Abstract

There are several neuropeptides, present in nerves supplying the rat adrenal zona glomerulosa, which have been shown to stimulate aldosterone secretion in the intact perfused rat adrenal preparation. The purpose of the present study was twofold: first, to determine whether these peptides acted directly on adrenocortical cells by examining their effects on collagenase-dispersed rat zona glomerulosa cells, and second, to investigate the likely physiological significance of these actions, by determining whether the responses of zona glomerulosa cells to neuropeptides were changed by prior sodium depletion.

Of the peptides tested, neuropeptide Y (NPY) and substance P had only a minor effect on aldosterone secretion, which was not substantially affected by sodium depletion. Corticotrophin-releasing hormone (CRH) had a significant stimulatory effect on aldosterone secretion, but neither the threshold concentration for significant stimulation nor the maximal response to stimulation were altered by prior sodium depletion.

Vasoactive intestinal peptide (VIP), on the other hand, had little effect on aldosterone secretion by cells from normal animals, but was a potent stimulus to aldosterone secretion in cells obtained from sodium-depleted animals. The response to the Met-enkephalin analogue, [d-Ala²-Met²]-enkephalinamide (DALA), was also significantly enhanced by prior sodium depletion. Experiments using the angiotensin II receptor blocker, saralasin, were carried out to determine whether the enhanced actions of DALA and VIP seen in sodium depletion may be a result of activation of angiotensin II receptors, known to be increased in sodium depletion. Saralasin did not affect the response to either peptide.

These data suggest that all the peptides tested may be able to stimulate aldosterone secretion. However, the data obtained with substance P, NPY and CRH do not support a major role for these peptides in the regulation of aldosterone secretion either under control conditions, or in sodium depletion. The finding that the responses to VIP and DALA were altered by sodium depletion suggests that the actions of VIP and opioid peptides may have physiological significance in the regulation of aldosterone secretion in response to sodium depletion. Furthermore, the observation that saralasin does not inhibit the responses to these peptides strongly suggests that they are not acting through angiotensin II receptors, and may indicate altered VIP- and opioid-receptor regulation in sodium depletion.

Journal of Endocrinology (1995) 146, 209–214

Previous studies, by this group and others, have shown that vasoactive intestinal peptide (VIP) stimulates aldosterone secretion, and that the actions of VIP on aldosterone secretion by the rat adrenal cortex are blocked by beta adrenergic antagonists, suggesting that VIP may act by the local release of catecholamines. The present studies were designed to test this hypothesis further, by measuring catecholamine release by adrenal capsular tissue in response to VIP stimulation. Using intact capsular tissue it was found that VIP caused a dose-dependent increase in aldosterone secretion, with a concomitant increase in both adrenaline and noradrenaline release. The effects of VIP on aldosterone secretion were inhibited by atenolol, a beta1 adrenergic antagonist, but not by ICI-118,551, a beta2 adrenergic antagonist. Binding studies were carried out to investigate VIP receptors. It was found that adrenal zona glomerulosa tissue from control rats contained specific VIP binding sites (Bmax 853+/-101 fmol/mg protein; Kd 2.26+/-0.45 nmol/l). VIP binding was not displaced by ACTH, angiotensin II or by either of the beta adrenergic antagonists. The response to VIP in adrenals obtained from rats fed a low sodium diet was also investigated. Previous studies have found that adrenals from animals on a low sodium diet exhibit increased responsiveness to VIP. Specific VIP binding sites were identified, although the concentration or affinity of binding sites in the low sodium group was not significantly different from the controls. In the low sodium group VIP was found to increase catecholamine release to the same extent as in the control group, however, in contrast to the control group, the adrenal response to VIP was not altered by adrenergic antagonists in the low sodium group. These data provide strong support for the hypothesis that VIP acts by the local release of catecholamines in adrenal zona glomerulosa tissue in normal animals. It does not appear that VIP acts through the same mechanism in animals maintained on a low sodium diet. The mechanism by which VIP stimulates aldosterone in this group remains to be determined.

Abstract

While there have been several studies on the actions of opioid peptides on adrenocortical steroidogenesis, the results of these studies have failed to resolve the question as to whether these peptides exert a direct action on the adrenal cortex. The present studies were designed to address this question directly, using collagenase-dispersed rat zona glomerulosa and zonae fasciculata/reticularis cells incubated in vitro. The results obtained clearly show that the opioid peptides tested (β-endorphin, Leu-enkephalin, Met-enkephalin, and its long-acting analogue, DALA) all exerted a significant stimulatory effect on aldosterone secretion by zona glomerulosa cells and all, except Leuenkephalin, stimulated corticosterone secretion by inner zone cells. The response was shown to be inhibited by naloxone. There did not appear to be a significant interaction between the effects of ACTH and the opioid peptides on adrenocortical cells.

Studies using specific agonists for opioid receptor subtypes (DAMGO, DPDPE and U-50488H, specific for μ, δ and κ receptors respectively) showed that the effect of opioid peptides on the zona glomerulosa appeared to be mediated exclusively by μ receptors while the response of inner zone cells was mediated by both μ and, to a lesser extent, κ receptors. Finally, studies on the second messenger systems activated by the opioid peptides and the receptor agonists showed that these peptides act to increase labelling of inositol trisphosphate, and strongly suggest that, in the rat adrenal cortex, both μ and κ opioid receptors are linked to the activation of phospholipase C.

Journal of Endocrinology (1995) 144, 503–510

This study was designed to investigate the synthesis and action of adrenomedullin in the rat adrenal gland. The results obtained from in situ hybridization and immunocytochemical studies suggest that adrenomedullin is synthesized not only in the medulla, but also within the zona glomerulosa of the rat adrenal cortex. Findings from in situ hybridization and binding studies also suggested that specific adrenomedullin receptors are expressed in the zona glomerulosa, and that low levels are present in the inner zones of the cortex. The Kd of the zona glomerulosa adrenomedullin receptor (5.5 nmol/l) suggests that it may respond to locally produced adrenomedullin rather than circulating concentrations of the peptide, which are in a lower range. It was found that adrenomedullin acted on zona glomerulosa cells in vitro to stimulate aldosterone release and cAMP formation, but in this tissue did not stimulate inositol phosphate turnover. The effect of adrenomedullin on aldosterone secretion was significantly attenuated by a protein kinase A inhibitor, suggesting that cAMP mediates the effects of adrenomedullin on aldosterone secretion. Adrenomedullin did not significantly affect the response of zona glomerulosa cells to stimulation by either ACTH or angiotensin II. Adrenomedullin did not affect the release of catecholamines, either adrenaline or noradrenaline, by intact adrenal capsular tissue. These data suggest that both adrenomedullin and its specific receptor are expressed in the rat adrenal zona glomerulosa, leading to the hypothesis that adrenomedullin may have an autocrine/paracrine role in the regulation of the rat adrenal zona glomerulosa.

Stimulation of aldosterone by a serine protease, trypsin, was first reported in 1982, although the mechanism of this effect was unclear. Recently, a family of protease-activated receptors (PARs) has been described and four members of the family characterised and cloned, including the previously recognised thrombin receptor. This study investigated whether PARs mediate the action of trypsin on aldosterone secretion. Using intact rat adrenal capsular tissue, thrombin was found to increase aldosterone secretion, and the effects of trypsin on aldosterone secretion were confirmed. Both trypsin and thrombin were shown to activate phospholipase C, as measured by an increase in inositol triphosphate turnover by adrenal capsular tissue. It was also shown that U73122, a phospholipase C inhibitor, attenuated the aldosterone response to trypsin. These effects were consistent with the activation of a PAR. Northern blot analysis revealed the presence of mRNA encoding PAR-1, but not PARs-2, -3 or -4 in the adrenal capsule/zona glomerulosa. Messenger RNA encoding PAR-1 was increased by dietary sodium depletion, consistent with previous reports of an increased response to trypsin after sodium depletion. These data suggest that the actions of trypsin on aldosterone secretion are mediated by PAR-1.

Previous studies from our laboratory have reported that adrenomedullin is synthesised in rat zona glomerulosa cells. In the present studies, it was found that the human adrenocortical cell line H295R expresses the gene encoding adrenomedullin, and that immunoreactive adrenomedullin is released into the culture medium. Furthermore, it was found that secretion of adrenomedullin is regulated by angiotensin II and forskolin. Studies on the actions of adrenomedullin and calcitonin gene-related peptide (CGRP) revealed a stimulatory effect of adrenomedullin, but not of CGRP, on aldosterone and cortisol secretion. These data suggest that adrenomedullin is not acting by a CGRP receptor-mediated mechanism in the H295R cell line. Adrenomedullin was also found to increase cAMP production, suggesting that in the adrenal, as in other cell types, cAMP is a second messenger for adrenomedullin action. However, the effects of adrenomedullin were not fully mimicked by forskolin, possibly suggesting a role for an additional second messenger. The presence of mRNA encoding both the putative adrenomedullin receptors, L1 and calcitonin receptorlike receptor/receptor-associated modulatory protein 2 (CRLR/RAMP-2), was demonstrated in H295R cells, but RAMP-1 was not detected, suggesting that these cells do not express the CGRPI receptor CRLR/RAMP-1. Taken together, these data have demonstrated that adrenomedullin is synthesised and secreted by H295R cells. The observed rate of adrenomedullin synthesis suggests that this peptide exerts a paracrine/autocrine effect in this adrenocortical cell line, probably acting through a specific adrenomedullin receptor, to stimulate steroidogenesis and increase aldosterone synthase expression.

ABSTRACT

The rate of blood flow through the intact adrenal gland is closely linked to steroid hormone secretion, and although the mechanism involved is unknown, it is thought to involve secretory products of the vascular endothelium.

In dispersed cell preparations, endothelin-1 and -3 both caused a dose-dependent and highly sensitive increase in steroid secretion by zona glomerulosa and zona fasciculata cells of the rat and human adrenal cortex. In addition, when the perfused rat adrenal was stimulated with ACTH, significant increases in steroid secretion and perfusion medium flow rate were accompanied by significantly increased secretion of immunoreactive endothelin into the adrenal vein. It is proposed that endothelin has a role in mediating the adrenocortical response to ACTH stimulation.

Journal of Endocrinology (1991) 128, 275–280

Abstract

Neuropeptide Y (NPY) has been identified in nerves supplying the adrenal cortex of several mammalian species, although its function in this tissue is unknown. The present studies, employing adrenocortical cells prepared by collagenase digestion, have shown that NPY, in the absence of other stimulants, has no effect on steroid secretion by the rat adrenal over a range of peptide concentrations (10⁻¹¹ to 10 ⁻⁶ mol/l). However, in the presence of physiological concentrations of ACTH, which are submaximal for the stimulation of aldosterone secretion, NPY (10⁻⁶ mol/l) significantly enhanced the secretion rate of aldosterone by rat zona glomerulosa cells in response to ACTH. This effect was specific to the rat zona glomerulosa as NPY had no effect on the response to ACTH in rat zona fasciculata cells. The effect of NPY appears to be biphasic, however, as NPY significantly attenuated the steroidogenic response to supramaximal ACTH concentrations: in rat zona glomerulosa cells the aldosterone response to 10 ⁻⁸ mol ACTH/l was significantly inhibited by NPY. The effect of NPY on the ACTH response appeared to be mediated by changes in the cAMP response. NPY had no effect on the steroidogenic response to potassium ions (K⁺), but enhanced the response to angiotensin II. NPY (10 ⁻⁶ mol/l) significantly stimulated inositol 1,4,5-trisphosphate (InsP₃) production although this concentration of peptide had no effect on steroid secretion. The effects of NPY on InsP₃ production were additive with those of angiotensin II. These results suggest that the role of NPY in the adrenal cortex may be to regulate the sensitivity of the zona glomerulosa to peptide stimulation.

Journal of Endocrinology (1995) 145, 283–289

Abstract

A range of neuropeptides has been identified in the adrenal glands of many mammalian species. In many cases these peptides have been located in nerves supplying the adrenal cortical cells, or within clusters of chromaffin cells within the zona glomerulosa. The function of these neuropeptides has yet to be determined, but from their location within the gland it is clearly possible that they may have a role in the regulation of aldosterone secretion. The effects of Met-enkephalin, Leu-enkephalin, neuropeptide Y, substance P, corticotrophin-releasing hormone (CRH) and neurotensin on aldosterone secretion were investigated using the intact perfused rat adrenal gland in situ. All the peptides tested, except CRH, caused a significant increase in aldosterone secretion over the dose range of 1 pmol to lOnmol, with a maximum response of about a twofold increase in secretion. Met-enkephalin, however, at a dose of 10 nmol caused a 350% increase in aldosterone secretion, a response comparable with that seen in response to angiotensin II in this preparation. These results suggest that, while substance P, neuropeptide Y, neurotensin and Leu-enkephalin all have the capacity to cause modest increases in the rate of steroid secretion by the zona glomerulosa, these neuropeptides probably do not have a major role in the acute regulation of aldosterone secretion, at least under basal conditions. Met-enkephalin, on the other hand, was a more potent stimulus to aldosterone secretion, and thus may have a role in the control of aldosterone secretion.

Journal of Endocrinology (1994) 140, 91–96

ABSTRACT

The effects of vasoactive intestinal peptide (VIP) on adrenocortical function were investigated using several different preparations of adrenocortical tissue. VIP caused a significant increase in perfusion medium flow rate and in aldosterone and corticosterone secretion by the isolated perfused rat adrenal gland, with a threshold of 1 pmol in 200 μl, but did not affect basal steroid secretion by collagenase-dispersed adrenocortical cells at any concentration used, from 10 pmol/l to 10 μmol/l. The presence of VIP (100 nmol/l) had no significant effect on the response of zona glomerulosa cells to stimulation by ACTH at any concentration. In incubations of intact adrenal capsular tissue, VIP (10 μmol/l) caused a significant stimulation of aldosterone secretion, and also induced a significant release of adrenaline into the incubation medium. Addition of (−)alprenolol (100 nmol/l), a βadrenergic antagonist, to the incubation medium significantly attenuated the response of capsular tissue to VIP. It is concluded that the effects of VIP on aldosterone, which are only seen when the architecture of the zona glomerulosa is preserved, may be mediated by the local release of adrenaline.

Journal of Endocrinology (1992) 133, 253–258