No abstract

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. Evidence has been previously provided that adrenal gland possesses abundant ghrelin-displaceable GHS-Rs, but nothing is known about the possible role of ghrelin in the regulation of adrenocortical function. Reverse transcription-polymerase chain reaction demonstrated the expression of ghrelin and GHS-R in the rat adrenal cortex, and high adrenal concentrations of immunoreactive ghrelin were detected by radioimmune assay (RIA). Autoradiography localized abundant [ 125 I]ghrelin binding sites in the adrenal zona glomerulosa (ZG) and outer zona fasciculata (ZF). Ghrelin (from 10 310 to 10 38 M) did not a¡ect either basal steroid hormone (pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone) secretion from dispersed ZG and zona fasciculata/reticularis (ZF/R) cells (as evaluated by quantitative high pressure liquid chromatography), or basal and agonist-stimulated aldosterone and corticosterone production from cultured ZG and ZF/R cells, respectively (as measured by RIA). Ghrelin (10 38 and 10 36 M) raised basal, but not agonist-stimulated, proliferation rate of cultured ZG cells (percent of cells able to incorporate 5-bromo-2P P-deoxyuridine), without a¡ecting apoptotic deletion rate (percent of cells able to incorporate biotinylated nucleosides into apoptotic DNA fragments). The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic e¡ect of 10 38 M ghrelin, while the protein kinase A and C inhibitors H-89 and calphostin-C were ine¡ective. Ghrelin (10 38 M) stimulated TK and MAPK activity of dispersed ZG cells, and the e¡ect was abolished by preincubation with tyrphostin-23 and PD-98059, respectively. Tyrphostin-23 annulled ghrelin-induced activation of MAPK activity. Taken together, the present ¢ndings indicate that (i) ghrelin and GHS-R are both expressed in the rat adrenal cortex, ghrelin binding sites being very abundant in the ZG; (ii) ghrelin does not a¡ect the secretory activity of rat adrenocortical cells, but signi¢cantly enhances the proliferation rate of cultured ZG cells, without a¡ecting apoptotic deletion rate; and (iii) the ZG proliferogenic action of ghrelin involves the TK-dependent activation of the p42/p44 MAPK cascade. ß

We investigated the gene expression and localization of endothelin-1 (ET-1) receptor subtypes ET(A) and ET(B) in the rat adrenal cortex as well as their involvement in the corticosteroid secretagogue effect of ET-1 in vitro. Reverse transcription-polymerase chain reaction with primers specific for ET(A) and ET(B) cDNAs demonstrated the expression of both receptor genes in homogenates of adrenocortical tissue. However, in isolated zona glomerulosa and zona fasciculata cells, only ET(B) mRNA was detected. Autoradiographic examination of the selective displacement of 125I-ET-1 binding by BQ-123 and BQ-788 (specific ligands for ET(A) and ET(B), respectively) indicated that zona glomerulosa possesses both ET(A) and ET(B), whereas zona fasciculata is exclusively provided with ET(B). ET-1 enhanced in a concentration-dependent manner aldosterone and corticosterone secretions of dispersed zona glomerulosa and zona fasciculata cells, respectively. The ET(B) antagonist BQ-788 markedly reduced the secretory response of zona glomerulosa cells and completely suppressed that of zona fasciculata cells, whereas the ET(A) antagonist BQ-123 was ineffective. These findings indicate that in the rat, the adrenocortical secretagogue action of ET-1 is mediated by the ET(B) receptor subtype and that the ET(A) receptor is not directly involved in such an effect.

Numerous lines of evidence indicate that adrenal medulla exerts a paracrine control on the secretory activity of the cortex by releasing catecholamines and several regulatory peptides. Adrenomedullin (ADM) is contained in adrenal medulla of several mammalian species, including humans. Thus, we investigated whether human ADM1-52 exerts a modulatory action on steroid secretion of human adrenal cortex in vitro. Dispersed adrenocortical cells (obtained from the gland tail deprived of chromaffin cells) and adrenal slices (including both capsule and medulla) were employed. ADM specifically inhibited angiotensin II-stimulated aldosterone secretion of dispersed cells and enhanced basal aldosterone production by adrenal slices, minimal effective concentrations being 10(-7) and 10(-9) mol/L, respectively. These effects of ADM were suppressed by the CGRP1 receptor antagonist CGRP8-37 (10(-5) mol/L). Neither basal and ACTH-stimulated aldosterone secretion of dispersed cells nor agonist-enhanced aldosterone production by adrenal slices were affected by ADM, which also did not alter cortisol secretion of both types of adrenal preparations. ADM (10(-6) mol/L) blunted the aldosterone secretagogue action of the Ca2+ ionophore A23187 (10(-5) mol/L) on dispersed cells and adrenal slices. The beta-adrenoceptor antagonist l-alprenolol (10(-6) mol/L) suppressed aldosterone response of adrenal slices to 10(-7) mol/L isoprenaline and ADM. ADM concentration dependently raised epinephrine and norepinephrine release by adrenal slices, minimal effective concentration being 10(-9) mol/L. Collectively, these findings suggest that ADM, acting via the CGRP1 receptor subtype, exerts a direct inhibitory effect on angiotensin II-stimulated aldosterone secretion, which, when the integrity of adrenal tissue is preserved, is overcome and reversed by an indirect stimulatory action, conceivably involving the release of catecholamines by adrenal chromaffin cells.

Interleukin-1 (IL-1), a monokine released by activated monocytes during the acute phase of the inflammatory responses, has been reported to enhance hypophyseal ACTH release mainly by stimulating hypothalamic CRF secretion. We investigated a possible direct effect of IL-1 beta on the adrenal gland of the rat. IL-1 beta was found to dose-dependently (4-8 micrograms/kg) raise corticosterone (B) blood concentration in hypophysectomized rats, without inducing any significant increase in the level of circulating ACTH. IL-1 beta did not affect B production by either isolated rat inner adrenocortical cells or fragments of adrenocortical autotransplants lacking chromaffin cells, but dose-dependently (10(-8)-10(-6) M) enhanced that by adrenal slices including both cortex and medulla. The secretory effect of IL-1 beta (10(-6) M) was completely blocked by both alpha-helical-CRF (10(-6) M) and corticotropin-inhibiting peptide (10(-6) M), two competitive inhibitors which (at these concentrations) were able to annul B response of adrenal slices to CRF (10(-6) M) and ACTH (10(-8) M), respectively. In light of many findings indicating that adrenal medulla contains and releases CRF and numerous POMC-derived peptides (including ACTH), the hypothesis is advanced that the mechanism underlying the direct secretory effect of IL-1 beta on the adrenal gland may involve the activation of an intraadrenal CRF/ACTH system.