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ABSTRACT

The intracellular mechanisms of action of α-MSH in rat adrenocortical cells were examined. When rat adrenal capsule (largely glomerulosa) cells were stimulated with a range of concentrations of α-MSH there was significant stimulation of aldosterone secretion at 10^-10 mol/l, although cyclic AMP was not increased until high concentrations of α-MSH were used (10^-6 mol/l and above). However, cells incubated with ACTH showed an increase in aldosterone secretion at 10^-11 mol/l and levels of cyclic AMP were elevated at 10^-9 mol ACTH/1.

When rat adrenal whole capsules were incubated with α-MSH, membrane-bound protein kinase C (PKC) activity was increased and cytosolic enzyme activity decreased, showing PKC activation. Stimulation with angiotensin II also induced translocation of PKC activity, but ACTH did not.

When [³H]inositol-loaded glomerulosa cells were stimulated with α-MSH there was significant generation of [³H]inositol trisphosphate (IP₃) at concentrations of α-MSH which stimulated secretion of aldosterone. Significantly increased levels of [³H]IP₃ were also measured when loaded cells were exposed to angiotensin II. ACTH did not cause any significant stimulation of [³H]IP₃ production at any concentration used. These results indicate that activation of PKC and phospholipase C is important in modulating the steroidogenic effect of α-MSH.

ABSTRACT

The involvement of the calcium messenger system in the control of steroidogenesis in the rat and bovine adrenal cortex has been studied extensively. However the role of these second messengers in the control of human adrenocortical function is not established. This was therefore studied by incubating collagenase-dispersed human adrenocortical cells with the calcium ionophore A23187 and the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). The effects of the calcium channel blocker verapamil on basal and stimulated steroidogenesis were also studied.

Both TPA (1 pmol/l–10 μmol/l) and A23187 (1 nmol/l–10 μmol/l) caused a dose-dependent increase in cortisol, aldosterone and corticosterone production. Verapamil (10 μmol/l) inhibited the increase in aldosterone, corticosterone and cortisol produced in response to ACTH(1–24), potassium, and desacetyl-αMSH. Unlike previous results in the rat, these effects were not specific for aldosterone secretion.

The results suggest that, as in other species, calcium mobilization and protein kinase C activation have a role in the control of steroidogenesis in the human adrenal cortex. However, in contrast to the rat, these mechanisms appear to be involved in the control of steroidogenesis in both the zona glomerulosa and inner zone cells.

ABSTRACT

It is well established that ACTH and angiotensin II (Ang II) stimulate aldosterone secretion from rat adrenal zona glomerulosa cells in vitro and mediate their steroidogenic effects via the cyclic AMP (cAMP) pathway and phosphoinositide turnover respectively. α-MSH also stimulates aldosterone secretion from zona glomerulosa cells in vitro, and recent studies from our laboratory have shown that its steroidogenic effects are mediated by increases in inositol 1,4,5-trisphosphate (IP₃) production. α-MSH also stimulates adenylyl cyclase activity, but only at concentrations that are supramaximal for stimulation of steroidogenesis. The observation that α-MSH-stimulated IP₃ accumulation declines as the activity of adenylyl cyclase increases prompted further studies on the interactions of cAMP and phosphoinositide production.

The effects of α-MSH and ACTH on Ang II-stimulated steroidogenesis and IP₃ accumulation were studied. On addition of increasing concentrations of ACTH, both the aldosterone and IP₃ responses to Ang II were significantly inhibited; however, only high concentrations of α-MSH achieved this effect. These results suggest that cAMP or a cAMP-dependent event is able to inhibit phospholipase C activity. This hypothesis was tested by measuring IP₃ production in Ang II-stimulated zona glomerulosa cells exposed to two different concentrations of α-MSH: 1 nmol/l, which stimulates the generation of IP₃, and 1 μmol/l, which activates adenylyl cyclase. It was found that this high concentration of α-MSH significantly inhibited Ang II-stimulated aldosterone secretion and IP₃ levels. In addition, α-MSH reduced ¹²⁵I-labelled Ang II binding to rat adrenal zona glomerulosa cells. ACTH and cAMP also inhibited Ang II binding, thus supporting the hypothesis that cAMP (or a cAMP-mediated event) inhibits Ang II receptor function.