Giuseppe Gottardo

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

Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: Comparison with calcitonin gene-related peptide effects

Adrenomedullin (ADM), a vasodilatatory peptide contained in adrenal medulla, was found to induce a dose-dependent increase in aldosterone (ALDO) and corticosterone (B) release by the in situ perfused rat adrenal gland, along with a rise in the flow rate of the perfusion medium. The minimal effective dose for ALDO response was three and two orders of magnitude less than those able to evoke B and medium flow rate responses. Calcitonin gene-related peptide (CGRP), another vasodilatatory peptide contained in adrenal medulla and showing a slight homology in its amino acid sequence with ADM, elicited similar effects. CGRP (8-37), a specific antagonist of CGRP1 receptors, annulled all the effects of both ADM and CGRP, whereas l-alprenolol, a beta-adrenoceptor antagonist, partially reversed only ALDO response to the peptides. In light of these findings the following conclusions are drawn: i) ADM and CGRP stimulate rat adrenals in vivo to release B by raising blood flow rate; ii) ADM and CGRP enhance ALDO secretion via an indirect mechanism probably requiring the release of catecholamines by medullary chromaffin cells; and iii) the effects of ADM and CGRP on the rat adrenal gland are mediated by a common receptor of the CGRP1 subtype.

Distribution and functional significance of angiotensin-II AT1-AND AT2-Receptor subtypes in the rat adrenal gland

The distribution and the functional significance of angiotensin-II (ANG-II) receptor subtypes, AT1 and AT2, in the rat adrenal gland has been investigated in vitro. Autoradiographic assessment of the selective displacement of [125I]ANG-II binding by selective ligands of the two receptor subtypes indicated that zona glomerulosa (ZG) was provided with both AT1 and AT2, and adrenal medulla (AM) almost exclusively with AT2 receptors. ANG-II (10(-9) M) evoked a marked rise in the secretion of aldosterone by dispersed ZG cells and catecholamines by AM fragments. The selective AT1-receptor antagonist DuP753 blocked aldosterone response to ANG-II, while the selective AT2-receptor antagonist PD123319 was ineffective. Catecholamine response to ANG-II was inhibited by PD123319 and only moderately affected by high concentrations of DuP753. The selective AT2-receptor agonist CGP42112 did not change basal aldosterone release of ZG cells, but concentration-dependently enhanced basal catecholamine release by AM fragments. In light of these findings the conclusion is drawn that in the rat the aldosterone secretagogue effect of ANG-II is exclusively mediated by the AT1 receptors present in the ZG, while the catecholamine secretagogue action preminently involves the activation of AT2 receptor located on medullary chromaffin cells.

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.

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.

Further investigations on the effects of neuropeptide Y on the secretion and growth of rat adrenal zona glomerulosa

NPY is a regulatory peptide, high levels of which are contained in adrenal glands of several mammals and which is co-released with catecholamines during various stressful conditions. The acute and chronic effects of NPY on adrenocortical secretion and growth were studied in the rat. NPY concentration-dependently increased aldosterone (ALDO), but not corticosterone (B) secretion of adrenal slices (maximal effective concentration was 10(-7) M). Two competitive inhibitors of NPY receptors, named PYX-1 and PYX-2, were found to dose-dependently inhibit ALDO response of adrenal preparations to 10(-7) M NPY; PYX-2 was more efficient than PYX-1, and at a concentration of 10(-5) M completely annulled the effect of 10(-7) M NPY. The acute bolus intraperitoneal (i.p.) injection of NPY (3 nmol/kg) raised plasma ALDO concentration (PAC), but not that of B (PBC); this effect of NPY was blocked by the simultaneous injection of PYX-2 (300 nmol/kg). The prolonged i.p. infusion with NPY (3 nmol/kg/h for 7 days) increased PAC (but not PBC) and induced a marked hypertrophy of the zona glomerulosa (ZG) and its parenchymal cells; dispersed ZG cells obtained from NPY-infused rats displayed a significantly enhanced basal and maximally agonist-stimulated ALDO production. The simultaneous infusion with PYX-2 (300 nmol/kg/h) completely annulled all these effects of NPY. The acute or chronic administration of PYX-2 alone did not evoke any apparent effect on the ZG secretion and growth. In light of these findings the following conclusions can be drawn: (i) NPY is able to stimulate not only the secretion, but also the growth of adrenal ZG in rats, via a receptor-mediated mechanism (since this effect is blocked by PYX-2); (ii) endogenous NPY does not play a prominent role in the physiological maintenance of secretion and growth of rat ZG (since PYX-2 alone is ineffective); (iii) NPY may play a crucial role in the fine tuning of the ZG functions in conditions requiring an increased release of mineralocorticoid hormones.

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.

Role of endothelins in regulation of vascular tone in the in situ perfused rat adrenals.

This study examined the role of endothelins (ETs) and their receptor subtypes ETA and ETB in the regulation of vascular tone in the in situ perfused rat left adrenal gland. Endothelin-1 (ET-1), which binds both ETA and ETB receptors, decreased adrenal flow rate of the perfusion medium, and its effect was reversed by the ETA antagonist BQ-123 and enhanced by the ETB antagonist BQ-788. ET-3, which preferentially binds ETB, and the selective ETB agonist BQ-3020 increased adrenal flow rate of perfusate, and their effects were annulled by BQ-788. BQ-123 magnified the effect of ET-3 and did not affect that of BQ-3020. The ETA-mediated decrease and the ETB-mediated rise in the rate of collection of perfusate were abolished by Ro-31-8220, an inhibitor of protein kinase C (PKC), and by N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS), respectively. Collectively, these findings suggest that ETs can regulate vascular tone in the in situ perfused rat adrenals via both PKC-coupled ETA and NOS-coupled ETB receptors, the activation of which evokes vasoconstriction and vasodilation, respectively.This study examined the role of endothelins (ETs) and their receptor subtypes ETAand ETB in the regulation of vascular tone in the in situ perfused rat left adrenal gland. Endothelin-1 (ET-1), which binds both ETA and ETB receptors, decreased adrenal flow rate of the perfusion medium, and its effect was reversed by the ETA antagonist BQ-123 and enhanced by the ETB antagonist BQ-788. ET-3, which preferentially binds ETB, and the selective ETB agonist BQ-3020 increased adrenal flow rate of perfusate, and their effects were annulled by BQ-788. BQ-123 magnified the effect of ET-3 and did not affect that of BQ-3020. The ETA-mediated decrease and the ETB-mediated rise in the rate of collection of perfusate were abolished by Ro-31-8220, an inhibitor of protein kinase C (PKC), and by N G-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS), respectively. Collectively, these findings suggest that ETs can regulate vascular tone in the in situ perfused rat adrenals via both PKC-coupled ETA and NOS-coupled ETB receptors, the activation of which evokes vasoconstriction and vasodilation, respectively.

Evidence that an extrahypothalamic pituitary corticotropin-releasing hormone (CRH)/adrenocorticotropin (ACTH) system controls adrenal growth and secretion in rats

Within two weeks, hypophysectomy induced in rats a striking decrease in the level of circulating ACTH (the concentration of which was at the limit of sensitivity of our assay system), coupled with a net reduction in the plasma corticosterone concentration and an evident adrenal atrophy. Zona fasciculata, the main producer of glucocorticoids, was decreased in volume, due to a lowering in both the number and average volume of its parenchymal cells. Subcutaneous ACTH infusion (0.1 pmol·min-1), administered during the last week following hypophysectomy, restored the normal blood level of ACTH and completely reversed all effects of hypophysectomy on the adrenals. Subcutaneous infusion for one week with α-helical-CRH or corticotropin-inhibiting peptide (1 nmol·min-1), which are competitive inhibitors of CRH and ACTH, evoked a further significant lowering of plasma corticosterone concentration and markedly enhanced adrenal atrophy in hypophysectomized rats. These findings strongly suggest that an extrahypothalamic pituitary CRH/ACTH system may be involved in the maintenance of the growth and steroidogenic secretory activity of the rat adrenal cortex.