Neuromedin U enhances proliferation of ACTH-stimulated adrenocortical cells in the rat.

Neuromedin U (NMU) is a brain-gut peptide involved in the regulation of the hypothalamic-pituitary-adrenal axis and adrenocortical cell proliferation. In this study, we investigated the effects of NMU8 (three subcutaneous injections of 6.0 nmol/100 g, 24, 16 and 8 h before autopsy) on the adrenal glands of rats treated for 2 or 4 days with a low (2 microg/100 g body weight/24 h) or a high (8 microg) dose of adrenocorticotropic hormone (ACTH). As revealed by RT-PCR, ACTH treatment did not prevent expression of NMUR1 in rat adrenal cortex. At day 4 of ACTH administration, the weight of adrenals was lower than at day 2. NMU8 administration prevented ACTH-induced increases of adrenal weight at day 2 of the experiment. ACTH plasma concentrations were increased in all ACTH-administered rats. NMU8 administration increased ACTH plasma concentration at day 2 of the lower ACTH dose-treated group while it reduced the ACTH plasma level at day 4 in the higher ACTH dose-administered rats. In all groups of ACTH-treated rats, NMU8 changed neither aldosterone nor corticosterone plasma concentrations. In the zona glomerulosa (ZG), NMU8 increased metaphase index at days 2 and 4 in the lower ACTH dose-treated group and had no statistically significant effect in rats treated with the higher ACTH dose. In the zona fasciculata (ZF), NMU8 administration increased metaphase index at day 2 in the lower ACTH dose-treated group but reduced metaphase index at day 4 in the higher dose ACTH-administered rats. NMU8 reduced the number of cells per unit area both in ZG and ZF at day 2 in the higher ACTH dose-treated rats. In the remaining groups NMU8 did not produce statistically significant changes in the number of cells per unit area. Thus, our findings demonstrate that exogenous NMU may stimulate proliferation primarily of the cortical ZG cells in rats administered with ACTH, although at high doses of exogenous corticotropin an opposite effect occurred.

Neuromedin-U stimulates enucleation-induced adrenocortical regeneration in the rat.

Neuromedin-U (NMU) is a brain-gut peptide, which has been previously found to stimulate hypothalamic-pituitary-adrenal axis in the rat. Enucleation-induced adrenal regeneration in rats with contralateral adrenalectomy is a well-established model of adrenal growth, that not only depends on the compensatory ACTH hypersecretion, but is also modulated by several regulatory peptides. Hence, we investigated whether NMU may be included in this group of bioactive molecules. Reverse transcription-polymerase chain reaction and immunocytochemistry showed that regenerating rat adrenocortical cells at days 5 and 8 after surgery express the NMU receptor NMUR1 as mRNA and protein. NMU8 administration to rats bearing regenerating adrenals markedly raised the plasma concentration of corticosterone and notably enhanced proliferative activity of adrenocortical cells. ACTH blood level was unchanged at day 5 and significantly decreased at day 8. The conclusion is drawn that NMU stimulates regeneration of rat adrenal cortex, via a mechanism independent of pituitary ACTH and involving the activation of NMUR1 located on adrenocortical cells.

Effects of proadrenomedullin N-terminal 20 peptide on the rat pituitary-adrenocortical axis under basal and stressful conditions.

Proadrenomedullin N-terminal 20 peptide (PAMP) derives, along with adrenomedullin (AM), from prepro-AM. AM has been reported to modulate the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and this study aimed at ascertaining whether PAMP exerts similar effects. PAMP was subcutaneously administered to non-stressed and stressed rats, and the plasma concentrations of ACTH and corticosterone were measured by radioimmune assay. In non-stressed rats, PAMP raised ACTH and corticosterone blood levels at 60 min, and ACTH plasma concentration at 120 min. Ether and cold stresses increased the plasma levels of both ACTH and corticosterone, and PAMP dampened HPA axis response to cold stress, without affecting that to ether stress. The conclusion is drawn that PAMP i) stimulates rat HPA axis, through a mechanism similar to that of ether stress; and ii) interferes with the neural pathways involved in the cold stress-induced activation of HPA axis.

Effects of some endocrine disruptors on the secretory and proliferative activity of the regenerating rat adrenal cortex.

The effects of some endocrine disruptors that possess estrogen-like activity on the secretion and growth of regenerating rat adrenal cortex have been investigated in ovariectomized (OVX) and sham-OVX rats. As reference groups, dexamethasone (Dx)-administered sham-OVX and 17beta-estradiol-administered OVX animals were used. Dx, estradiol and endocrine disruptors were subcutaneously injected daily at a dose of 3 nmoles/100 g for 10 consecutive days after surgery, and adrenal enucleation was performed on day 5 of the experiment. Dx and genistein significantly decreased corticosterone plasma concentration (as measured by RIA) in sham-OVX rats with regenerating adrenals, while other disruptors (eusolex, procymidone, linurone, resveratrol, bisphenol-A and and silymarin) were ineffective. Mitotic index (as assayed by the stachmokinetic method with vincristine) was not changed by either Dx or disruptors. Estradiol significantly increased and genistein significantly lowered corticosterone blood level in OVX rats; similar effects were induced in the mitotic index of regenerating adrenals, but the changes were not significant. Eusolex increased the mitotic index, without altering the level of circulating corticosterone. Collectively, our findings allow us to conclude that, of the endocrine disruptors tested, only genistein is able to suppress the secretory activity of regenerating adrenal cortex, this Dx-like effect being apparently unrelated to its estrogen-like activity, and only eusolex enhances the proliferation rate of regenerating adrenal, the effect being conceivably connected with its estrogen-like activity.

Accumulation of steroidogenic acute regulatory protein mRNA, and decrease in the secretory and proliferative activity of rat adrenocortical cells in the presence of proteasome inhibitors.

Sporadic findings indicate that proteolysis may affect steroid secretion in rat ovary granulosa cells. We examined the effects of the proteasome inhibitors MG115 and MG101 on the in vitro secretion and growth of rat adrenocortical cells. MG115 and/or MG101 decreased within 120 min the aldosterone and corticosterone secretion from freshly dispersed zona glomerulosa and zona fasciculata-reticularis (ZF/R) cells. After a 24-h incubation MG115 alone or with MG101 lowered corticosterone production and enhanced proliferation rate of cultured ZF/R cells, while MG101 was per se ineffective. Real-time polymerase chain reaction demonstrated that MG101 decreased steroidogenic acute regulatory protein (StAR) mRNA in cultured cells. MG115 was per se ineffective, but when added together with MG101 evoked a marked rise in StAR mRNA content. In light of the present findings, we conclude that i) protein breakdown by proteasomes is required for the maintenance of a normal secretory and proliferative activity of freshly dispersed or cultured rat adrenocortical cells; and ii) in long-term experiments, great caution must be taken in correlating StAR mRNA content and steroidogenic capacity.

Acute in vivo effects of leptin and leptin fragments on corticosteroid hormone secretion and entero-insular axis in the rat.

Leptin is a 147-amino acid adipose tissue-secreted hormone, which acts via several subtypes of receptors, the main and better known variants of which are named Ob-Ra and Ob-Rb. Structure-activity relationship studies pointed out the importance of the N-terminal and C-terminal amino-acid sequences 22-115 and 116-166, respectively, for the biological and receptor binding activities of leptin. Evidence has been provided that leptin affects corticosteroid-hormone and insulin secretion, and therefore we have investigated in the rat the expression of leptin receptor expression in adrenal cortex and pancreatic islets, as well as the effects of the acute treatment with leptin and leptin fragments 150-167, 138-167, 93-105, 22-56 and 26-39 on the plasma concentrations of aldosterone, corticosterone, insulin and glucagon. Reverse transcription-polymerase chain reaction showed the expression of both Ob-Ra and Ob-Rb mRNAs in adrenal cortex and pancreatic islets, the Ob-Rb expression in pancreas being 2-fold higher than in adrenals. Radioimmuno assay demonstrated that leptin enhanced plasma aldosterone and corticosterone concentrations, decreased plasma insulin concentration, and did not significantly affect glucagon plasma concentration. All leptin fragments tested exerted a corticosteroid-hormone secretagogue action, while only leptin fragments 116-130, 138-167 and 93-105 elicited a sizeable insulin antisecretagogue effect. Taken together these findings suggest that: i) the in vivo acute stimulating effect of leptin on adrenocortical hormone secretion is not connected to specific sequences of its molecule, while the insulinostatic effect is probably mediated by the sequence 93-105; and ii) the secretagogue and antisecretagogue effect of leptin are prevalently mediated by Ob-Ra and Ob-Rb, respectively.

Effects of arginine vasopressin (AVP) on the pituitary-thyroid axis in the rat: Evidence that endogenous AVP, acting via V1 receptors, lowers TSH blood concentration.

We have investigated the effects of the prolonged administration (2 or 8 days) of arginine-vasopressin (AVP), alone or with antagonists of its V1 and V2 receptors (V1-Ra and V2-Ra), on the rat pituitary-thyroid axis. In the 8-day, but not 2-day experiments, AVP raised thyroid weight, and the effect was prevented by V1-Ra. Morphometry showed that the AVP-induced increase in thyroid weight was mainly due to a rise in the stroma volume. In the 2-day, but not 8-day experiments, AVP lowered TSH plasma concentration, and the effect was annulled by V1-Ra. V1-Ra was ineffective per se, while V2-Ra lowered TSH blood level. AVP administration increased the level of circulating thyroid hormones, especially thyroxine (free and total T4). In the 2-day, but not 8-day experiments, this effect of AVP was blocked by both V1-Ra and V2-Ra. When administered alone V1-Ra and V2-Ra induced a small, but significant rise in T4 plasma concentration in both the 2-day and 8-day experiments. Collectively, these findings indicate that: i) AVP administration exerts a transient inhibitory effect on TSH secretion, and a more prolonged stimulatory action on thyroid secretion and growth, both of which are mainly mediated by the V1-R subtype; and ii) endogenous AVP exerts a clearcut tonic inhibitory effect on pituitary TSH release and doubtful regulatory actions on the thyroid gland.

Effects of leptin and leptin fragments on steroid secretion of freshly dispersed rat adrenocortical cells.

The biological actions of leptin on target tissues are mediated via several isoforms of receptors (Ob-Rs), which may differently interact with native leptin and its fragments. Based on the presence in the rat adrenals of at least two Ob-R isoforms and the conflicting findings on the effect of leptin on adrenocortical secretion, we investigated the effects of the native leptin and several leptin fragments (10(-8) and 10(-6)M) on aldosterone and corticosterone secretion from freshly dispersed rat zona glomerulosa (ZG) and zona fasciculata-reticularis (ZF/R) cells. Reverse transcription (RT)-polymerase chain reaction (PCR) showed the expression of Ob-Ra and Ob-Rb mRNAs in both ZG and ZF/R cells. Native murine leptin (1-147) enhanced aldosterone and corticosterone secretion from dispersed ZG and ZF/R cells, and similar effects were elicited by murine leptin fragment 116-130, and human leptin fragments 138-167, 150-167 and [Tyr] 26-39. Human leptin fragment 93-105 was ineffective, while fragment 22-56 decreased corticosterone output without affecting aldosterone secretion. Taken together, our findings indicate that in rat adrenocortical cells leptin and leptin fragments may differently interact with Ob-Rs or interact with different Ob-R isoforms. Moreover, they suggest that (1) the direct adrenocortical secretagogue effect of leptin mainly depends on the C-terminal sequence 116-166; and (2) the N-terminal sequence is not needed for leptin to activate Ob-Rs positively coupled to steroidogenesis, but is possibly responsible for a direct inhibitory effect on glucocorticoid secretion.

Effects of prolonged cholecystokinin administration on rat pituitary-adrenocortical axis: role of the CCK receptor subtypes 1 and 2.

Many lines of evidence indicate that cholecystokinin (CCK) and its receptors, named CCK1-R and CCK2-R, are expressed in the hypothalamo-pituitary-adrenal (HPA) axis, the function of which they acutely stimulate. However, the role of endogenous CCK system in the regulation of HPA axis is still unknown. To address this issue we investigated the effect of the prolonged (6-day) administration of CCK, CCK-R antagonists (CCK-RAs) and pentagastrin (PG), a CCK2-R agonist, on adult rat HPA axis. Semiquantitative reverse transcription-polymerase chain reaction showed that CCK treatment lowered the expression of CCK1-R and CCK2-R mRNAs in the pituitary, but not adrenal gland. ACTH plasma concentration was not affected by any treatment. Neither CCK nor PG administration induced significant changes in the blood levels of aldosterone and corticosterone. CCK1-RA, although being per se ineffective, in the presence of CCK raised plasma levels of aldosterone and corticosterone; conversely, CCK2-RA, either alone or in the presence of agonists, lowered the blood concentrations of the two hormones. CCK, but not PG, treatment decreased relative adrenal weight, and morphometry showed that CCK-induced adrenal atrophy was coupled to decreases in the volume of adrenocortical zones, which in turn mainly depended on the lowering in the volume and number of adrenocortical cells. PG administration raised and CCK2-RA per se decreased the volume and number of adrenocortical cells. Taken together, these findings allow us to draw the following main conclusions: i) the prolonged exposure to elevated CCK concentrations down-regulates CCK-R expression in the pituitary gland, which accounts for the lack of effect of CCK on ACTH secretion; ii) adrenal CCK1-Rs and CCK2-Rs inhibit and stimulate, respectively, corticosteroid secretion; and iii) endogenous CCK system plays a minor role in the physiological regulation of rat HPA axis, its main effect being the CCK2-R-mediated maintenance of adrenocortical-cell number.