INTRODUCTION

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THE PRESENCE OF A COMPLETE renin-angiotensin system (RAS) in the adrenal gland, probably involved in the local paracrine regulation of aldosterone secretion, is well known (for review, see Refs. 12, 13, 19). However, the direct in vivo demonstration that adrenal RAS plays a physiological role in the control of zona glomerulosa secretory activity is not yet available.

Therefore, we decided to address this issue by pharmacologically manipulating adrenal RAS in in situ-perfused left rat adrenals. In fact, this experimental model allows for the delivery of the chemicals directly to the adrenal gland without any possible interference with the systemic mechanisms regulating mineralocorticoid secretion.

	MATERIALS AND METHODS

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Reagents. The nonselective angiotensin II (ANG II) antagonist [Sar¹,Val⁵,Ala⁸]-ANG II (saralasin) was obtained from Peninsula Laboratories (St. Helens, UK), the angiotensin-converting enzyme (ACE) inhibitor captopril (Capoten) was obtained from Squibb (Milan, Italy), and the AT₁ receptor antagonist losartan (DuP753) was obtained from Merck Sharp & Dohme (Rome, Italy). Human serum albumin (HSA) was from Sigma Chemical (St. Louis, MO), and Medium 199 was from DIFCO (Detroit, MI). The sodium-deprived diet (<0.01 meq Na⁺/g) was purchased from Dr. Piccioni Laboratory (Milan, Italy), and RIA kits for aldosterone and corticosterone were from IRE Sorin (Vercelli, Italy) and Eurogenetix (Milan, Italy), respectively.

Animal treatment. Adult male Wistar rats (260 ± 30 g body wt) were purchased from Charles River (Como, Italy). A group of rats was sodium restricted (sodium-deprived diet and demineralized water as drinking fluid) for 14 days, and another group was maintained on a standard diet and tap water.

In situ adrenal perfusion. Sodium-deprived and normally fed rats were anesthetized with pentobarbital sodium, and the left adrenal gland was perfused in situ, as previously detailed (11). Perfusion medium was introduced via a cannula inserted in the celiac artery into an isolated segment of aorta from which the adrenal arteries arise. After flowing through the adrenal gland, medium was collected by a cannula inserted in the renal vein. Perfusion medium (tissue culture Medium 199, modified by dilution with KCl-free Krebs-Ringer bicarbonate to give a final K⁺ concentration of 3.9 mM and containing 0.2% glucose and 5 mg/ml HSA) was gassed with 95% air-5% CO₂, maintained at 37°C, and delivered by peristaltic pump at a constant rate of 2 ml/10 min for 90 min. Perfusion pressure was monitored by a pressure transducer inserted in the arterial cannula and was found to average 30 ± 3 mmHg. After an initial equilibration period of 30 min, three 10-min samples were collected, and then the perfusion medium was substituted with one in which the chemicals to be tested were dissolved to the required concentration, and three more 10-min samples were collected. Two experiments were performed. In the first experiment, captopril, saralasin, or losartan was added to the perfusion medium in concentration ranging from 10⁹ to 10⁴ M. In the second experiment, perfusion medium contained 10⁶ M captopril, 10⁶ M saralasin, or 10⁷ M losartan, and 10 mM K⁺ was added after the first three sample collections. This concentration of K⁺ is the maximally effective one in eliciting aldosterone secretion (6).

Hormone assays. Aldosterone and corticosterone were extracted from perfusion media and purified by HPLC (15). Their concentrations were measured by RIA with the following commercial kits: ALDOCTK2 (sensitivity, 5 pg/ml; intra- and interassay variations, 5.8 and 7.5%, respectively), and CTRX-RIA (sensitivity, 50 pg/ml; intra- and interassay variations, 6.6 and 8.2, respectively).

Statistics. For each rat, the rate of hormone output was calculated as the average of the three 10-min collection periods before (control value) and after addition of the chemicals to the perfusion medium (experimental value). In the first experiment, for each experimental point five rats were perfused, control and experimental values were averaged, and their statistical significance was assessed. The data were graphically expressed as the means ± SE of the percent change from the group control value. Baseline (control) values from sodium-restricted and normally fed rats were averaged and expressed as picomoles per 10 minutes. In the second experiment, for each experimental point six rats were perfused, and data were expressed as the means ± SE of the average secretion rate (pmol/10 min) before and after 10 mM K⁺ addition. The statistical comparison of the results was done by ANOVA, followed by Duncan's multiple range test. A value of P < 0.05 was considered significant.

	RESULTS

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Pilot experiments showed that, during the 60 min of sample collection, the basal rate of hormone production remained satisfactorily constant in both normally fed and sodium-restricted rats (Fig. 1). Sodium restriction raised aldosterone output by perfused rat adrenal (~80%), without affecting corticosterone release (Fig. 2).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Representative experiment showing that after initial period of 30 min, aldosterone secretory rate (left panel) becomes satisfactorily constant over the next 60 min of perfusion in both normally fed (bottom line) and sodium-restricted rats (top line). After 90 min of perfusion, rate of secretion starts to decline, probably because of functional alteration of the gland. The same behavior is shown by corticosterone secretion rate in both normally fed (right panel) and sodium-restricted rats (not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of sodium restriction on basal aldosterone (left panel) and corticosterone (right panel) output by in situ perfused rat adrenals. Bars are means ± SE; n = 72. * P < 0.01 from controls.

Captopril, saralasin, or losartan did not evoke significant changes in aldosterone production by perfused adrenal of normally fed rats (Fig. 3). In contrast, they markedly lowered aldosterone output in sodium-restricted animals in a concentration-dependent manner with a maximally effective concentration ranging from 10⁷ to 10⁶ M (Fig. 3). Corticosterone production was not affected in either group of rats (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of captopril (A), saralasin (B), and losartan (C) on basal aldosterone output by in situ perfused adrenals of normally fed and sodium-restricted rats. Values are percentage changes (means ± SE; n = 5) from respective baseline value (B). + P < 0.05 and * P < 0.01 from baseline.

K⁺ (10 mM) increased aldosterone production in both normally fed and sodium-restricted rats (three- and fivefold rise, respectively, Fig. 4). The presence of captopril (10⁶ M), saralasin (10⁶ M), or losartan (10⁷ M) in the perfusion medium did not significantly affect either the basal rate of aldosterone production or aldosterone response to 10 mM K⁺ in normally fed animals (Fig. 4A). Conversely, in sodium-restricted rats, they lowered the basal rate of aldosterone secretion (by 48-62%) and aldosterone response to 10 mM K⁺ (from a five- to about a threefold rise, Fig. 4B).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of captopril (106 M), saralasin (106 M), and losartan (107 M) on 10 mM K+-stimulated aldosterone output by in situ perfused adrenals of normally fed (A) and sodium-restricted (B) rats. Aldosterone responses to 10 mM K+ in absence of renin-angiotensin system inhibitors are also shown. Bars are means ± SE; n = 6. * P < 0.01 from respective control value; A P < 0.01 from the baseline control value.

	DISCUSSION

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Many lines of in vitro evidence (obtained by the use of capsular zona glomerulosa strips, adrenal slices, or zona glomerulosa cell cultures) suggest that adrenal RAS controls aldosterone secretion. ACE inhibitors were found to lower either basal (20) or K⁺- and ACTH-stimulated aldosterone output by rat zona glomerulosa (2, 14, 17, 18, 21) and basal aldosterone yield by cultured bovine (9) and human adrenal tissue (5), as well as by K⁺-stimulated aldosterone release by human adrenocortical NCI-H295 cell line (8). A selective AT₁ receptor antagonist was reported to block both basal and agonist-stimulated aldosterone secretion from cultured bovine zona glomerulosa cells (7) and K⁺-enhanced aldosterone production from the NCI-H295 cell line (8).

Our study, although partly confirming these observations, casts serious doubts on the possibility that adrenal RAS in vivo plays a major role in the regulation of aldosterone secretion under basal conditions. In fact, both ACE inhibition by captopril and ANG II receptor blockade by saralasin or losartan were ineffective on basal or K⁺-stimulated aldosterone secretion from in situ perfused adrenals in rats kept on a normal diet. This discrepancy stresses that marked differences occur in the adrenal cortex physiology between in vivo and in vitro conditions. In fact, when the structural integrity of the entire adrenal gland is preserved, several complex paracrine interactions between cortex and medulla are operative (for review, see Ref. 16), which conceivably may obscure under basal conditions the stimulatory effect of adrenal RAS on the zona glomerulosa secretory activity.

Conversely, our investigation strongly suggests that adrenal RAS may be involved in enhancing aldosterone secretion under pathophysiological conditions leading to prolonged stimulation of zona glomerulosa, such as those elicited by sodium intake restriction. Sodium restriction was found to increase adrenal renin mRNA and protein (1, 3). Hence, our observations could suggest that, only when overexpressed, adrenal RAS plays a role in enhancing aldosterone secretion. This contention appears to be in keeping with the fact that the transgenic rat strain TGR(mREN2)27, which overexpresses the murine Ren-2^d gene in adrenal glands, secretes elevated amounts of aldosterone (for review, see Refs. 4, 10). Further studies are underway to see whether captopril and the ANG II receptor antagonists alter aldosterone output by in situ perfused adrenals of TGR(mREN2)27 rats kept on a normal diet, as well as those of animals with prolonged K⁺ intake.