Effects of neurotensin and bombesin on the secretory and proliferative activity of regenerating rat adrenal cortex.

Neurotensin (NT) and bombesin (BM)-like peptides are known to be involved in the regulation of the rat hypothalamo-pituitary-adrenal axis. By using selective NT- and BM-receptor antagonists (NT-A and BM-A, respectively) we investigated whether endogenous NT and BM-like peptides play a role in the control of rat adrenal secretion and growth during enucleation-induced regeneration. At day 5 of regeneration, NT-A did not affect the plasma concentrations of aldosteronc (PAC) and corticosterone (PBC), but at day 8, it raised both PAC and PBC over the respective baseline value; the simultaneous administration of NT abolished this effect of NT-A. BM-A did not alter PAC and PBC at day 5 of regeneration, while at day 8 it enhanced PBC, an effect reversed by BM. NT-A did not alter mitotic index, and BM-A lowered it at both day 5 and day 8 of regeneration, an effect suppressed by the simultaneous administration of BM. Collectively, these findings allow us to draw the following conclusions: 1) endogenous NT and BM-like peptides influence adrenocortical regeneration in rats; 2) NT exerts a tonic inhibitory action on both aldosterone and corticosterone secretion, without affecting cell-proliferation rate; and 3) BM-like peptides exert a tonic suppressive effect on corticosterone production, coupled with a clear-cut stimulating effect on cell proliferation.

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.

Adrenomedullin and calcitonin gene-related peptide (CGRP) interact with a common receptor of the CGRP1 subtype in the human adrenal zona glomerulosa.

Frozen sections of normal adrenal glands, obtained from patients undergoing unilateral nephrectomy for kidney cancer, were labeled in vitro with human [125I]ADM(1-52). Autoradiography showed the presence of abundant ADM binding sites in the zona glomerulosa (ZG) and the outermost portion of the zona fasciculata, which were completely displaced by the addition of an excess of cold ADM(1-52). Calcitonin gene-related peptide (CGRP) and the non-selective ligand of the CGRP-receptor subtypes 1 and 2 CGRP(8-37) eliminated [125I]ADM(1-52) binding in the ZG, while the selective ligand of CGRP receptor subtype 2 [Cys(acm)2,7]-CGRP and CGRP(1-8) were ineffective. These findings confirm the presence of ADM binding sites in the human ZG, and provide the first morphological evidence that ADM and CGRP interact with a common receptor of the CGRP1 subtype.

Autoradiographic evidence that zona glomerulosa and capsular vessels of the human adrenal cortex are provided with different subtypes of adrenomedullin receptors.

Frozen sections of normal adrenal glands, obtained from patients undergoing unilateral nephrectomy for kidney cancer, were labeled in vitro with human [125I]ADM(1-52). Autoradiography and quantitative densitometry showed the presence of abundant ADM(1-52) binding sites in both zona glomerulosa (ZG) and capsular vessels, which were displaced with about the same efficiency by cold ADM(1-52) and rat ADM(1-50). The selective calcitonin gene-related peptide type 1 (CGRPI) ligand CGRP(8-37) eliminated, although less efficiently than ADMs, [125I]ADM(1-52) binding in the ZG, but not in the capsular vessels. These findings suggest the existence of different receptor subtypes for ADM in the human adrenal cortex. The CGRP(8-37)-sensitive receptors located in the ZG may mediate the well-known inhibitory effect of ADM on aldosterone secretion, while the CGRP(8-37)-insensitive receptors present in the capsular vessel may be involved in the ADM-induced rise in adrenal blood flow.

Pancreatic polypeptide stimulates rat adrenal glucocorticoid secretion by activating the adenylate cyclase-dependent signaling pathway.

Pancreatic polypeptide (PP) concentration-dependently raised basal corticosterone and cyclic-AMP production of dispersed rat zona fasciculata/reticularis adrenocortical cells, maximal effective concentration being 10(-7) M. 10(-7) M PP also significantly enhanced submaximally (10[-12]/10[-11] M), but not maximally (10[-9]/10[-8] M) ACTH-stimulated corticosterone and cyclic-AMP release. Corticosterone responses to PP were abolished by the specific protein kinase A (PKA) antagonist H-89 (10[-5] M). The selective ACTH-receptor antagonist corticotropin-inhibiting peptide (10[-6] M) annulled corticosterone response to 10(-9) M ACTH, but not to 10(-7) M PP. Collectively, our present findings indicate that PP stimulates glucocorticoid secretion of rat adrenal glands, acting through specific receptors coupled, like those of ACTH, with the adenylate cyclase/PKA-dependent signaling pathway.

Distribution and functional significance of the endothelin receptor subtypes in the rat adrenal gland.

Endothelins (ET) are a family of vasoactive peptides that act via two subtypes of receptors, named ETA and ETB. ET-1 binds to both ETA and ETB, whereas the isopeptide ET-3 preferentially binds to ETB. The localization of ETA and ETB receptors in the rat adrenal gland and their involvement in the adrenal secretagogue effect of ETs has been studied in vitro. Autoradiographic assessment of the selective displacement of [125I]ET-1, [125I]ET-3 and [125I]BQ-3020 (an ETB agonist) by BQ-123 or BQ-788 (specific antagonists of ETA and ETB, respectively) indicates that the zona glomerulosa and adrenal medulla possess both ETA and ETB, whereas the zona fasciculata/reticularis is exclusively provided with ETB. ET-1, ET-3 and BQ-3020 enhance aldosterone and corticosterone secretion by dispersed cells of the zona glomerulosa and zona fasciculata/reticularis, respectively. BQ-123 does not affect the secretagogue action of these three agonists, whereas BQ-788 completely annuls it. ET-1 induces a marked rise in catecholamine release by fragments of the adrenal medulla, and both BQ-123 and BQ-788 partially reverse this effect. ET-3 and BQ-3020 elicit a catecholamine release that is less intense than that produced by ET-1; this response is unaffected by BQ-123 and abolished by BQ-788. Thus, in the rat, the corticosteroid secretagogue effect of ETs seems to be exclusively mediated by the ETB receptor subtype, and the catecholamine secretagogue action by both ETA and ETB. The functional relevance of ETA receptors present in the zona glomerulosa remains to be investigated.

[From the anatomical desk to virtual anatomy]. / Dal tavolo anatomico all'anatomia per immagini.

Teaching of gross Anatomy, the oldest between medical sciences, today suffers the lack of cadavers for notomization, therefore this subject is more theoretical than practical in medical school. The computer techniques could be very useful in this field. Is it possible nowadays to get a software of Virtual Anatomy? The answer is yes. We present in this work a review of the state-of-art of these techniques mainly based on data acquired by computer tomography (CT) or magnetic resonance imaging (MRI) from human. Serial slices obtained from imaging (CT or MRI) can be reconstructed using computers in order to generate a realistic view of the surface of an anatomical object.

Effects of endothelin-1 on the rat pituitary-adrenocortical axis under basal and stressful conditions.

Endothelins (ETs) and their receptor subtypes A and B (ETA and ETB) are expressed in the various components of the mammalian hypothalamo-pituitary-adrenal (HPA) axis, but their involvement in the functional regulation of HPA is controversial. To gain insight into this topic, we have investigated the effects of ET-1 and/or the specific antagonists of ETA and ETB receptors (BQ-123 and BQ-788, respectively) on the plasma concentrations of ACTH, corticosterone and aldosterone of non-stressed (control) and ether- or cold-stressed rats. The study of the effects of the administration of the two ET-receptor antagonists alone could provide informations about the possible action of endogenous ETs on the HPA axis. Exogenous ET-1 increased ACTH, corticosterone and aldosterone blood levels in control rats, as well as evoked a sizable enhancement of the HPA axis response to ether stress and a marked depression of the response to cold stress. BQ-123 and BQ-788 did not prevent the stimulatory effect of exogenous ET-1 in control rats, but when administered alone, raised the plasma concentrations of ACTH, corticosterone and aldosterone. Both ET-receptor antagonists magnified the HPA axis response to ether and cold stresses, but their effect was not counteracted by exogenous ET-1. Although very difficult to interpret, our present findings allow us to conclude that endogenous ETs play a role in the maintenance of the basal activity of rat HPA axis acting through ETA and ETB receptor subtypes, which are partially insensitive to BQ-123 and BQ-788. Conversely, the involvement of ETs in the modulation of the HPA axis responses to various stresses is very doubtful.

Evidence that both ETA and ETB receptor subtypes are involved in the in vivo aldosterone secretagogue effect of endothelin-1 in rats.

Endothelins (ET) are a family of vasoconstrictor peptides, secreted by vascular endothelium, which act through two main subtypes of receptors: ETA and ETB. ET-1 is known to stimulate aldosterone (ALDO) secretion by adrenal zona glomerulosa (ZG), and in vitro its effect was recently found to be exclusively mediated by ETB receptors. In this study the involvement of ETA and ETB in the mediation of the in vivo acute ALDO secretagogue action of ET-1 was investigated by the use of their selective antagonists BQ-123 and BQ-788, respectively. The bolus intraperitoneal administration of ET-1 dose-dependently raised both basal and angiotensin II (ANG II)-enhanced plasma ALDO concentration (PAC) in rats. Both antagonists counteracted the stimulatory effect of ET-1 on basal PAC, and when administered together completely annulled it. Conversely, only BQ-788 reversed the effect of ET-1 on ANG II-enhanced PAC. ET-1 increased systolic blood pressure (BP) in normal rats, but not in animals simultaneously administered ANG II. The hypertensive effect of ET-1 was completely abolished by BQ-123, and not affected by BQ-788. In light of these findings the following conclusions can be drawn: (i) the in vivo ALDO secretagogue action of ET-1 is mediated by both ETA and ETB, this latter subtype of ET receptors playing a major role; and (ii) the mechanism whereby ETA participates in this in vivo effect of ET-1 is indirect, and probably connected with the ET-1-induced rise in BP and adrenal blood flow.

In-vitro and in-vivo studies of the effects of arginine-vasopressin on the secretion and growth of rat adrenal cortex.

Arginine-vasopressin (AVP) markedly increased basal aldosterone (ALDO) secretion by dispersed zona-glomerulosa (ZG) cells, and its effect was selectively reversed by V1-receptor antagonists (AVP-A1). Corticosterone (B) production by dispersed zona fasciculata (ZF) cells was not affected. The bolus intraperitoneal (i.p.) administration of AVP acutely raised the plasma concentrations of both ALDO and B in normal rats, but only that of ALDO in bilaterally adrenalectomized animals bearing regenerated adrenocortical autotransplants, which are deprived of medullary chromaffin cells. Accordingly, AVP raised ALDO and B secretions by adrenal slices (including both cortical and medullary tissues), and only ALDO production by autotransplant quarters. The B response of adrenal slices to AVP was blocked by alpha-helical-CRH and corticotropin-inhibiting peptide (two competitive inhibitors of CRH and ACTH, respectively), but not by 1-alprenolol (a beta-adrenoreceptor antagonist); ALDO response was not affected by any of these antagonists. A 7-day i.p. infusion with AVP increased the volume of ZG cells and ZG-like cells of autotransplants, as well as their basal and maximally angiotensin-II-stimulated ALDO secretory capacity; it also raised the volume, and basal and maximally ACTH-stimulated B secretory capacity of ZF cells, but it did not affect ZF-like cells of autotransplants. The simultaneous administration of AVP-A1 annulled all these effects of AVP. When infused alone, AVP-A1 caused a marked atrophy of ZG cells, coupled with a net drop in their steroidogenic capacity; however, AVP-A1 infusion did not change the morphology and function of either ZF cells or ZG-like and ZF-like cells of autotransplants. Taken together,, our findings allow us to draw the following conclusions: (i) AVP plays an important physiological role in the maintenance and stimulation of ZG growth and mineralocorticoid secretory activity in rats, the source of endogenous AVP exerting adrenoglomerulotropic action probably being adrenal chromaffin cells; and (ii) AVP indirectly stimulates the growth and glucocorticoid secretory activity of rat ZF cells, by activating intramedullary CRH/ACTH system; however, the physiological relevance of this effect of AVP appears to be doubtful.

Pancreatic polypeptide enhances plasma glucocorticoid concentration in rats: possible role in hypoglycemic stress.

The acute bolus intraperitoneal (i.p.) administration of pancreatic polypeptide (PP) dose-dependently enhanced the plasma concentration of corticosterone (PBC) in hypophysectomized/ACTH replaced rats, but not that of aldosterone. Minimal and maximal effective doses were 10(-12) and 10(-10) mol/rat, respectively, and maximal PBC increase occurred between 60 and 120 min after PP injection. Insulin (1 U/kg, i.p.) evoked a net decrease in the blood glucose concentration, and marked rises in the plasma levels of PP and PBC, that attained their maximum at 60 and 120 min, respectively. The effects of insulin were annulled by the simultaneous injection of 0.5 mg/kg atropine. The effects of 1 U/kg insulin and 10(-10) mol/rat PP on PBC were not additive; atropine did not affect PBC response to PP or PP plus insulin, though annulling that to insulin alone. Taken together these findings suggest that PP plays a physiologic role in the rat as modulator of the adrenal response to the insulin-induced hypoglycemic stress.

Neuropeptide K enhances glucocorticoid release by acting directly on the rat adrenal gland: the possible involvement of zona medullaris.

Neuropeptide K (NPK), a member of the kassinin-like tachykinin family, is contained in the rat hypothalamus and is known to stimulate pituitary ACTH release. The intraperitoneal bolus administration of NPK dose-dependently enhanced corticosterone blood level not only in intact rats, but also in hypophysectomized/ACTH replaced animals. NPK did not affect corticosterone secretion of dispersed rat adrenocortical cells; however, it concentration-dependently raised basal corticosterone production by decapsulated adrenal quarters (including both cortical and medullary tissues). Minimal and maximal effective concentrations were 10(-9) and 10(-8) M, respectively. 10(-8) M NPK potentiated corticosterone response of adrenal quarters elicited by 10(-12) M ACTH, but not that evoked by higher concentrations of ACTH. The direct corticosterone secretagogue effect of 10(-8) M NPK is annulled by 10(-6) M alpha-helical-CRH or corticotropin-inhibiting peptide, competitive inhibitors of CRH and ACTH, respectively. In light of these findings, the hypothesis is advanced that NPK exerts a direct stimulatory action on adrenocortical secretion and that the mechanism underlying this effect of NPK may involve the activation of the intra-medullary CRH/ACTH system.

Evidence that endogenous arginine-vasopressin (AVP) is involved in the maintenance of the growth and steroidogenic capacity of rat adrenal zona glomerulosa.

A 7-day subcutaneous infusion with the AVP antagonist [Deamino-Pen1, Val4, D-Arg8]-vasopressin (AVP-A; 3 nmol.kg-1 x min-1) significantly lowered plasma aldosterone concentration in rats, without affecting the plasma levels of ACTH and corticosterone. Prolonged AVP-A treatment caused a marked atrophy of adrenal zona glomerulosa (ZG) and its parenchymal cells, without inducing any significant change in zona fasciculata morphology. Isolated ZG cells from AVP-A-infused rats evidenced a notable decrease in both their basal and maximally-stimulated aldosterone production. The simultaneous infusion of rats with AVP (3 nmol.kg-1 x min-1) completely reversed all these effects of AVP-A. These findings suggest that endogenous AVP may be specifically involved in the maintenance of the growth and steroidogenic capacity of rat adrenal ZG. Moreover, they seem to indicate that under basal conditions the pituitary-adrenal-glucocorticoid axis is independent of AVP release.

Morphology and functional responses of isolated inner adrenocortical cells of rats infused with interleukin-beta.

The effects of the prolonged infusion with interleukin-1 beta (IL-1 beta) (20 pM.kg-1.min-1) on the function and morphology of the isolated inner cells of the rat adrenal cortex were investigated. After 3 and 5 days of IL-1 beta infusion, the level of circulating ACTH was below the control level, while the plasma concentration of corticosterone was strikingly elevated. After 5 days of infusion, isolated inner adrenocortical cells showed an enhanced basal and ACTH-stimulated corticosterone secretion, and showed a conspicuous hypertrophy. The acute exposure to IL-1 beta 10(-6) M did not affect the secretory activity of dispersed cell from either control or IL-1 beta-infused rats. These findings indicate that the prolonged exposure to high levels of circulating IL-1 beta, like those occurring during chronic inflammatory diseases, is able to enhance the growth and steroidogenic (glucocorticoid) capacity of the rat inner adrenocortical zones. Moreover, they suggest that the mechanism underlying this adrenocorticotrophic effect of IL-1 beta does not involve either a stimulation of the hypophyseal ACTH release or a direct stimulatory effect of monokine on adrenocortical cells. It is suggested that IL-1 beta may activate an intra-adrenal paracrine regulatory mechanism.

Calcitonin gene-related peptide depresses the growth and secretory activity of rat adrenal zona glomerulosa.

The bolus ip. injection of rat calcitonin gene-related peptide (CGRP) (5 pm. kg-1) significantly lowered plasma aldosterone concentration (PAC) in rats, despite a mild rise in plasma renin activity. Natremia, kalaemia and the blood levels of ACTH or corticosterone were not affected. Similar results were obtained after prolonged (5 days) sc. infusion of rats with CGRP (1 pm. kg-1. h-1). Moreover, CGRP infusion caused a notable atrophy of the zona glomerulosa (ZG) and its parenchymal cells, as well as a clearcut reduction in the surge of PAC evoked by a bolus injection of a high dose of angiotensin-II (100 micrograms. kg-1). From these results it is suggested that CGRP exerts an inhibitory effect on the growth and secretory activity of ZG in rats.

Evidence that endogenous somatostatin (SRIF) exerts a tonic inhibitory effect on the rat renin--angiotensin--aldosterone system.

The bolus ip. administration of a SRIF antagonist (SRIF-A) (60 nM/rat) significantly increased renin activity (PRA) and plasma aldosterone concentration (PAC) in rats, without affecting natremia, kalaemia and the blood levels of ACTH or corticosterone. SRIF-A also raised PAC in rats whose renin-angiotensin system had been pharmacologically interrupted by combined captopril/angiotensin-II infusion and in which PRA was very low. The ip. injection of an equimolar dose of SRIF completely reversed these effects of SRIF-A, but the administration of SRIF alone did not affect either PRA or PAC. Taken together, these data would suggest that, in the rat, endogenous SRIF exerts, under basal conditions, a two-fold maximum tonic inhibitory effect on both renin release by kidneys and aldosterone secretion by zona glomerulosa cells.

Effects of interleukin-1 beta on the renin-angiotensin-aldosterone system in rats.

A bolus IP injection of interleukin-1 beta (IL-1 beta) (8 micrograms.kg-1) increased blood pressure and PRA without affecting plasma aldosterone (ALDO) concentration. IL-1 beta strongly attenuated angiotensin-II (ANG-II, 10(-8) M)-stimulated ALDO secretion by both isolated zona glomerulosa (ZG) cells and capsular strips. These findings suggest that IL-1 beta exerts a twofold opposite action on the main components of the rat renin-angiotensin-aldosterone system: simultaneous stimulation of renin release by kidneys and inhibition of the stimulatory effect of ANG-II on ALDO production. At the highest concentrations (10(-6)/10(-5) M), IL-1 beta was found to lower also basal ALDO output by isolated ZG cells, but not by capsular strips. However, in the presence of saralasin 10(-8) M (a competitive inhibitor of ANG-II) and captopril 10(-8) M (an angiotensin-I-converting enzyme inhibitor), IL-1 beta significantly reduced basal ALDO yield of capsular strips. These last results would suggest that IL-1 beta could also similarly affect the intra-adrenal renin-angiotensin system, which seems to be involved in the local regulation of ZG secretory activity.

Endothelin-1 stimulates mitotic activity in the zona glomerulosa of the rat adrenal cortex.

Subcutaneous infusion with endothelin-1 (ET-1; 30 pM min-1) for 24 h induced a 9-fold increase in the mitotic index (% of metaphase-arrested cells) of rat adrenal zona glomerulosa (ZG). Infusions with adrenocorticotrophic hormone (ACTH) angiotensin-II (ANG-II) or arginine vasopressin (AVP) (30 pM min-1/24 h) raised the ZG mitotic index 13-fold, 9-fold and 10-fold, respectively. Combined infusion with ET-1 and ACTH increased the ZG mitotic index 20-fold, while the effects of ET-1 and ANG-II or AVP were not additive. These findings suggest that ET-1 exerts a strong proliferogenic effect on the rat ZG, by a mechanism probably similar to that underlying the adrenoglomerulotrophic actions of ANG-II and AVP.

Neurotensin enhances plasma adrenocorticotropin concentration by stimulating corticotropin-releasing hormone secretion.

The systemic administration of neurotensin (NT) dose-dependently increased plasma adrenocorticotropin (ACTH) concentration in rats, and this effect was annulled by (alpha-helical)-CRH9-41, an antagonist of corticotropin-releasing hormone (CRH). The systemic administration of [D-Trp11]-neurotensin (NT-A), a specific NT antagonist, dose-dependently reduced the basal level of circulating ACTH, and this effect was blunted by NT injection. The ACTH inhibitory action of NT-A was completely overcome by the administration of CRH. Taken together, our findings suggest that NT plays a physiologic role in rats, as ACTH secretagogue, and that the mechanism underlying this action of NT involves the stimulation of CRH release.

Lipid droplets in the secretory response of Leydig cells of normal and hCG-treated rats.

Prolonged (7-day) hCG infusion caused a remarkable hypertrophy of rat Leydig cells, coupled with a notable enhancement of their basal and stimulated testosterone production in vitro. It also provoked a conspicuous accumulation of lipid droplets in the cytoplasm of hypertrophic cells. The administration of 4-aminopyrazolo-pyrimidine (4APP), at a dose causing a significant hypocholesterolaemia, did not affect the morphology and function of Leydig cells in normal rats. Conversely, in chronically hCG-infused animals, 4APP reversed lipid-droplet accumulation and significantly depressed stimulated testosterone production. The following conclusions are drawn: 1) chronic hCG treatment enhances the ability of rat Leydig cells to take-up exogenous cholesterol from circulating lipoproteins, and to store it in lipid droplets as a reserve material; 2) lipid-droplet stored cholesterol is employed only in the case of intense acute stimulation, since, under basal conditions, endogenous cholesterol synthesis is able to meet the requirements for testosterone production alone.