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.

Expression of neuromedins S and U and their receptors in the hypothalamus and endocrine glands of the rat.

Neuromedin S (NMS) and neuromedin U (NMU) are regulatory peptides that share the C-terminal amino-acid sequence and act via common G protein-coupled receptors called NMUR1 and NMUR2. Semiquantitative real time-PCR showed that in the rat hypothalamus and testis NMS gene expression was markedly higher than that of the NMU gene, while the reverse occurred in the anterior pituitary and thyroid gland. Low expression of both genes was detected in the thymus, adrenal gland and ovary, whereas in the pancreatic islets only the expression of NMU mRNA was detected. In the rat hypothalamus the expression of the NMUR2 gene was strikingly higher than that of the NMUR1 gene; in contrast, in the testis and ovary the very low expression of NMUR2 contrasted with the relatively high expression of the NMUR1 gene. In the other glands examined only expression of the NMUR1 gene was found. The marked differences in the level of expression of NMU, NMS and their receptors in the hypothalamus and endocrine glands of the rat suggest that in this species such neuromedins may play different roles in the functional regulation of neuroendocrine axes.

IGF-I enhances cortisol secretion from guinea-pig adrenal gland: in vivo and in vitro study.

Insulin-like growth factor (IGF)-I is a ubiquitously synthesized peptide that, along with IGF-II, acts via the IGF-R type I receptor. IGF-I and its receptor are expressed in the adrenal gland of humans and bovines, the secretion of which they seem to stimulate. As in humans and cows, the main glucocorticoid hormone secreted by guinea-pig adrenals is cortisol, and hence we have studied the adrenocortical effects of IGF-I in this species. In vivo experiments showed that prolonged IGF-I administration raised the plasma concentration of cortisol in both normal and dexamethasone/captopril-treated guinea pigs, thereby ruling out the possibility that IGF-I may act by activating the hypothalamic-pituitary-adrenal axis and the renin-angiotensin system. In vitro experiments demonstrated that IGF-I enhanced basal, but not maximally agonist [ACTH and angiotensin-II (Ang-II)]-stimulated, cortisol secretion from freshly dispersed guinea-pig inner adrenocortical cells. The IGF-I immuno-neutralization suppressed the IGF-I secretagogue effect, without altering the cortisol response to both ACTH and Ang-II. IGF-I raised cyclic-AMP and inositol triphosphate release from dispersed guinea-pig cells, and the effect was reversed by the adenylate cyclase inhibitor SQ-22536 and the phospholipase-C (PLC) inhibitor U-73122. SQ-22536, U-73122, the protein kinase (PK) A inhibitor H-89 and the PKC inhibitor calphostin-C decreased by approximately 50% the cortisol response of dispersed cells to IGF-I, and the combined exposure to SQ-22536 and U-73122 abolished it. We conclude that IGF-I stimulates glucocorticoid secretion from guinea-pig adrenocortical cells, acting via selective receptors coupled to both the adenylate cyclase/PKA- and PLC/PKC-dependent signaling cascades.

On the role of receptor-receptor interactions and volume transmission in learning and memory.

Learning and memory seem to be inherent to a biological neural network. To emerge, they need an extensive functional connectivity, enabling a large repertoire of possible responses to stimuli, and sensitivity of the connectivity to activity, allowing for the selection of adaptive responses. According to the classical view about the organization of the CNS, the connectivity issue is realized by the huge amount of synaptic contacts each neuron establishes, while the adaptation of the network to specific tasks is obtained by mechanisms of activity-dependent synaptic plasticity. The discovery of direct receptor-receptor interactions at the level of the plasma membrane and the existence in the brain of two main modes of communication, the wiring transmission (such as the synaptic transmission) and the volume transmission (based on the diffusion of signals in the extracellular space), provided a broader view of the functional organization of the CNS with potential important consequences on the understanding of learning and memory processes. Owing to receptor-receptor interactions clusters of receptors, the receptor mosaics (RM), can be formed at the plasma membrane where they can work as collective functional units. As a consequence, the connections between the cells become themselves networks (molecular networks) able to adapt their function according to the stimuli they receive. Learning, therefore, can occur also at the level of RMs. Thus, memory formation seems not only to be a distributed process, but also to follow a hierarchical morpho-functional organization. Furthermore, the combination of the two different forms of transmission could allow processes of correlation and coordination to be established between networks and network elements without the need of additional physical connections, leading to a significant increase of the degrees of freedom available to the CNS for learning.

Galanin in the regulation of the hypothalamic-pituitary-adrenal axis (Review).

Galanin is a regulatory 30- or 29-amino acid peptide, widely distributed in the nervous system and gut, that acts via three subtypes of G protein-coupled receptors, named GAL-R1, GAL-R2 and GAL-R3. Findings have been accumulated that galanin regulates neuroendocrine hypothalamic axes, including the hypothalamic-pituitary-adrenal (HPA) one. Galanin and its receptors are expressed in the hypothalamic paraventricular and supraoptic nuclei, anterior pituitary and adrenal medulla. Adrenal cortex does not express galanin, but is provided with GAL-R1 and GAL-R2. The bulk of evidence indicates that galanin stimulates the activity of the central branch of the HPA axis (i.e. the release of corticotropin-releasing hormone and ACTH), thereby enhancing glucocorticoid secretion from the adrenal cortex. Investigations carried out in the rat show that galanin is also able to directly stimulate corticosterone (glucocorticoid) secretion from adrenocortical cells, through GAL-R1 and GAL-R2 coupled to the adenylate cyclase-protein kinase A signaling cascade, and nor-epinephrine release from adrenal medulla. There is indication that galanin may also enhance corticosterone release via an indirect paracrine mechanism involving the local release of catecholamines, which in turn activate beta-adrenoceptors located on adrenocortical cells. The physiological relevance in the rat of the glucocorticoid secretagogue action of galanin is suggested by the demonstration that the blockade of galanin system significantly lowers basal corticosterone secretion. There is also evidence that galanin plays a role in the modulation of HPA-axis response to stress, as well as in the pathogenesis of pituitary adenomas and perhaps of pheochromocytomas.

Evidence for a paracrine role of endogenous adrenomedullary galanin in the regulation of glucocorticoid secretion in the rat adrenal gland.

Previous investigations have shown that rat adrenocortical cells are provided with galanin receptors, and galanin stimulates glucocorticoid secretion from dispersed cells. The present study aimed to clarify the possible role of galanin in the physiological regulation of rat adrenal secretory activity. Reverse transcription-polymerase chain reaction detected galanin mRNA expression in the adrenal medulla, but not in the cortex. Sizeable concentrations of galanin-immunoreactivity were measured by radioimmune assay only in the adrenomedullary tissue. Galanin raised norepinephrine, but not epinephrine, release from adrenomedullary tissue. Galanin immunoneutralization (obtained with concentrations of anti-galanin antibody able to block the galanin glucocorticoid secretagogue effect on dispersed adrenocortical cells) decreased basal corticosterone production from adrenal slices containing adrenomedullary tissue, without affecting that from dispersed adrenocortical cells. The beta-adrenoceptor antagonist l-alprenolol partially prevented galanin-stimulated corticosterone secretion from adrenal slices, without per se altering basal secretion. Taken together, our findings allow us to conclude that endogenous galanin, produced in adrenal medulla, is involved in the regulation of adrenocortical glucocorticoid secretion acting via a two-fold paracrine mechanism: i) direct activation of adrenocortical galanin receptors; and ii) stimulation of adrenomedullary release of catecholamines, which in turn activate beta-adrenoceptors located on adrenocortical cells.

Ouabain chronic infusion enhances the growth and steroidogenic capacity of rat adrenal zona glomerulosa: the possible involvement of the endothelin system.

Ouabain, an inhibitor of the Na+/K+-ATPase, has been reported to affect the secretory activity of the adrenal cortex, and especially of zona glomerulosa (ZG). However, conflicting results were obtained, depending on the experimental condition used since ouabain appears to interact with angiotensin-II (Ang-II) and its action to be influenced by the electrolyte balance. Hence, we investigated the effects of prolonged (4-month) infusion with ouabain on the rat adrenal cortex. Ouabain raised the plasma concentrations of aldosterone, corticosterone and endothelin-1 (ET-1), without affecting either systolic blood pressure (SBP) or plasma renin activity (PRA). The treatment caused a marked hypertrophy of ZG and ZG cells, which mainly ensued from increases in the volume of the mitochondrial and smooth-endoplasmic-reticulum compartments, where the enzymes of steroid synthesis are located. Conversely, the volume of the lipid-droplet compartment, which stores cholesterol utilized in steroid-hormone production, underwent a striking decrease. Zona fasciculata and its parenchymal cells were not affected. Basal and maximally agonist (ACTH, Ang-II and ET-1)-stimulated in vitro mineralocorticoid secretion from adrenal slices was also notably enhanced by ouabain administration. Collectively, these findings indicate that prolonged treatment with ouabain selectively stimulates the growth and steroidogenic capacity of the rat adrenal ZG. The possibility that the activation of the renin-angiotensin system may be involved in this effect of ouabain is ruled out by the lack of significant changes in SBP and PRA. Instead, our results suggest the possible involvement of ET-1, the plasma level of which is elevated in ouabain-infused rats.

In vitro culture on Matrigel favors the long-term maintenance of rat zona glomerulosa-cell differentiated phenotype.

Zona glomerulosa (ZG) cells cultured on plastic within few days dedifferentiate losing their capacity to secrete aldosterone (ALDO) in appreciable amounts. Evidence indicates that extracellular matrix modulates the secretory behavior of adrenocortical cells cultured in vitro. Hence, we compared the morphology and function of rat ZG cells grown on plastic and Matrigel basement membrane matrix (hereinafter Matrigel) for up to 12 days. At day 3, no significant differences were observed between cells cultured on plastic and Matrigel. Starting from day 6, ZG cells cultured on plastic lost their ultrastructural differentiated features (mitochondria with tubular cristae, smooth endoplasmic reticulum cisternae and lipid droplets), exhibiting a fibroblast-like appearance. The mRNA expression of the main steroidogenic enzymes, as evaluated by real-time polymerase chain reaction, the baseline secretion of ALDO and other post-pregnenolone hormones, as evaluated by high pressure liquid chromatography, and the secretory response to ACTH, angiotensin-II and K(+), as evaluated by radioimmunoassay, displayed a time-dependent decrease. Matrigel was found to maintain unchanged both the ultrastructure and the expresion of steroidogenic enzymes of ZG cells until day 12 of culture. Baseline and agonist-stimulated steroid-hormone secretion decreased with the duration of culture on Matrigel, but was always higher than that of ZG cells grown on plastic. Hence, our study clearly indicates that the culture on Matrigel favors the maintenance of rat ZG-cell differentiated phenotype, allowing the conclusion that this technique is suitable for long-term in vitro investigations.

Atrial natriuretic peptide enhances cortisol secretion from guinea-pig adrenal gland: evidence for an indirect paracrine mechanism probably involving the local release of medullary catecholamines.

Atrial natriuretic peptide (ANP) is a regulatory hormone widely expressed, along with its receptors, in organs and body tissues. ANP is well known to inhibit aldosterone secretion from mammalian adrenals, but its effect on glucocorticoid-hormone production is controversial. In vivo experiments showed that prolonged ANP administration raised the plasma concentration of cortisol in both normal and dexamethasone/captopril-treated guinea pigs (i.e. in animals with pharmacologically interrupted hypothalamic-pituitary-adrenal axis and renin-angiotensin system). ANP did not affect cortisol secretion from dispersed guinea pig zona fasciculata-reticularis cells, but enhanced catecholamine release from adrenomedullary cells. ANP stimulated cortisol output from guinea pig adrenal slices containing medullary chromaffin tissue, and the beta-adrenoceptor antagonist l-alprenolol blocked this effect. The conclusion is drawn that ANP, when the structural integrity of the adrenal gland is preserved, is able to enhance glucocorticoid secretion in guinea pigs, through an indirect mechanism involving the rise in the catecholamine release, which in turn, acting in a paracrine manner, stimulate secretion of inner adrenocortical cells.

Neuropeptides B and W enhance the growth of human adrenocortical carcinoma-derived NCI-H295 cells by exerting MAPK p42/p44-mediated proliferogenic and antiapoptotic effects.

Neuropeptides B and W (NPB and NPW) are endogenous ligands of two G protein-coupled receptors, named GPR7 and GPR8. GPR7 and GPR8 are expressed in the adrenal cortex, and there is evidence that NPB and NPW stimulate glucocorticoid secretion from human adrenocortical cells by activating protein kinase (PK) A and PKC signaling. To gain insight into the role of NPB and NPW in human adrenal functional regulation, we have investigated their effects on the secretion and growth of the human adrenocortical carcinoma-derived NCI-H295 cell line. NCI-H295 cells were found to express both GPR7 and GPR8 mRNAs, but neither NPB nor NPW (up to 10(-6) M) affected their secretory activity. In contrast, both peptides (from 10(-10) to 10(-6) M) enhanced the growth of NCI-H295 cells, by raising their proliferative activity and lowering their apoptotic deletion rate. NPB and NPW (10(-6) M) stimulated tyrosine kinase (TK) and mitogen-activated PK (MAPK) p42/p44 activities in NCI-H295 cells. Both these effects were blocked by the TK inhibitor tyrphostin-23, while the MAPK p42/p44 inhibitor PD-98059 annulled only MAPK p42/p44 activation. The growth-stimulating effect of 10(-6) M NPB and NPW were not affected by either the PKA and PKC inhibitors H-89 and calphostin-C or the MAPK p38 antagonist SB-293580, but were abolished by both tyrphostin-23 and PD-98059. Taken together, our findings allow us to conclude that GPR7 and GPR8 expressed in NCI-H295 cells: i) are, at variance with those present in normal human adrenocortical cells, uncoupled to PKA- and PKC-dependent cascades, thereby explaining the absence of any secretory response to NPB and NPW; and ii) are coupled to the TK-dependent MAPK p42/p44 signaling, whose activation mediates the proliferogenic and antiapoptotic effect of NPB and NPW.

Orexins modulate the growth of cultured rat adrenocortical cells, acting through type 1 and type 2 receptors coupled to the MAPK p42/p44- and p38-dependent cascades.

Orexin A and B are hypothalamic peptides that act through two subtypes of receptors named OX1-R and OX2-R. The OX1-R almost exclusively binds orexin-A, whereas OX2-R is non-selective for both orexins. We previously found that rat adrenocortical cells express both orexin-receptor subtypes, and orexin-A stimulates corticosterone secretion from dispersed adrenocortical cells acting via the OX1-R. Here, we examined the possibility that orexins, acting through both their receptor subtypes, modulate the growth of adrenocortical cells. Reverse transcription-polymerase chain reaction showed that rat adrenocortical cells cultured in vitro for four days expressed OX1-R and OX2-R mRNAs. Orexin-A increased the proliferation rate (PR) of cultured cells, while orexin-B lowered it. Using selective antibodies, we demonstrated that OX1-R immuno-blockade reversed the proliferogenic action of orexin-A, causing a sizeable decrease in PR. In contrast, OX2-R immuno-blockade magnified the proliferogenic effect of orexin-A and annulled the antiproliferogenic action of orexin-B. The proliferogenic effect of orexin-A in the presence of OX2-R immuno-blockade was abrogated by the MAPK p42/p44 inhibitor PD-98059, while the antiproliferogenic effect of orexin-A in the presence of OX1-R immuno-blockade was annulled by the MAPK p38 inhibitor SB-203580. Neither inhibitor altered per se the basal PR of cultured cells. Taken together, our present findings allow us to conclude that i) orexins modulate the growth of rat adrenocortical cells cultured in vitro, by exerting both proliferogenic and antiproliferogenic effects, which are mediated by OX1-Rs and OX2-Rs, respectively; and ii) OX1-R and OX2-R growth effects involve the activation of the MAPK p42/p44 and p38 signaling cascades, respectively.

Endothelin-1 and adrenomedullin enhance the growth of human adrenocortical carcinoma-derived SW-13 cell line by stimulating proliferation and inhibiting apoptosis.

The human adrenocortical carcinoma-derived SW-13 cell line is currently used to study the interrelationships occurring between cytokines and growth factors and endothelins (ET) and adrenomedullin (AM). SW-13 cells express either ET-1 and AM or growth factors, and several cytokines stimulate ET-1 and AM release from SW-13 cells. However, neither the morphology and steroid-hormone secretion of SW-13 cells nor the expression of ET and AM receptors and the effects of ET and AM on SW-13 cell growth have been investigated. Electron microscopy showed that SW-13 cells were deprived of the typical organelles involved in steroid-hormone synthesis (i.e. mitochondrial with tubular cristae, smooth endoplasmic reticulum and lipid droplets), their prominent ultrastructural features being rough endoplasmic reticulum cisternae, free ribosomes and mitochondria with laminar cristae. Accordingly, steroid-hormone secretion was very low: no cortisol was produced and only very small amounts of aldosterone and its precursors were released. No appreciable secretory response to physiological concentrations of ACTH was observed. Reverse transcription-polymerase chain reaction showed the expression of pro ET-1 and proAM genes, as well as detected the mRNAs of only the ET- and AM-receptor subtypes, which are currently thought to mediate the growth-promoting action of these peptides: i.e. the ETA and AM2 receptors. In keeping with these observations, both ET-1 and AM markedly stimulated the growth of SW-13 cells, by enhancing the proliferation and lowering the apoptosis rate. Taken together, our findings allow us to conclude that SW-13 cannot be used for investigating the mechanisms involved in the regulation of steroid-hormone secretion, but are a suitable and useful model to study the role of endogenous ET and AM systems in the autocrine-paracrine control of human adrenocortical-cell growth.

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.

Up-regulation of the adrenomedullin system mediates hypotension and hypoaldosteronism induced by simulated microgravity.

We recently demonstrated that prolonged simulated microgravity (SMG) induced hypotension and hypoaldosteronism in rats, and gathered preliminary evidence for an involvement of circulating adrenomedullin (AM). Thus, we aimed to investigate whether short-term SMG elicits the same effects, and whether up-regulation of adrenal AM system plays a relevant role. Rats were exposed for 8 days to SMG in the form of hindlimb unweighting, and then, along with control animals, were given an intraperitoneal injection of AM22-52 and/or angiotensin-II (Ang-II) (100 nmoles/kg) or the saline vehicle. Systolic blood pressure (SBP) was measured by tail-cuff sphygmomanometry. The adrenal expression of AM was assayed by semiquantitative RT-PCR. The plasma concentrations of aldosterone (PAC) and AM, and adrenal AM content were measured by RIA. Short-term SMG induced significant decreases in SBP and PAC. Conversely, both the plasma and adrenal levels of AM, and adrenal AM mRNA were enhanced in SMG-exposed animals. The SMG-induced hypotension and hypoaldosteronism were reversed by AM22-52, an AM-receptor antagonist, thereby demonstrating a causal link between these effects and the up-regulation of AM system. SMG hampered SBP and PAC responses to Ang-II; the co-administration of AM22-52 restored these responses. These findings accord well with the known ability of AM to counteract the effects of Ang-II on both blood vessels and adrenocortical cells. Taken together, our findings allow us to conclude that up-regulation of the adrenal AM system i) occurs early and takes part in the adaptative changes occurring during SMG conditions; and ii) may account for both hypotension and hypoaldosteronism on returning to the normogravitational environment.

Expression of the beacon gene in the rat adrenal gland: direct inhibitory effect of beacon[47-73] on aldosterone secretion from dispersed adrenal zona glomerulosa cells.

Beacon gene was recently identified in the rat hypothalamus, and there is evidence that beacon may be involved in the functional regulation of neuroendocrine axes. Reverse transcription-polymerase chain reaction and immunocytochemistry showed the expression of beacon mRNA and protein in the rat adrenal gland, especially in the cortex. Beacon[47-73], at a concentration over 10(-7) M decreased basal aldosterone secretion from dispersed rat zona glomerulosa (ZG) cells, without affecting the ACTH-stimulated one. Basal and agonist-stimulated corticosterone secretion from dispersed zona fasciculata-reticularis cells and catecholamine release from adrenomedullary slices were unaffected by beacon[47-73]. The suppressive effect of beacon[47-73] on aldosterone secretion from ZG cells was abolished by either H-89 or calphostin-C, which are inhibitors of protein kinase A and C signaling cascades. Taken together, these findings allow us to suggest that beacon can be included in the group of regulatory peptides involved in the fine tuning of ZG secretory activity.

Effects of leptin and leptin fragments on steroid secretion and proliferative activity of regenerating rat adrenal cortex.

Leptin, an adipose tissue-secreted hormone, acts via several isoforms of specific receptors (Ob-Rs), which may variously interact with the native leptin molecule and its fragments. Evidence has been provided that leptin affects rat adrenal functions, but the results were rather conflicting depending on the experimental condition examined (e.g. regenerating vs. mature or immature adrenal gland). Hence, we investigated the effects of three subcutaneous injections of murine leptin(1-147) and several leptin fragments (3 nmol/100 g body weight; 28, 16 and 4 h before the sacrifice) on the secretory activity and growth of regenerating rat adrenal cortex. The following leptin fragments were tested: murine leptin(116-130), and human leptin fragments 150-167, 138-167, 93-105, 22-56 and [Tyr]26-39. Leptin(1-147) enhanced plasma concentration of both aldosterone and corticosterone. The blood level of aldosterone was raised by leptin(116-130), leptin(138-167) and leptin(93-105), and that of corticosterone by leptin(93-105) and Tyr-leptin(26-39). Metaphase index (stachmokinetic method with vincristine) was unaffected by leptin(1-147), and lowered by leptin(116-130), leptin(150-167) and leptin(138-167). Collectively, our findings allow us to conclude that leptin and leptin fragments enhance the secretory activity and inhibit the growth of regenerating rat adrenal cortex, the biological activity of leptin being located in the C-terminal segment of its molecule.

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.

Prolonged exendin-4 administration stimulates pituitary-adrenocortical axis of normal and streptozotocin-induced diabetic rats.

Evidence is available that exendin-4 (EX4), a glucagon-like peptide-1 receptor (GLP-1R) agonist acutely stimulates hypothalamo-pituitary-adrenal (HPA) axis in the rat. EX4 is a potent insulinotropic agent, which is currently under clinical trial for treatment of type 2 diabetes. Since diabetes is known to affect adrenal function, we investigated the effects of the prolonged administration of EX4 and/or the GLP-1R antagonist EX4(9-39) (EX4-A) (daily subcutaneous injections of 1 nmol/kg EX4 and/or EX4-A, for 7 days) on the HPA axis of normoglycemic and streptozotocin (STZ)-induced diabetic rats. In STZ-untreated rats, chronic EX4 treatment did not change the blood level of ACTH. In contrast, it evoked a marked rise in the plasma concentrations of aldosterone and corticosterone, these effects being reversed by EX4-A. In STZ-induced diabetic rats, prolonged EX4 administration increased the plasma levels of ACTH, aldosterone and corticosterone. EX4-A did not prevent the first two effects of EX4, and annulled the latter one. These findings allow us to draw the following conclusions: i) EX4 prolonged exposure desensitizes hypothalamo-hypophyseal GLP-1R in normal rats, and exerts an ACTH-independent GLP-1R-mediated aldosterone and corticosterone secretagogue effect; and ii) experimental diabetes induces the expression of EX4 receptors other than the classic GLP-1R, whose activation mediate the ACTH and aldosterone, but not corticosterone, secretagogue effects. Our study provides evidence that metabolic dysregulations occurring in STZ-induced diabetic rats are able to profoundly affect the response of the HPA axis to GLP-1.

Norbormide enhances late steps of steroid-hormone synthesis in rat and mouse adrenal cortex.

Norbormide (N) is a vasoconstrictor agent, which acts selectively on the peripheral arteries of the rat, through the activation of the phospholipase C (PLC) cascade and the stimulation of Ca(2+) entrance in the vascular myocytes. Several endogenous vasoconstrictor agent (e.g. angiotensin-II (ANG-II) and endothelin-1 (ET-1)), that stimulate PLC pathway, are also able to enhance aldosterone secretion by the adrenal gland. Hence, we examined the effects of norbormide ((0.5, 1.0 or 5) x 10(-5)M) on corticosteroid-hormone secretion from adrenal slices of rats and mice. Quantitative HPLC assay showed that under basal conditions rat and mouse adrenal quarters secreted progesterone (PROG), 11-deoxycorticosterone (DOC), 18-hydroxy-DOC (18OH-DOC), corticosterone (CORT), 18-hydroxy-corticosterone (18OH-CORT) and aldosterone (ALDO), as well as large amounts of pregnenolone (PREG) when its metabolism was blocked by 10(-5)M cyanoketone. Norbormide concentration-dependently raised the secretion of all post-DOC steroids assayed, decreased progesterone and DOC production, and did not affect pregnenolone release. In conclusion, norbormide is able to enhance late steps of steroid synthesis, i.e. those leading to the transformation of DOC to corticosterone and aldosterone, without affecting early steps. This is an interesting finding because the other main endogenous adrenal secretagogues are known to stimulate both early and late steps of steroid synthesis. The mechanism underlying the selective activating action of norbormide on 11beta- and 18-hydroxylation remains to be investigated.

Ghrelin and growth hormone secretagogue receptor are expressed in the rat adrenal cortex: Evidence that ghrelin stimulates the growth, but not the secretory activity of adrenal cells.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. Evidence has been previously provided that adrenal gland possesses abundant ghrelin-displaceable GHS-Rs, but nothing is known about the possible role of ghrelin in the regulation of adrenocortical function. Reverse transcription-polymerase chain reaction demonstrated the expression of ghrelin and GHS-R in the rat adrenal cortex, and high adrenal concentrations of immunoreactive ghrelin were detected by radioimmune assay (RIA). Autoradiography localized abundant [(125)I]ghrelin binding sites in the adrenal zona glomerulosa (ZG) and outer zona fasciculata (ZF). Ghrelin (from 10(-10) to 10(-8) M) did not affect either basal steroid hormone (pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone) secretion from dispersed ZG and zona fasciculata/reticularis (ZF/R) cells (as evaluated by quantitative high pressure liquid chromatography), or basal and agonist-stimulated aldosterone and corticosterone production from cultured ZG and ZF/R cells, respectively (as measured by RIA). Ghrelin (10(-8) and 10(-6) M) raised basal, but not agonist-stimulated, proliferation rate of cultured ZG cells (percent of cells able to incorporate 5-bromo-2'-deoxyuridine), without affecting apoptotic deletion rate (percent of cells able to incorporate biotinylated nucleosides into apoptotic DNA fragments). The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of 10(-8) M ghrelin, while the protein kinase A and C inhibitors H-89 and calphostin-C were ineffective. Ghrelin (10(-8) M) stimulated TK and MAPK activity of dispersed ZG cells, and the effect was abolished by preincubation with tyrphostin-23 and PD-98059, respectively. Tyrphostin-23 annulled ghrelin-induced activation of MAPK activity. Taken together, the present findings indicate that (i) ghrelin and GHS-R are both expressed in the rat adrenal cortex, ghrelin binding sites being very abundant in the ZG; (ii) ghrelin does not affect the secretory activity of rat adrenocortical cells, but significantly enhances the proliferation rate of cultured ZG cells, without affecting apoptotic deletion rate; and (iii) the ZG proliferogenic action of ghrelin involves the TK-dependent activation of the p42/p44 MAPK cascade.