Time Course of Enhanced Adrenal Responsiveness to Angiotensin on a Low Salt Diet. Suzanne Rogacz, Gordon H. Williams, and Norman K.

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1 376 Time Course of Enhanced Adrenal Responsiveness to Angiotensin on a Low Salt Diet Suzanne Rogacz, Gordon H. Williams, and Norman K. Hollenberg To assess the rate of activation of the renin-angiotensin-aldosterone axis and enhancement of adrenal responsiveness to angiotensin II (Ang II) with restriction of sodium intake, 16 healthy male subjects were placed initially on a 2 meq daily sodium intake; adrenal responsiveness to Ang II was assessed, and then daily sodium intake was reduced abruptly to 1 meq. Adrenal responses to Ang II were assessed again during the non-steady state interval 24 and 48 hours later, and after balance was achieved in 5-7 days. Renin-angiotensin system activation was evident within 24 hours after sodium intake was restricted. The increase in basal plasma aldosterone concentration and enhancement of the adrenal response to Ang II, on the other hand, tended to lag. Within 24 hours of restricting sodium intake, despite a significant increase in both plasma renin activity (1.±.2 vs. 2.4±.7 ng/ml/hr,p<.1) and Ang II concentration (22.±1.9 vs. 29.5±1.3 pg/ml, p<.5), there was no increase in basal plasma aldosterone concentration (1.4±1.3 vs. 11.7±1.2 ng/dl). At 48 hours, despite little further change in plasma renin activity or plasma Ang II concentration, there was a sharp increase in basal plasma aldosterone concentration (22.5±3.6 ng/dl, /><.1). The adrenal response to Ang II was increased significantly at 24 hours, evident at only a 1 ng/kg/min dose, but showed progressive further enhancement with time. Early enhancement was not related to shifts in potassium balance as none occurred, but later progressive enhancement could reflect in part negative potassium balance, as a small but consistent element of negative balance occurred. These observations add further support to the concept that some unidentified factor other than plasma Ang II concentration or potassium balance, but related to sodium balance, modifies the adrenal response to Ang II. Available evidence suggests that enhancement reflects events at the terminal step of aldosterone biosynthesis. (Hypertension 199;15:376-38) Shifts in sodium intake are accompanied by reciprocal shifts in vascular and adrenal responsiveness to angiotensin II (Ang II) in normal humans 12 and in animals. 3-4 On a high sodium intake, the adrenal response is suppressed, whereas the vascular response is enhanced. Conversely, on a low sodium intake the adrenal response is enhanced and vascular responses blunted. There is From the Departments of Medicine and Radiology, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts. Supported by the National Institutes of Health (grants HL , HL-2995, 2T32HL-7236, HL-3316, and HL-361) and a SCOR in Hypertension (HL-33697). The studies were carried out in a Clinical Research Center supported by a grant (RR 2635) from the Division of Research Resources, and the statistical analyses were performed in a CLINFO facility supported by the same grant. Address for correspondence: Norman K. Hollenberg, MD, PhD, Brigham and Women's Hospital, 75 Francis Street, Boston, MA Received June 12,1989, accepted in revised form December 15, substantially greater unanimity on the responsible mechanism for shifts in vascular smooth muscle responsiveness than for adrenal responsiveness. In the case of vascular smooth muscle, all of the available evidence from the more direct studies possible in animals, 3 ' 5 " 7 and less direct studies in humans, 8-1 suggest a mechanism involving the number or availability of angiotensin receptors. For the adrenal, some studies have supported a receptor mechanism, 3-7 and others have not. 4 In the normal human, converting enzyme inhibition modified vascular responsiveness to Ang II 8-1 but not that of the adrenal responsiveness This observation suggests a different responsible mechanism in vascular smooth muscle than in the adrenal in humans. All of the reported studies on adrenal responsiveness have been performed under steady-state conditions of sodium balance. In the present study, we assessed the adrenal response to Ang II during the first several days of a shift from a high to a low sodium intake. If factors in the transduction process are responsible for the

2 Rogacz et al Aldosterone and Sodium Balance 377 TABLE 1. Measurements Urine Na (meq/24 hr) Urine K (meq/24 hr) Aldosterone (ng/dl) PRA (ng/ml/hr) Plasma Ang II (pg/ml) Serum Na (meq/1) Serum K (meq/1) Baseline Values During Non-Steady State Study High salt diet in balance (H = 18) 217±11 83±5 1.4 ±1.3 1.±.2 22.± after 24 hours (n=1) ± ±.7* 29.5±1.3t 139±.4 after 48 hours (n=6) 59±7 85±4 22.5±3.6* 2.4±.7* 3.8±2.5t 139±.8 Values shown are mean±sem. PRA, plasma renin activity; Ang II, angiotensin II. *p<.1; 1p<.5 significantly different vs. high salt balance. in balance (n-16) * 92±4 37.4±6.6* 4.3±.7* 38.8±3.4* 137.6±.5 enhanced adrenal response to Ang II with reduced sodium intake, it would be anticipated that time would be required to increase adrenal sensitivity to Ang II, as suggested by studies in animals. 12 The present study demonstrates that is indeed the case. Methods Subjects and Protocol Sixteen normal male subjects ranging in age from 19 to 44 years and in weight from 68 to 9 kg were admitted to the Clinical Research Center of the Brigham and Women's Hospital. During their entire stay, subjects were maintained on isocaloric-constant diets containing 1 meq potassium. The subjects were first brought into balance on a 2 meq sodium intake, and their adrenal responsiveness was assessed. Sodium intake was decreased abruptly to 1 meq daily; adrenal responsiveness was assessed again, either at 24 or 48 hours after the dietary shift, and repeated when 1 meq sodium balance was attained in 5-7 days. Daily 24-hour urine collections were made for measurement of volume, sodium, potassium, and creatinine content. Because of known fecal losses of potassium, potassium excretion on the day before the shift from a high sodium to a low sodium intake was used for calculation of net cumulative potassium balance (Table 3). Angiotensin II Infusion Each infusion of Ang II amide (Hypertensin, CIBA-GEIGY Corp., Summit, New Jersey) was begun between 8:3 and 9: AM with an electronic infusion pump (Harvard Apparatus Co., Millis, Massachusetts) at doses of 3 and 1 ng/kg/min for 45 minutes at each dose. All subjects remained supine. Blood samples were obtained for measurement of potassium, sodium, aldosterone, cortisol, and Ang II concentration at the start of the infusion and at the completion of each dose of Ang II. Laboratory Procedures Blood samples were collected on ice, immediately cold centrifuged, and the plasma separated and frozen until the time of assay. Sodium and potassium were measured by means of an ion-selective electrode system (Nova I, Nova Biomed, Waltham, Massachusetts). Serum creatinine was measured by an autoanalyzer technique (Beckman Creatinine Analyzer II, Beckman Instrs., Inc., Fullerton, California). Ang II, plasma renin activity, aldosterone, and cortisol were analyzed by radioimmunoassay Group means are presented with the standard error of the mean as the index of dispersion. Statistical probability was assessed with linear regression and analysis of variance. The null hypothesis was rejected when p achieved a level of.5 or less. The protocol was approved by the Committee for the Protection of Human Subjects from research risks at the Brigham and Women's Hospital and written consent was obtained from each subject. Results During the first 24 hours of restricted sodium intake, sodium excretion fell from 217±11 to 15±7 meq/24 hr, so that during that day excretion exceeded intake by 95 meq (Table 1). The 95 meq negative sodium balance was associated with a sharp increase in plasma renin activity (1.±.2 to 2.4±.7 ng/ml/hr, p<.1) and plasma Ang II concentration (22.±1.9 to 29.5±1.3 pg/ml, p<.1). Basal plasma aldosterone concentration, however, did not increase (1.4±1.3 to 11.7±1.2 ng/dl). During the next 24 hours, sodium excretion fell to 59 ±7 meq/24 hours, exceeding intake by 49 meq. During that interval, neither plasma renin activity (2.4±.7 ng/ml/hr) nor plasma Ang II concentration (3.8±2.5 pg/ml) increased further, but plasma aldosterone concentration rose sharply (22.5±3.6 ng/dl, p<.1). When low salt balance had been achieved in 5-7 days,

3 (IP/I 378 Hypertension Vol 15, No 4, April 199 TABLE 2. Diet Aldosterone Response to Intravenous Angiotensin II Angiotensin II dose (ng/kg/min) 3 1 High salt Low salt: 24 hours Low salt: 48 hours Low salt: hours 1.4± ± ± ± ± ± ± ±7.1 Values are plasma aldosterone concentration (ng/dl)+sem. /><.5;p<.1 significantly different from high salt diet. plasma renin activity, plasma Ang II concentration, and plasma aldosterone all showed a further rise (Table 1). Ang II infusion brought out the anticipated difference in response between the subjects when in balance on a high salt intake and on a low salt intake (Table 2). Plasma aldosterone rose from 1.4 ±1.3 to 27.2±2.9 ng/dl with a 1 ng/kg/min Ang II infusion on a high sodium intake and from 35.7±5.5 to 72.8±7.1 ng/dl at that angiotensin dose on a low salt intake. The 3 ng/kg/min Ang II dose induced an intermediate response. Although basal plasma aldosterone had not increased 24 hours after instituting a restricted sodium intake, the Ang II infusion brought out evidence of sensitization. Not only was the increment in plasma aldosterone concentration larger at the 1 ng/kg/min Ang II infusion rate (Table 2), but the slope relating plasma Ang II concentration to plasma aldosterone concentration was increased significantly (/?<.1). By 48 hours, the basal plasma aldosterone concentration had increased, and the responses to Ang 11 infusion increased further (Table 2). The anticipated differences in the relation between plasma Ang II concentration and plasma aldosterone concentration in the steady-state low salt and high salt balance studied were found (Figure 1). Regression analysis revealed a significant difference in both the intercept ( vs , p<.1) and in the slope (.67±.26 vs ,/><.1). All of the points during the assessment on the low salt diet lay above the line relating plasma Ang II and aldo *>^ \ / Low Salt Balance +/ A A High Salt Balance 1OO 2 3 PLASMA AH (pg/ml) FIGURE 1. Scatterplot showing relation of plasma aldosterone to plasma angiotensin II (All) concentration at steady state on a very low (1 meq) and high (2 meq) NaCl intake. The two regression relations differ in both slope and intercept. c g H g < Low Salt Balance High Salt Balance PLASMA AH (pg/ml) FIGURE 2. Scatterplot showing relation between plasma angiotensin II (All) aldosterone concentration 24 hours after initiating a restricted NaCl intake. Regression relations defined in Figure 1 are shown as well. At 24 hours, slope is increased significantly in comparison with a high salt diet (p<.25), but intercept was unchanged. sterone concentration on a high salt diet. At 24 hours, the intercept of the line relating plasma Ang II and aldosterone concentration (16.7±3.46) did not differ from that on a high salt diet, but the slope had increased significantly (.11±.2,/?<.25) (Figure 2). At the lower end of the plasma Ang II concentration range (below 75 pg/ml) the values were randomly distributed around the regression line for high salt balance. Above that plasma Ang II concentration, 14 of 15 points were above the line (p<.1). At 48 hours, intermediate values were found. Changes in potassium balance during the first 48 hours were modest and tended to be slightly positive (Table 3). Thereafter, net potassium balance became progressively more negative (Table 3). Although the changes were small, potassium excretion increased in 15 of the 16 subjects during the third to the seventh day after institution of a restricted sodium intake. Discussion In the first 24 hours after a shift to a very low sodium intake, with a reduction in total body sodium of over 1 meq, there was a sharp increase in plasma renin activity and plasma Ang II concentration but no increase in plasma aldosterone concentration. TABLE 3. Day Sodium Potassium Excretion and Balance Excretion (meq/24 hr)* Na K ±7 2 59±7 3 3±3 4 18±2 5 12± ±4 Values are mean±sem. 83±5 74± ±3 9±3 92±4 94±4 93±7 Cumulative balance (meq) Na K

4 Ang II infusion at that time, however, brought out a subtle but clear enhancement of the adrenal response to Ang II that was evident in an increase in the values of plasma aldosterone concentration with increasing doses of administered Ang II (Table 2) and in an increase in the slope relating plasma Ang II to plasma aldosterone concentration at that time (Figure 2). With a continued but more limited reduction in total body sodium over several days, basal plasma aldosterone concentration rose, and there was a further increase in the slope relating plasma Ang II and plasma aldosterone concentration. The shift in the slope relating angiotensin and aldosterone concentration at extremes of sodium intake confirms earlier reports and provides the most compelling evidence favoring sensitization of the adrenal response as the analysis was performed over a wide and overlapping range of plasma Ang II concentration. This is important as analyses of the Ang II dose-adrenal response relation potentially could have been altered by the rise in basal plasma aldosterone concentration with time. The factors responsible for increasing adrenal sensitivity remain obscure, reflecting the complexity of the regulatory processes involved in aldosterone biosynthesis. All factors that acutely modify aldosterone secretion increase the rate of conversion of cholesterol to pregnenolone (early pathway) and may also modify the late pathway (the conversion of corticosterone to aldosterone) In both human and rat tissue, sodium restriction does not modify early pathway activity but does substantially increase the rate of activity of the late pathway. 16 " 2 Thus, this change alone could account for the enhanced adrenal response to Ang II with sodium restriction. Whether the adrenal Ang II receptor also participates in this change in sensitivity remains uncertain. In the rat, the number of binding sites for Ang II increases with sodium restriction However, in the primate sodium restriction reduces the number of adrenal Ang II receptors. 7 Furthermore, functional assessment of the Ang II receptors in sodium-restricted rats suggests that they are reduced. 3 Finally, when humans or rats are sodium restricted and given converting enzyme inhibitors sufficient to substantially reduce blood pressure, there is no change in the adrenal responsiveness to exogenously administered Ang II We also have considered the possibility that the state of potassium balance played a role, and compatible data are available from other studies, but the results of this study make this less likely. There was essentially no change in potassium excretion or in potassium balance during the first 48 hours (Tables 1 and 3). Indeed, overall cumulative potassium balance tended to be somewhat positive during the early 48 hours when clear enhancement of the adrenal response occurred. Later, the small but consistent negative cumulative potassium balance that followed could have contributed to the progressive enhancement of the adrenal response that occurred when the shift in sodium Rogacz et al Aldosterone and Sodium Balance 379 balance had largely occurred. On the other hand, the negative potassium balance is as likely to reflect the influence of aldosterone on renal potassium handling as the cause of shifts in adrenal responsiveness. The often capricious relation between sodium intake and the state of potassium balance, seen in the context of the precise relation between sodium balance and adrenal responsiveness, makes potassium an unlikely candidate. Although changes in the level of other hormonal factors sensitive to sodium intake, such as dopamine and atrial natriuretic factor, both of which inhibit aldosterone secretion, could mediate the change in sensitivity, decisive evidence in favor of either of these as the determinants of adrenal sensitivity is not yet available This study confirms a remarkable sensitivity of the adrenal response to Ang II to the state of sodium homeostasis. Small shifts in sodium balance are especially important at the low end of the sodium intake scale, reflecting a relation with the logarithm of urine sodium. 25 A delay in the enhancement of the adrenal response to Ang II has also been suggested in the rat. 12 As a reflection of the extremely rapid rate at which the rat achieves external sodium balance, plasma renin activity and Ang II concentration are increased within 8 hours of restricting sodium intake, but plasma aldosterone concentration rises more gradually over a 48-hour period. Subsequent in vitro studies confirm the delay in sensitization and are compatible with a gradual, progressive enhancement of adrenal biosynthetic capacity for aldosterone. 28 Whatever the responsible mechanism for the shift in adrenal sensitivity, the results of this study indicate that there is a lag in the increased sensitivity of the adrenal responsiveness to Ang II with restriction of sodium intake, as opposed to the rapid adaptation of the renin-angiotensin system to the state of sodium balance, consistent with a crucial role for biosynthetic elements in the late pathway of aldosterone synthesis. Acknowledgments We acknowledge the expert assistance of Diana Capone, Joan Munroe, Diane Passan, and the nursing and dietary staff of the Clinical Research Center in the performance and preparation of this study. References 1. Hollenberg NK, Chenitz WR, Adams F, Williams GH: Reciprocal influence of salt intake on adrenal glomerulosa and renal vascular responses to angiotensin II in normal man. / Clin Invest 1974;54: Oelkers W, Brown JJ, Fraser R, Lever AF, Morton JJ, Robertson JIS: Sensitization of the adrenal cortex to angiotensin II in sodium-depleted man. Ore Res 1974;34: Williams GH, Hollenberg NK, Braley LM: Influence of sodium intake on vascular and adrenal angiotensin II receptors. Endocrinology 1976;98: Aguilera G, Hauger RL, Catt KJ: Regulation of aldosterone secretion by the renin-angiotensin system during sodium restriction in rats. Proc NatlAcad Sci USA 1978;74:

5 38 Hypertension Vol 15, No 4, April Devynck MA, Pemollet GG, McDonald DJ, Matthews PG, Raisman RS, Meyer P: Alterations of adrenal and uterine angiotensin II receptors during variations of sodium intake and/or experimental hypertension. Clin Sci Mol Med 1978; 55: Gunther S, Gimbrone Jr MA, Alexander RW: Regulation by angiotensin II of its receptors in resistance blood vessels. Nature 198;287:23 7. Platia MD, Catt KJ, Hodges GD, Aguilera G: Regulation of primate angiotensin II receptors during altered sodium intake. Hypertension 1986;8: Dawson-Hughes BF, Moore TJ, Dluhy RG, Hollenberg NK, Williams GH: Plasma angiotensin II concentration regulates vascular but not adrenal responsiveness to restriction of sodium intake in normal man. Clin Sci 1981;61: Shoback DM, Williams GH, Hollenberg NK, Davies RO, Moore TJ, Dluhy RG: Endogenous angiotensin II as a determinant of sodium-modulated changes in tissue responsiveness to angiotensin II in normal man. / Clin Endocrinol Metab 1983;57: Redgrave J, Rabinowe S, Hollenberg NK, Williams GH: Correction of abnormal renal blood flow response to angiotensin II by converting-enzyme inhibition in essential hypertensives. J Clin Invest 1985;75: Rogacz S, Hollenberg NK, Williams GH: Role of angiotensin II in the hormonal, renal, and electrolyte response to sodium restriction. Hypertension 1987;9: Holtzman EJ, Braley LM, Menachery A, Williams GH, Hollenberg NK: Rate of renin-angiotensin-aldosterone axis activation and sodium intake in the rat. Am J Physiol 1989; 256:H Emanuel RL, Cain JP, Williams GH: Double antibody radioimmunoassay of renin activity and angiotensin II in human peripheral plasma. / Lab Clin Med 1973;81: Underwood RH, Williams GH: The simultaneous measurement of aldosterone, cortisol, and corticosterone in human peripheral plasma by displacement analysis. / Lab Clin Med 1972;79: Williams GH, McDonald LM, Tait SAS, Tait JF: The effect of medium composition and in vitro stimuli on the conversion of corticosterone and aldosterone in rat glomerulosa tissue. Endocrinology 1972;91: Mueller J: Regulation of Aldosterone Biosynthesis. New York, Springer-Verlag, New York, Inc, 1971, p Haning R, Tait SAS, Tait JF: In vitro effects of ACTH, angiotensin, serotonin, and potassium on steroid output and conversion of corticosterone to aldosterone by isolated adrenal cells. Endocrinology 197;87: Davis WW, Burwell LR, Bartter PC: Inhibition of the effects of angiotensin II on adrenal steroid production of dietary sodium. Proc NatlAcad Sci USA 1969;63: Marusic ET, Mulrow PJ: Simulation of aldosterone biosynthesis in adrenal mitochondria by sodium depletion. / Clin Invest 1967;46: Muller J, Huber R: Effects of sodium deficiency, potassium deficiency and uremia upon steroidogenic response of rat adrenal tissue to serotonin, potassium ions and adrenocorticotrophin. Endocrinology 1969;85: Douglas J, Catt KJ: Regulation of angiotensin II receptors in the rat adrenal cortex by dietary electrolytes. / Clin Invest 1976;58: Bradshaw B, Moore TJ: Abnormal regulation of adrenal angiotensin II receptors in spontaneously hypertensive rats. Hypertension 1988;ll: Baumber JS, Davis JO, Johnson JA, Witty RT: Increased adrenocortical potassium in association with increased biosynthesis of aldosterone. Am J Physiol 1971;22: Hollenberg NK, Williams GH, Burger B, Hooshmand I: The influence of potassium on the renal vasculature and the adrenal gland, and their responsiveness to angiotensin II in normal man. Clin Sci Mol Med 1975;49: Adler GK, Moore TJ, Hollenberg NK, Williams GH: Changes in adrenal responsiveness to potassium balance with shifts in sodium intake. Endocr Res 1987;13: Van RC, Freeman RH, Davis JO, Villarreal D, Verburg KM: Effect of synthetic atrial natriuretic factor on aldosterone secretion in the rat. Am J Physiol 1986;251:R48-R Gordon MB, Moore TJ, Dluhy RG, Williams GH: Dopaminergic modulation of aldosterone responsiveness to angiotensin II with changes in sodium intake. / Clin Endocrinol Metab 1983;56: Menachery A, Braley LM, Williams GH: The association between renin activity and aldosterone response to sodium restriction. Abstracts of the 1989 Endocrine Society Meeting, CL Washington KEY WORDS aldosterone adrenal glands potassium angiotensin II sodium