Piera Rebuffat

Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) did not affect either basal or ACTH-stimulated secretion of a1dosterone and corticosterone by dispersed rat capsular and inner adrenocortical cells, respectively. However, both peptides strongly depressed angiotensin-II (ANG- II)-stimulated a1dosterone production by capsular cells, the minimal effective concentration was 10(-7) M. The inhibitory effect of both ADM and CGRP was reversed by CGRP8-37, a specific CGRP1 receptor antagonist; a complete reversal was obtained with a CGRP8-37 concentration of 10(-6) M. Our findings indicate that ADM and CGRP specifically interfere with the intracellular mechanisms transducing the secretagogue signal of ANG-II, and suggest that the ADM effect is mediated by CGRP receptors

Ghrelin inhibits FGF-2-mediated angiogenesis in vitro and in vivo.

Recent evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is highly expressed in the cardiovascular system, and in this study we addressed the possibility that ghrelin may affect angiogenesis in vitro and in vivo. Reverse transcription-polymerase chain reaction showed that human umbilical vein endothelial cells (HUVECs) express ghrelin and GHS-R mRNAs. Ghrelin inhibited FGF-2-induced proliferation of HUVECs cultured in vitro, the maximal effective concentration being 10(-8) M, and this effect was annulled by the GHS-R antagonist D-Lys3-growth hormone releasing peptide-6. FGF-2 stimulated HUVEC cultured on Matrigel to form capillary-like structures, and ghrelin (10(-8) M) suppressed this effect. In the chick embryo chorioallantoic membrane in vivo assay, FGF-2 induced a strong angiogenic response, which was counteracted by ghrelin (500 ng). Taken together, these findings suggest that ghrelin acts as an angiostatic molecule and indicate that its activity is comparable to that of a well-known angiostatic agent, i.e., vinblastine. The antiangiogenic activity of ghrelin deserves further investigations, alone or together with other antiangiogenic agents, for the treatment of pathological conditions characterized by enhanced angiogenesis.

Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), two subtypes of which have been identified and named GHS-R1a and GHS-R1b. Evidence has been provided that ghrelin and its receptors are expressed in the adrenal gland, and we have investigated the possible role of the ghrelin system in the functional regulation of the human adrenal cortex. Reverse transcription-polymerase chain reaction detected the expression of both subtypes of GHS-Rs exclusively in the zona glomerulosa (ZG). Ghrelin did not significantly affect either basal or agonist-stimulated aldosterone secretion from cultured ZG cells. In contrast, ghrelin raised proliferative activity and decreased apoptotic deletion rate of ZG cells, the maximal effective concentration being 10(-8) M. The growth effects of 10(-8) M ghrelin on cultured ZG cells were not affected by either the protein kinase (PK)A and PKC antagonists H-89 and calphostin-C or the mitogen-activated PK (MAPK) p38 antagonist SB-293580, but were abolished by both the tyrosine kinase (TK) and MAPK p42/p44 antagonists tyrphostin-23 (10(-5) M) and PD-98059 (10(-4) M), respectively. Ghrelin (10(-8) M) enhanced TK and MAPK p42/p44 activities of ZG cells. Preincubation with 10(-5) M tyrphostin-23 blocked the ghrelin-induced stimulation of both TK and MAPK p42/p44, while preincubation with 10(-4) M PD-98059 only annulled MAPK p42/p44 stimulation. Collectively, our findings allow us to conclude that ghrelin, acting via GHS-Rs exclusively located in the ZG, enhances the growth of human adrenal cortex, through a mechanism involving the activation of the TK-dependent MAPK p42/p44 cascade.

Effects of adrenomedullin and proadrenomedullin N-terminal 20 peptide on rat zona glomerulosa cells

Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) derive from a 185-amino acid prohormone, called preproadrenomedullin, which is highly expressed in rat adrenal medulla. ADM and PAMP did not affect either basal or ACTH-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells In contrast, both peptides markedly suppressed angiotensin-II-stimulated aldosterone production, PAMP being much more effective than ADM (minimal effective concentration, 10(-10) M versus 10(-8) M. IC50, 2.0 +/- 0.17 x 10(-9) M versus 3.1 +/- 0.22 x 10(-8) M; P<0.01. Maximum inhibition, 80% versus 43%, respectively). The inhibitory effect of 10(-7) M ADM was completely reversed by the competitive antagonist of type 1 calcitonin gene-related peptide (CGRP) receptors CGRP(8-37) (10(-6) M), while that of 10(-7) M PAMP did not, thereby suggesting that this last peptide acts through specific receptors. Collectively, these findings may suggest that of the two main preproadrenomedullin derived peptides is PAMP which has probably to be considered a physiologic inhibitor of mineralocorticoid secretion in rats.

Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat

SummaryShort-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3β-hydroxysteroid dehydrogenase (3βHSD), 11β-hydroxylase (11βOH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the “aldosterone-escape” phenomenon). The activities of 3βHSD and 11βOH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata celltypes. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11βOH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the “aldosterone-escape” phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3βHSD and 11βOH.

Long-term stimulatory effect of neuropeptide-Y on the growth and steroidogenic capacity of rat adrenal zona glomerulosa.

A prolonged infusion with neuropeptide-Y (NPY) caused a notable hypertrophy of the adrenal zona glomerulosa and its parenchymal cells in rats whose hypothalamo-hypophyseal axis and renin-angiotensin system were pharmacologically interrupted. Zona glomerulosa hypertrophy was associated with a significant rise in both basal and stimulated plasma levels of aldosterone. Zona fasciculata cells and the blood concentration of corticosterone were not affected. These findings suggest that NPY is specifically involved in the stimulation of the growth and steroidogenic capacity of rat adrenal zona glomerulosa.

Effects of substance P on the rat adrenal zona glomerulosa in vivo

Substance P (SP) acutely enhanced the plasma concentration of aldosterone in rats whose hypothalamo-hypophyseal-adrenal axis and renin-angiotensin system were pharmacologically interrupted. The maximal response was obtained with a dose of 100 micrograms/kg. A prolonged (7 days) subcutaneous infusion with SP (50 micrograms/kg/hr) caused a notable hypertrophy of zona glomerulosa cells associated with significant rises in both basal and angiotensin-stimulated plasma levels of aldosterone. Zona fasciculata and the blood concentration of corticosterone were not affected. These findings suggest that SP is specifically involved in the stimulation of the growth and secretory activity of the rat zona glomerulosa.

Inhibitory effect of somatostatin on the growth and steroidogenic capacity of rat adrenal zona glomerulosa

Chronic administration of somatostatin induced atrophy of zona glomerulosa cells of rat adrenals and lowering of plasma aldosterone concentration. Zona fasciculata cells did not display any significant change and corticosterone plasma concentration was not significantly affected. These findings are interpreted to indicate that somatostatin exerts a direct inhibitory effect on the growth and steroidogenic capacity of rat adrenal zona glomerulosa.

Investigations on the turnover of adrenocortical mitochondria

SummaryThe effects of chronic administration of dexamethasone (for up to 15 consecutive days) on both the morphology and DNA-synthesis of the mitochondria of the rat adrenal zona fasciculata were investigated by stereologic and autoradiographic techniques. Up to the 3rd day of continuous dexamethasone treatment, the average volume of mitochondria did not change, whereas the number of mitochondria per cell was significantly decreased. From the 3rd to the 15th day of hormonal administration both the volume and number of mitochondria were found to decrease in proportion to the duration of treatment. Autoradiography showed that after the 3rd day of dexamethasone administration there is virtually no incorporation of 3H-thymidine into the mitochondrial compartment. These findings are discussed in the light of evidence indicating that dexamethasone blocks ACTH-release by inhibiting the hypothalamo-hypophyseal axis. The results confirm the view that ACTH controls the maintenance of growth and proliferation of rat adrenocortical mitochondria.

The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex

SummaryThe morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.