Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [¹²⁵I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K⁺-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca²⁺-channel activator BAYK-8644, and blocked K⁺- and BAYK-8644-evoked rise in the intracellular Ca²⁺ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca²⁺ channel-mediated Ca²⁺ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.     

Effects of prolonged treatment with adrenocorticotropin on the morphology and function of rat adrenocortical autotransplants.

Regenerated adrenocortical nodules were obtained by implanting in the musculus gracilis of rats fragments of the capsular tissue of their excised adrenal glands. Five months after operation, transplanted rats showed a slightly elevated blood concentration of adrenocorticotropin (ACTH), a moderately reduced plasma level of corticosterone (PBC) and a very low concentration of circulating aldosterone (PAC). Regenerated nodules were well encapsulated, and from the connective capsule some septa dipped into the parenchyma. Subcapsular-outer (OZ) and inner (IZ) cells were similar to those of the zona fasciculata/zona reticularis (ZF/ZR) of the normal gland; juxta-septal (JZ) cells resembled those of the zona glomerulosa (ZG). Prolonged (14 days) ACTH infusion normalized PBC and caused a conspicuous hypertrophy of transplanted tissue, which was coupled with a marked hypertrophy of ZF/ZR-like OZ and IZ cells and a notable rise in the basal in vitro production of corticosterone. Conversely, ACTH infusion strikingly lowered PAC, reduced the number of ZG-like JZ cells, and decreased both basal and stimulated secretion of 18-hydroxylated steroids by transplants in vitro.     

Stimulation of endogenous nitric oxide production is involved in the inhibitory effect of adrenomedullin on aldosterone secretion in the rat

Adrenomedullin (AM) (10(-8) M) partially suppressed aldosterone response of dispersed rat zona glomerulosa (ZG) cells to 10 mM K+, and the nitric oxide (NO) synthase inhibitors L-NAME (10(-3) M) and 1400W (10(-4) M) effectively counteracted this effect of AM. The NO donor L-Arginine (L-Arg) (10(-5) M) decreased both basal and K+ -stimulated aldosterone secretion. The guanylate-cyclase inhibitor Ly-83583, at a concentration (10(-4) M) abolishing either the guanylate-cyclase activator guanylin- or L-Arg-induced cGMP release from dispersed ZG cells, did not affect the aldosterone antisecretagogue action of AM and L-Arg. AM (10(-8) M) evoked a moderate increase in cGMP release by dispersed ZG cells, and the effect was blocked by both 10(-4) M Ly-83583 and 10(-3) M L-NAME. Collectively, these findings allow us (1) to confirm that NO inhibits aldosterone secretion through a cGMP-independent mechanism; and (2) to suggest that stimulation of endogenous NO synthesis plays a role in the mechanisms underlying the inhibitory effect of AM on K+ -stimulated aldosterone secretion from rat ZG cells.     

Effects of prolactin on aldosterone secretion in rat zona glomerulosa cells

Acute effects and action mechanisms of prolactin (PRL) on aldosterone secretion in zona glomerulosa (ZG) cells were investigated in ovariectomized rats. Administration of ovine PRL (oPRL) increased aldosterone secretion in a dose-dependent manner. Incubation of [3H]-pregnenolone combined with oPRL increased the production of [3H]-aldosterone and [3H]-deoxycorticosterone but decreased the accumulation of [3H]-corticosterone. Administration of oPRL produced a marked increase of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in ZG cells. The stimulatory effect of oPRL on aldosterone secretion was attenuated by the administration of angiotensin II (Ang II) and high potassium. The Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 10(-2) M), inhibited the basal release of aldosterone and completely suppressed the stimulatory effects of oPRL on aldosterone secretion. The stimulatory effects of oPRL on aldosterone secretion were attenuated by the administration of nifedipine (L-type Ca2+ channel blocker) and tetrandrine (T-type Ca2+ channel blocker). These data suggest that the increase of aldosterone secretion by oPRL is in part due to (1) the increase of cAMP production, (2) the activation of both L- and T-type Ca2+ channels, and (3) the activation of 21-hydroxylase and aldosterone synthase in rat ZG cells.     

Simulated microgravity impairs aldosterone secretion in rats: possible involvement of adrenomedullin

The prolonged exposure to microgravity (MG) or simulated MG (SMG) has been reported to cause hypotension, mainly due to reduced vascular contractility, and dysregulation of fluid and electrolyte balance. However, the mechanism(s) involved in these MG- or SMG-induced effects is not yet completely elucidated. Hence, we investigated in the rat the effect of prolonged (15 day) SMG, in the form of hindlimb unweighting, on the renin-angiotensin-aldosterone system (RAAS), as well as on atrial natriuretic peptide (ANP) and adrenomedullin (ADM), two hypotensive peptides that play a major role in the regulation of RAAS activity by inhibiting adrenal aldosterone secretion. SMG caused a mild hypotension in rats, associated with the blockade of body weight gain. Plasma aldosterone concentration and basal and agonist-stimulated in vitro aldosterone secretion from adrenal slices were decreased, and plasma renin activity was moderately increased. Neither Na(+) and K(+) serum concentrations nor ACTH and corticosterone blood levels were significantly affected. Plasma ANP concentration did not display significant alterations, while ADM blood concentration underwent a marked rise. The administration of the ADM-receptor antagonist ADM-(22-52) during the last 3 days of hindlimb unweighting reversed the SMG-induced hypotension and hypoaldosteronism. Collectively, these findings allow us to suggest that prolonged SMG impairs RAAS activity in rats, through a mechanism probably involving upregulation of the ADM system. Both hypoaldosteronism and increased ADM secretion may contribute to the development of hypotension during prolonged exposure to SMG.     

Local inhibition of nitric oxide temporarily stimulates aldosterone secretion in conscious sheep in vivo

The effect of localized blockage of endogenous nitric oxide (NO) on basal aldosterone secretion was studied in conscious sheep with autotransplanted adrenal glands. We have shown that infusion of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 130 microg/l blood flow) significantly stimulated basal aldosterone secretion rate (ASR). This stimulatory effect was seen up to 4 h of infusion. Beyond this time point, however, the elevated ASR level was not sustained, and it was seen to drop markedly to lower than control values at 5 h. L-NAME had no effect on cortisol secretion rate (FSR) during the first 4 h of infusion, but a significant reduction in FSR was seen by the 8-h time point. Adrenal blood flow was consistently decreased in association with long L-NAME infusion. Additionally, L-NAME was shown to have no effect on aldosterone secretion when infused systemically. We conclude that the relationship between NO and aldosterone secretion is an inhibitory one, in which NO seems to have a negative effect on basal aldosterone secretion.     

Age-related changes in the morphology and function of the zona glomerulosa of the rat adrenal cortex

The age-related changes in the morphology and function of rat adrenal zona glomerulosa (ZG) were investigated by coupled stereological and radioimmunological techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Aging caused a notable lowering in the plasma aldosterone concentration and a marked decrease in both basal and ACTH- or angiotensin II (ANG-II)-stimulated secretion of collagenase-dispersed ZG cells. Plasma renin activity (PRA) underwent an age-dependent decrease, while the plasma level of ACTH displayed a significant rise. ZG and its parenchymal cells did not evidence any age-related morphologically demonstrable alteration in their growth, nor ZG cells showed any marked ultrastructural change, with the exception of a severe lipid-droplet repletion. This last finding is in keeping with the aging-induced decrease in the secretory activity of ZG cells, inasmuch as lipid droplets are the intra-cellular stores of cholesterol esters, the obligate precursors of steroid hormones in rat adrenals. ACTH and ANG-II are well known to be involved in the maintenance of the growth of rat ZG; thus, the combined impairment of ANG-II production (as evidenced by PRA lowering) and increase in ACTH secretion may maintain unchanged ZG growth during aging.     

Effects of fasting on aldosterone secretion in ovariectomized rats

The present study was designed to assess the effect of fasting on aldosterone secretion in ovariectomized (Ovx) rats. Ovx rats were divided into fed (allowed access to food ad libitum) and fasted (deprived of food for 24 hours) groups. The trunk blood of fed and fasted rats was collected after decapitation. In the in vitro study, adrenal zona glomerulosa (ZG) cells from fed or fasted rats were incubated with angiotensin II (Ang II, 10(-6) M), adrenocorticotropic hormone (ACTH, 10(-9) M), or forskolin (an activator of adenylyl cyclase, 10(-6) M) at 37 degrees C for 30 min. The levels of aldosterone in medium and plasma extracts were measured by radioimmunoassay. Results showed that the levels of plasma aldosterone in fasted rats were lower than those in fed rats. There were no significant differences in basal and Ang II-stimulated aldosterone secretion between fed and fasted groups. The increment of aldosterone induced by ACTH in fasted group was significantly less than that in fed group. Administration of forskolin led to a significant increase in aldosterone secretion in both fed and fasted groups. Fasted group had a decreased aldosterone secretion in response to forskolin as compared with fed group. In summary, these results suggest that fasting decreases aldosterone secretion in Ovx rats through a mechanism in part involving a reduction of aldosterone production in response to ACTH, a decreased activity of adenylyl cyclase, and/or an inhibition of post-cAMP pathway in ZG cells.     

Role of endogenous PACAP in catecholamine secretion from the rat adrenal gland

We elucidated the contribution of endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) to neurally evoked catecholamine secretion from the isolated perfused rat adrenal gland. Infusion of PACAP (100 nM) increased adrenal epinephrine and norepinephrine output. The PACAP-induced catecholamine output responses were inhibited by the PACAP type I receptor antagonist PACAP- (6-38) (30-3,000 nM) but were resistant to the PACAP type II receptor antagonist [Lys1,Pro2,5,Ara3,4,Tyr6]-vasoactive intestinal peptide (LPAT-VIP; 30-3,000 nM). Transmural electrical stimulation (ES; 1-10 Hz) or infusion of ACh (6-200 nM) increased adrenal epinephrine and norepinephrine output. PACAP-(6-38) (3,000 nM), but not LPAT-VIP, also inhibited the ES-induced catecholamine output responses. However, PACAP-(6-38) did not affect the ACh-induced catecholamine output responses. PACAP at low concentrations (0.3-3 nM), which had no influence on catecholamine output, enhanced the ACh-induced catecholamine output responses, but not the ES-induced catecholamine output responses. These results suggest that PACAP is released from the nerve endings to facilitate the neurally evoked catecholamine secretion through PACAP type I receptors in the rat adrenal gland.     

Role of the pituitary in the effects of tonin on adrenal secretion of the rat

Chronically catheterized conscious rats were infused intravenously with tonin at 2.4 and 12 micrograms x kg-1 x min-1 for 2 h. Plasma aldosterone concentration (PAC) at the end of the experiment was 11.2 +/- 2.4 ng% in controls, 8.5 +/- 2.8 ng% in rats infused with tonin at the lower rate, and 26.2 +/- 3.6 ng% (p less than 0.01 vs. controls) in rats infused at the higher rate. Plasma corticosterone (PC) was significantly higher (p less than 0.05) in the group infused at the high rate while plasma renin activity (PRA) was significantly reduced in this group of rats. Plasma angiotensin II (AII) concentration was similar in all three groups. PAC was elevated after tonin infusion in the presence of AII blockade. PAC in conscious sodium-depleted rats infused with tonin was not significantly changed, but PRA was significantly reduced (p less than 0.01). In chronically hypophysectomized rats, PAC remained unchanged by tonin infusion. The failure of tonin to stimulate aldosterone in hypophysectomized animals indicates a role of a pituitary hormone (probably ACTH) in the effect of tonin on adrenal secretion.     

Possible hyperaldosteronism and discrepancy in enzyme activity deficiency in adrenal and gonadal glands in Japanese patients with 17 alpha-hydroxylase deficiency

We reviewed the pathophysiology of our previously reported female patient who had glucocorticoid-responsive hyperaldosteronism and was treated successfully with daily dose of dexamethasone (Dex) for 21 years. In this present study, the possibility that the patient may have 17 alpha-hydroxylase deficiency (17-OH-D) mainly in the adrenal could not be ruled out. We therefore reviewed 31 Japanese patients diagnosed as having 17-OH-D with suppressed plasma renin activity reported in Japan. Among these patients, 9 were found to have a high plasma aldosterone (Ald) concentration (PAC) (group I). Twenty-one patients had either normal or low-normal PAC and the remaining patient had low urine Ald (group II). The slight cross-reactivity of the anti-Ald-antibodies used with 17-deoxy-steroids such as progesterone, 11-deoxycorticosterone and corticosterone which were increased in both groups did not explain the increased PAC in group I. In the patients in group I and group II with high-normal basal PAC, PAC further increased after ACTH and was suppressed by Dex. PAC in 2 group I patients, however, did not respond to angiotensin-II or angiotensin-III infusion. PAC in patients in group II with low or low-normal basal PAC responded equivocally to ACTH and Dex. The basal plasma cortisol in group I was lower than in group II, and plasma cortisol level after ACTH in group I appeared to remain at a lower level than that in group II patients. Among the study subjects, 28 showed a negative correlation between basal PAC and plasma cortisol. A possible discrepancy in the deficiency of 17 alpha-hydroxylase activity in adrenal and gonadal glands was also suggested in three 17-OH-D patients. The pathophysiology of Ald secretion and discrepancy in the deficiency of the enzyme activities in both glands in 17-OH-D patients was discussed.     

Dissociated response of aldosterone from plasma renin activity during prolonged exercise under hypoxia

6 healthy male subjects on a fixed salt-diet performed 1 hour ergocycle exercise at 65% of VO2 max in normoxic (N) and hypoxic (H) conditions. Blood samples were taken at intervals for estimations of plasma aldosterone (PAC), angiotensin converting enzyme (ACE), adrenocorticotrophic hormone (ACTH) and catecholamine concentrations. Plasma volume reductions with exercise were similar in N (4.3 +/- 1%) and H (4.0 +/- 1%). PRA response to exercise was increased by hypoxia while PAC and plasma catecholamine rose to a similar extent in both conditions. Increases in ACTH concentration occurred at the end of exercise but no difference was found between high and low altitudes. Plasma ACE remained unchanged throughout exercise in either condition. These results indicate that hypoxemia interferes with PRA-mediated aldosterone secretion. The variations in plasma ACTH levels during exercise in hypoxia do not appear responsible for this interference.     

Effects of prolonged cysteamine administration on the rat adrenal cortex: evidence that endogenous somatostatin is involved in the control of the growth and steroidogenic capacity of zona glomerulosa.

A week daily administration of cysteamine (CYS, 300 mg kg-1) lowered plasma aldosterone concentration in rats, without affecting PRA, kalaemia and the plasma levels of ACTH and corticosterone. Prolonged CYS treatment caused a notable hypertrophy of adrenal zona glomerulosa (ZG) and its parenchymal cells, without inducing any apparent change in zona fasciculata morphology. Isolated ZG cells from CYS-treated rats evidenced a notable enhancement in their basal and maximally-stimulated productions of aldosterone and corticosterone. All these effects of chronic CYS administration were completely reversed by the simultaneous infusion of rats with somatostatin (SRIF, 12 micrograms kg-1 h-1). CYS exposure was not found to directly affect the secretory activity of isolated ZG cells from normal rats. Since CYS is known to be a specific depletor of SRIF in different organs of rats, these findings suggest that endogenous SRIF may be involved in the modulation of ZG function.     

Role of adrenal renin-angiotensin system in the control of aldosterone secretion in sodium-restricted rats

This study examined the effect of the pharmacological manipulation of adrenal renin-angiotensin system (RAS) on aldosterone secretion from in situ perfused adrenals of rats kept on a normal diet and sodium restricted for 14 days. Neither the angiotensin-converting enzyme inhibitor captopril nor the nonselective angiotensin II receptor antagonist saralasin and the AT(1) receptor-selective antagonist losartan affected basal aldosterone output in normally fed rats. In contrast, they concentration dependently decreased aldosterone secretion in sodium-restricted animals, with maximal effective concentration ranging from 10(-7) to 10(-6) M. Captopril (10(-6) M), saralasin (10(-6) M), and losartan (10(-7) M) counteracted aldosterone response to 10 mM K(+) in sodium-restricted rats but not in normally fed animals. Collectively, these findings provide evidence that adrenal RAS plays a role in the regulation of aldosterone secretion, but only under conditions of prolonged stimulation of zona glomerulosa probably leading to overexpression of adrenal RAS.     

cGMP-dependent protein kinase type II regulates basal level of aldosterone production by zona glomerulosa cells without increasing expression of the steroidogenic acute regulatory protein gene

The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of salt and water homeostasis. Here, we demonstrate the expression and functional role of cGMP-dependent protein kinases (PKGs) in rat adrenal cortex. Expression of PKG II is restricted to adrenal zona glomerulosa (ZG) cells, whereas PKG I is localized to the adrenal capsule and blood vessels. Activation of the aldosterone system by a low sodium diet up-regulated the expression of PKG II, however, it did not change PKG I expression in adrenal cortex. Both, activation of PKG II in isolated ZG cell and adenoviral gene transfer of wild type PKG II into ZG cells enhanced aldosterone production. In contrast, inhibition of PKG II as well as infection with a PKG II catalytically inactive mutant had an inhibitory effect on aldosterone production. Steroidogenic acute regulatory (StAR) protein that regulates the rate-limiting step in steroidogenesis is a new substrate for PKG II and can be phosphorylated by PKG II in vitro at serine 55/56 and serine 99. Stimulation of aldosterone production by PKG II in contrast to stimulation by PKA did not activate StAR gene expression in ZG cells. The results presented indicate that PKG II activity in ZG cells is important for maintaining basal aldosterone production.     

Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway.

Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide, belonging to the VIP-secretin-glucagon superfamily, some members of this group are able to regulate adrenocortical function. GIP-receptor mRNA has been detected in the rat adrenal cortex, but investigations on the effect of GIP on steroid-hormone secretion in this species are lacking. Hence, we have investigated the distribution of GIP binding sites in the rat adrenal gland and the effect of their activation in vivo and in vitro. Autoradiography evidenced abundant [125I]GIP binding sites exclusively in the inner adrenocortical layers, and the computer-assisted densitometric analysis of autoradiograms demonstrated that binding was displaced by cold GIP, but not by either ACTH or the selective ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP). The intraperitoneal (IP) injection of GIP dose-dependently raised corticosterone, but not aldosterone plasma concentration: the maximal effective dose (10 nmol/rat) elicited a twofold increase. GIP did not affect aldosterone and cyclic-AMP release by dispersed zona glomerulosa cells. In contrast, GIP enhanced basal corticosterone secretion and cyclic-AMP release by dispersed inner adrenocortical cells in a concentration-dependent manner, and the maximal effective concentration (10(-7) M) evoked 1.5- and 2.4-fold rises in corticosterone and cyclic-AMP production, respectively. GIP (10(-7) M) did not display any additive or potentiating effect on corticosterone and cyclic-AMP responses to submaximal or maximal effective concentrations of ACTH. The corticosterone secretagogue action of 10(-7) M GIP was abolished by the protein kinase A (PKA) inhibitor H-89 (10(-5)M), and unaffected by CIP (10(-6)M). Collectively, these findings indicate that GIP exerts a moderate but statistically significant stimulatory effect on basal glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.     

Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37°C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca²⁺ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes. Ling-Ling Chang and Paulus S. Wang contributed equally to this work.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.