Ludwik K. Malendowicz

Effects of pituitary adenylate-cyclase activating peptide (PACAP) on the rat adrenal secretory activity: Preliminary in-vitro studies

PACAP did not affect secretory activity of dispersed rat adrenocortical cells, but it markedly raised aldosterone (ALDO) and corticosterone (B) production by adrenal slices, containing both medullary and cortical tissues. The secretagogue effects of PACAP were suppressed by PACAP(6-38), a specific competitive antagonist. Isoprenaline (IP) enhanced ALDO, but not B secretion of adrenal slices, and l-alprenolol (AL) completely blocked IP effect. AL and corticotropin-inhibiting peptide (CIP) partially reversed ALDO response to a maximal effective concentration of PACAP; AL did not affect B response to a maximal effective concentration of PACAP, while CIP completely annulled it. Quarters of regenerated adrenocortical autotransplants, that are completely deprived of chromaffin cells, though displaying ALDO and B responses to IP and ACTH, were insensitive to PACAP. The hypothesis is advanced that adrenal medulla plays a pivotal role in the mechanism(s) underlying the adrenocortical secretagogue action of PACAP, being mineralocorticoid and glucocorticoid responses probably mediated by the release by chromaffin cells of catecholamine and ACTH or exclusively ACTH, respectively.

Effects of oxytocin on the function and morphology of the rat adrenal cortex: in vitro and in vivo investigations

The effects of oxytocin (OX) on the function and morphology of the rat adrenal cortex were studied in vivo and in vitro. OX exerted a potent stimulatory action on basal, but not 10−8 M ACTH-stimulated corticosterone (B) secretion of dispersed rat inner (zona fasciculata and zona reticularis) adrenocortical cells (maximal effective concentration: 10−9 M); in contrast, at higher concentrations (10−7/10−6 M) OX inhibited maximally ACTH-stimulated B output. A single subcutaneous (s.c.) injection of 1.2 nmol/100 g body weight OX resulted in a longlasting (up to 12 h) rise in plasma B concentration (PBC). The prolonged administration of OX (daily s.c. injections of 0.6 or 1.2 nmol/100 g for 10 days) caused a marked lowering in the adrenal weight and volume of all adrenocortical zones, that in turn was due to a decrease in the number of their parenchymal cells; however, the average volume of inner adrenocortical cells was significantly increased. Basal PBC was lowered, but its response to ether stress was unchanged in comparison with control rats. Prolonged OX treatment did not change B secretion by adrenal slices, but it markedly raised that of dispersed inner adrenocortical cells. Our present findings clearly show that the effects of OX on the adrenal cortex depend on the experimental model employed (in vitro versus in vivo) and the duration of treatment (acute versus chronic). Taken together they allow us to conclude that OX exerts an acute direct stimulatory effect on the rat adrenal cortex, and a chronic inhibitory one, that at least in part could be due to the interference of OX with the mechanism(s) of intracellular transduction of the ACTH secretagogue signal.

Endothelin-1[1–31], acting as an ETA-receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells

Endothelin‐1 (ET‐1)[1–31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET‐1[1–21]. However, at variance with ET‐1[1–21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET‐1[1–31] did not elicit such effect. Both ET‐1[1–21] and ET‐1[1–31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10−8 M. This effect was blocked by the ETA‐receptor antagonist BQ‐123 and unaffected by the ETB‐receptor antagonist BQ‐788. Quantitative autoradiography showed that ET‐1[1–21] displaced both [125I]PD‐151242 binding to ETA receptors and [125I]BQ‐3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET‐1[1–31] displaced only [125I]BQ‐3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin‐23 and the p42/p44 mitogen‐activated protein kinase (MAPK) inhibitor PD‐98059 abolished the proliferogenic effect of ET‐1[1–31], while the protein kinase‐C (PKC) inhibitor calphostin‐C significantly reduced it. ET‐1[1–31] (10−8 M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ‐123. The stimulatory action of ET‐1[1–31] on TK activity was annulled by tyrphostin‐23, while that on MAPK activity was reduced by calphostin‐C and abolished by either tyrphostin‐23 and PD‐98059. These data suggest that ET‐1[1–31] is a selective agonist of the ETA‐receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC‐ and TK‐dependent activation of p42/p44 MAPK cascade.

Cerebellin stimulates the secretory activity of the rat adrenal gland: in vitro and in vivo studies.

Cerebellin is a 16-aminoacid peptide widely distributed in the central nervous system, where it exerts neuromodulatory functions. Cerebellin is contained in human adrenal medulla, and it has been recently demonstrated that cerebellin elicits catecholamine release by human adrenal in vitro. Aim of the present study was to ascertain whether cerebellin affects adrenal function in the rat. Cerebellin concentration-dependently (from 10(-9)to 10(-7)M) increased norepinephrine (but not epinephrine) and cyclic-AMP production by adrenomedullary tissue in vitro. The norepinephrine response to 10(-7)M cerebellin was blocked by the protein kinase (PK) A inhibitor H-89, but not by the phospholipase C inhibitor U-73122 or the PKC inhibitor calphostin-C. Cerebellin did not affect aldosterone and corticosterone secretion of dispersed zona glomerulosa and zona fasciculata-reticularis adrenocortical cells. Cerebellin concentration-dependently (from 10(-8)to 10(-7)M) enhanced norepinephrine release by in situ perfused rat adrenals. Cerebellin (10(-7)M) also elicited a significant rise in aldosterone and corticosterone output, and this effect was annulled by either the beta1-adrenoceptor antagonist l -alprenolol or H-89. Collectively, the present findings allow us to conclude that cerebellin 1) directly stimulates norepinephrine release via the adenylate cyclase/PKA-dependent signaling pathway; and 2) indirectly enhances adrenocortical secretion in vivo, through a paracrine mechanism involving medullary catecholamine release.

Arginine-Vasopressin Stimulates CRH and ACTH Release by Rat Adrenal Medulla, Acting Via the V1 Receptor Subtype and a Protein Kinase C-Dependent Pathway

Arginine-vasopressin (AVP) is a hypothalamic hormone that, like CRH, stimulates the pituitary release of ACTH, thereby activating adrenal glucocorticoid secretion. Evidence indicates that rat adrenal medulla contains a CRH-ACTH system duplicating that existing at the hypothalamo-pituitary level and involved in the paracrine stimulation of the cortex secretion. Therefore, we investigated by RIA the effect of AVP on the release of CRH and ACTH immunoreactivities (IR) by rat adrenal medulla in vitro. AVP concentration-dependently enhanced the release of both CRH-IR and ACTH-IR, and the effect was blocked by a selective antagonist of the V1 subtype of AVP receptors. The CRH receptor antagonist alpha-helical-CRH partially reversed AVP-evoked rise in ACTH-IR release, without altering either CRH response or basal secretions of CRH and ACTH. The specific inhibitors of protein kinase C Ro31-8220 and calphostin C abolished both CRH and ACTH responses to AVP. In conclusion, our present findings suggest that AVP stimulates intramedullary the CRH-ACTH system, acting via V1 receptors and activating protein kinase C.

Endothelins stimulate aldosterone secretion from dispersed rat adrenal zona glomerulosa cells, acting through ETB receptors coupled with the phospholipase C-dependent signaling pathway

Compelling evidence indicates that endothelins (ETs) stimulates aldosterone secretion from rat zona glomerulosa (ZG) cells, acting through the ETB receptor subtype. We have investigated the mechanisms transducing the aldosterone secretagogue signal elicited by the pure activation of ETB receptors. Aldosterone response of dispersed rat ZG cells to the selective ETB-receptor agonist BQ-3020 was not affected by inhibitors of adenylate cyclase/protein kinase (PK)A, tyrosine kinase-, mitogen-activated PK-, cyclooxygenase- and lipoxygenase-dependent pathways. In contrast, the inhibitor of phospholipase C (PLC) U-73122 abrogated, and the inhibitors of PKC, phosphatidylinositol trisphosphate (IP(3))-kinase and calmodulin (calphostin-C, wortmannin and W-7, respectively) partially prevented aldosterone response to BQ-3020. When added together, calphostin-C and wortmannin or W-7 abolished the secretagogue effect of BQ-3020. BQ-3020 elicited a marked increase in the intracellular Ca2+ concentration ([Ca2+]i) in dispersed rat ZG cells, and the effect was abolished by the Ca(2+)-release inhibitor dantrolene. The Ca2+ channel blocker nifedipine affected neither aldosterone nor Ca2+ response to BQ-3020. Collectively, our findings suggest that (1) ETs stimulate aldosterone secretion from rat ZG cells through the activation of PLC-coupled ETB receptors; (2) PLC stimulation leads to the activation of PKC and to the rise in [Ca2+]i with the ensuing activation of calmodulin; and (3) the increase in [Ca2+] is exclusively dependent on the stimulation of IP(3)-dependent Ca2+ release from intracellular stores.

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.

Effects of neuromedin U-8 on the secretory activity of the rat adrenal cortex: evidence for an indirect action requiring the presence of the zona medullaris

The acute effect of increasing concentrations (from 10−8 to 10−6M) of neuromedin U-8 (NMU-8) on steroid secretion of rat adrenal gland was investigated in vitro by high-pressure liquid chromatography. The production of the following steroids was measured: pregnenolone (PREG), progesterone (PROG), 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18OH-DOC), 18-hydroxycorticosterone (18OH-B) and aldosterone (ALDO). NMU-8 had no effects on either dispersed adrenocortical cells or fragments of adrenocortical autotransplants lacking medullary chromaffin cells. Conversely, NMU-8 exerted concentration-dependent secretagogue effects on adrenal slices, including both cortex and medulla. At all concentrations tested, NMU-8 increased the production of both PREG and total post-PREG steroids. The increase in total post-PREG steroid output induced by low concentrations of NMU-8 (10−8M) was due to similar rises in the production of non-18-hydroxylated steroids (PROG, DOC and B) and 18-hydroxylated hormones (18OH-DOC, 18OH-B and ALDO); conversely, that provoked by higher concentrations of the neuropeptide (10−7 to 10−6M) was almost exclusively caused by the rise in the yield of 18-hydroxylated steroids. The stimulating effect of NMU-8 on PREG output was blocked by both α-helical-CRH and corticotropin-inhibiting peptide, which are competitive inhibitors of CRH and ACTH, respectively. The following conclusions have been drawn: (1) NMU-8 affects adrenal steroid secretion indirectly by acting on the medullary chromaffin cells, which in turn may paracrinally stimulate the cortical ones; (2) at all concentrations tested, NMU-8, by stimulating the intramedullary CRH/ACTH system, causes a net rise in the activity of the early ratelimiting step of steroidogenesis, with the consequent increase in the output of the entire spectrum of post-PREG steroids; and (3) at higher concentrations (over 10−8M), NMU-8 also elicits the release from chromaffin cells of a factor (not yet known) that specifically enhances 18-hydroxylase activity.

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.

Effects of neuromedin-N on the pituitary-adrenocortical axis of dexamethasone-suppressed rats

Neuromedin-N (NMN) (6 micrograms/100 g body weight for 2 d) partially reversed the dexamethasone (Dx)-induced inhibition of ACTH release and the consequent adrenal atrophy and decrease in glucocorticoid (corticosterone) plasma concentration in rats. Dx administration did not alter the level of circulating mineralocorticoid (aldosterone), but NMN (2 or 6 micrograms/100 g body weight for 2 d) significantly increased it. These findings suggest that the mechanism underlying the glucocorticoid (but not the mineralocorticoid) secretagogue action of NMN involves the stimulation of hypophyseal ACTH release. The hypothesis is advanced that the potent mineralocorticoid secretagogue effect of NMN may be mediated either by a direct action on zona glomerulosa cells or by the enhanced release of other regulatory peptides exerting aldosterone stimulating effect.