supported by results obtained from studies done by Hirata et al. (1987), Kihara et al., (1985) and Ben-Ishay et al. (1973). This being the case, then, a down-regulation of C-receptor density may, in part, explain the experimental data that elderly subjects are more likely to develop exaggerated natriuresis and diuresis compared to younger subjects following a water or saline load (Lindeman et al., 1970; Schalekamp et al., 1971), infusion of a small dose of a-ANP (Heim et al., 1989), or subjected to head-out water immersion (Tajima et al., 1988; Epstein et al., 1987). Then, a reduction in the number of C-receptors would allow the increase in endogenous ANP to interact with B-receptors, thereby producing exaggerated responses. - 81 -Therefore, If down-regulation of the ANP receptor observed in the present study represents a decrease in the number of clearance receptors, then this may partially explain the age-related accumulation of ANP in the plasma of the 20-month-old rats because less ANP has been removed from the circulation. The fact that chronically elevated circulating ANP levels in the 20-month-old rats correspond with diminished glomerular filtration rate and reduced natriuresis in the kidney suggests the existence of age-related alterations in receptors. Decreases in receptor regulation, either in synthesis or degradation, changes in structure composition and function of the membrane, or decreases in responsiveness to stimulation by hormones may play an important part in the hormone-receptor interaction and in the generation of functional responses. - 82 -5.0 SUMMARY In summary, data obtained from this study suggests that age may affect the processing, secretion, circulating levels, and receptor binding of ANP on 4 levels (Fig. 28). The first level of modification may occur at the site of intracellular storage. A depletion of ANP concentration in the storage granules within the aging atria may occur as a result of reduced synthesis. The major molecular form of ANP in the atrium was y-ANP and remained unchanged with age. The second level of alteration may reside in the secretion of ANP. A relationship was established between the rate of ANP release and the atrial tissue level, suggesting that reduced ANP secretion is a result of lower atrial content. Although the molecular forms of ANP in the atrium were not altered by age, ANP released in the perfusate showed an increasing amount of y-ANP with the progression of age. This was taken to indicate an impaired post-translational processing of y-ANP to active a-ANP, which seems to take place prior to secretion. A third site of modulation may take place at the circulating levels of ANP. The plasma ANP levels correlated positively with age. An increased plasma ANP concentration is not a consequence of inappropriate secretion, but instead may be caused by an age-related reduction in the renal clearance of ANP. The elevation in total ANP concentration may, in part, be due to the rise in y-ANP concentration. Since y-ANP appears to have a greater half-life in the circulation, this - 83 -ANP Accumulation i ANP Degradation Secretion Receptor I ANP Content i ANP Release t y-ANP/a-ANP Ratio t Total ANP Level T y-ANP/a-ANP Ratio IB max Compensation IK l Clearance C-ANP B-ANP Receptor Receptor No Change Fig. 28 Summary of possible age-related alterations in the homeostatic balance of ANP in aged rats - 84 -may partially contribute to the accumulation of plasma ANP. A fourth site of age-associated alteration may occur at the level of receptor binding. An elevation of plasma ANP in the aging rats caused a down-regulation of the glomerular receptor density, and the observed increase in binding affinity of the receptors may represents its own compensatory response. 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