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Background Nicotinamide phosphoribosyltransferase (Nampt/visfatin/PBEF) acts both as an enzyme in the nicotinamide adenine dinucleotide (NAD) synthesis pathway as well as an extracellular hormone (eNampt). Among its effects, eNampt exerts potent pro-inflammatory effects. We have recently shown that, in rats, eNampt stimulates corticosterone secretion by acting through the pituitary rather than the hypothalamus. Objectives To investigate the mechanism of action of eNampt on the secretion of adrenocorticotropic hormone (ACTH) and chemokine (C-C motif) ligand 2 (CCL2), which are cytokines secreted by pituitary neuroendocrine tumors. Material and methods The research was carried out on the AtT-20 murine cell line, primary rat pituitary cell culture, isolated pituitary corticotropes, and in vivo. The effects of the performed experiments were examined using the following methods: gene expression profiling using microarrays, quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Results The results suggest that eNampt stimulates ACTH secretion from rat corticotropes both directly and indirectly. Indirect action most likely occurs through interleukin (IL)-6 secreted by folliculostellate cells of the pituitary gland. In isolated ACTH cells of the rat pituitary gland, eNampt stimulates the expression of genes involved in the immune response. Among them, the protein encoded by the CCL2 gene seems to also be involved in the regulation of corticotropin-releasing hormone (CRH)-dependent metabolism. Unlike rat corticotropes, murine AtT-20 corticotropic cells do not react to either eNampt or Fk866 (the inhibitor of Nampt enzymatic action). Conclusions The eNampt stimulates the secretion of ACTH from rat corticotropes indirectly and directly, likely by stimulating IL-6 secretion from folliculostellate cells of the pituitary gland. This effect was not observed in the AtT-20 corticotropic cell cancer cell line.

More than 35 years have passed since the identification of neuromedin U (NMU). Dozens of publications have been devoted to its physiological role in the organism, which have provided insight into its occurrence in the body, its synthesis and mechanism of action at the cellular level. Two G protein-coupled receptors (GPCRs) have been identified, with NMUR1 distributed mainly peripherally and NMUR2 predominantly centrally. Recognition of the role of NMU in the control of energy homeostasis of the body has greatly increased interest in this neuromedin. In 2005 a second, structurally related peptide, neuromedin S (NMS) was identified. The expression of NMS is more restricted, it is predominantly found in the central nervous system. In recent years, further peptides related to NMU and NMS have been identified. These are neuromedin U precursor related peptide (NURP) and neuromedin S precursor related peptide (NSRP), which also exert biological effects without acting via NMUR1, or NMUR2. This observation suggests the presence of another, as yet unrecognized receptor. Another unresolved issue within the NMU/NMS system is the differences in the effects of various NMU isoforms on diverse cell lines. It seems that development of highly specific NMUR1 and NMUR2 receptor antagonists would allow for a more detailed understanding of the mechanisms of action of NMU/NMS and related peptides in the body. They could form the basis for attempts to use such compounds in the treatment of disorders, for example, metabolic disorders, circadian rhythm, stress, etc.

Background Galanin-like peptide (Galp) and alarin (Ala) are 2 new members of the galanin peptide family. Galanin (Gal), the "parental" peptide of the entire family, is known to regulate numerous physiological processes, including energy and osmotic homeostasis, reproduction, food intake, and secretion of adrenocortical hormones. Galp and Ala are known to regulate food intake. In the rat, Galp mRNA has been found in the brain, exclusively in the hypothalamic arcuate nucleus (ARC) and median eminence, which are involved in the regulation of energy homeostasis. Alarin-like immunoreactivity is present in the locus coeruleus (LC) and the ARC of rats and mice. Objectives The aim of the study was to investigate the expression of Ala, Galp and their receptors in the organs of the hypothalamo-pituitary-adrenal (HPA) axis of the rat. Material and methods The expression of the examined genes was measured in different models of adrenal growth of the rat in vivo (postnatal ontogenesis, compensatory adrenal growth, adrenocortical regeneration, adrenocorticotropic hormone (ACTH) administration). The expression was evaluated using the Affymetrix® microarray system or quantitative polymerase chain reaction (qPCR). Results The expression of Ala gene was observed in each organ of the HPA axis (the hypothalamus, hypophysis and adrenal gland). The elevated level of expression of this gene was observed in the pituitary of 2-day rats, while very low levels of Ala mRNA were observed in the adrenals. Galp mRNA expression was observed only in the hypothalamus and the hypophysis during postnatal ontogenesis. The expression of Gal receptors was demonstrated in the hypothalamus, the hypophysis and the adrenal gland. In different compartments of the adrenal glands of adult, intact male and female rats, the expression of Ala, Galp and galanin receptor 1 (Galr1) genes was negligible, but the expression of galanin receptor 2 (Galr2), galanin receptor 3 (Galr3) and neurotrophic receptor tyrosine kinase 2 (Ntrk2) genes was noticeable. Conclusions The examined genes showed different expression levels within the studied HPA axis; some of them were neither expressed in the hypothalamus or the pituitary gland, nor in the adrenal gland.

Compensatory adrenal growth evoked by unilateral adrenalectomy (hemiadrenalectomy) constitutes one of the most frequently studied in vivo models of adrenocortical enlargement. This type of growth has been quite well characterized for its morphological, biochemical, and morphometric parameters. However, the molecular basis of compensatory adrenal growth is poorly understood. Therefore, the aim of this study was to investigate the rat adrenal transcriptome profile during the time of two previously described adrenocortical proliferation waves at 24 and 72 h after unilateral adrenalectomy. Surgical removal of the left adrenal or a sham operation was accomplished via the classic dorsal approach. As expected, the weight of the remaining right adrenal glands collected at 24 and 72 h after hemiadrenalectomy increased significantly. The transcriptome profile was identified by means of Affymetrix® Rat Gene 2.1 ST Array. The general profiles of differentially expressed genes were visualized as volcano plots and heatmaps. Detailed analyzes consisted of identifying significantly enriched gene ontological groups relevant to adrenal physiology, by means of DAVID and GOplot bioinformatics tools. The results of our studies showed that compensatory adrenal growth induced by unilateral adrenalectomy exerts a limited influence on the global transcriptome profile of the rat adrenal gland; nevertheless, it leads to significant changes in the expression of key genes regulating the circadian rhythm. Our results confirm also that regulation of compensatory adrenal growth is under complex and multifactorial control with a pivotal role of neural regulatory mechanisms and a supportive role of other components.

Introduction. Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. Material and methods. Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix ® Rat Gene 1.1 ST Array Strip and QPCR. Results. By means of Affymetrix ® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ER α , ER β and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ER α genes were the highest. In studied adrenal samples the relative expression of ER α mRNA was higher than ER β mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERR α and ERR β were similar, and only in the ZF/R of female rats ERR γ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5 α -reductase (Srd5a1, Srd5a2 and Srd5a3) and aromatase (Cyp19a1) and expression levels of all these genes were similar in ZG and ZF/R of male and female rats. Conclusions. In contrast to Affymetrix microarray data QPCR revealed higher expression levels of AR gene in adrenal glands of the male rats. In adrenals of both sexes expression levels of ERa, ERb, non-genomic GPR30 (GPER-1), ERR α and ERR β receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

NUCB2/nesfatin and its proteolytically cleaved product nesfatin-1 are recently discovered anorexigenic hypothalamic neuroproteins involved in energy homeostasis. It is expressed both centrally and in peripheral tissues, and appears to have potent metabolic actions. NUCB2/nesfatin neurons are activated in response to stress. Central nesfatin-1 administration elevates circulating ACTH and corticosterone levels. Bilateral adrenalectomy increased NUCB2/nesfatin mRNA levels in rat paraventricular nuclei. To date, studies have not assessed the effects of nesfatin-1 stimulation on human adrenocortical cells. Therefore, we investigated the expression and effects of nesfatin-1 in a human adrenocortical cell model (H295R). Our findings demonstrate that NUCB2 and nesfatin-1 are expressed in human adrenal gland and human adrenocortical cells (H295R). Stimulation with nesfatin-1 inhibits the growth of H295R cells and promotes apoptosis, potentially via the involvement of Bax, BCL-XL and BCL-2 genes as well as ERK1/2, p38 and JNK1/2 signalling cascades. This has implications for understanding the role of NUCB2/nesfatin in adrenal zonal development. NUCB2/nesfatin may also be a therapeutic target for adrenal cancer. However, further studies using in vivo models are needed to clarify these concepts.

Notable sex-related differences exist in mammalian adrenal cortex structure and function. In adult rats, the adrenal weight and the average volume of zona fasciculata cells of females are larger and secrete greater amounts of corticosterone than those of males. The molecular bases of these sex-related differences are poorly understood. In this study, to explore the molecular background of these differences, we defined zone- and sex-specific transcripts in adult male and female (estrous cycle phase) rats. Twelve-week-old rats of both genders were used and samples were taken from the zona glomerulosa (ZG) and zona fasciculata/reticularis (ZF/R) zones. Transcriptome identification was carried out using the Affymetrix(®) Rat Gene 1.1 ST Array. The microarray data were compared by fold change with significance according to moderated t-statistics. Subsequently, we performed functional annotation clustering using the Gene Ontology (GO) and Database for Annotation, Visualization and Integrated Discovery (DAVID). In the first step, we explored differentially expressed transcripts in the adrenal ZG and ZF/R. The number of differentially expressed transcripts was notably higher in the female than in the male rats (702 vs. 571). The differentially expressed genes which were significantly enriched included genes involved in steroid hormone metabolism, and their expression levels in the ZF/R of adult female rats were significantly higher compared with those in the male rats. In the female ZF/R, when compared with that of the males, prevailing numbers of genes linked to cell fraction, oxidation/reduction processes, response to nutrients and to extracellular stimuli or steroid hormone stimuli were downregulated. The microarray data for key genes involved directly in steroidogenesis were confirmed by qPCR. Thus, when compared with that of the males, in the female ZF/R, higher expression levels of genes involved directly in steroid hormone synthesis were accompanied by lower expression levels of genes regulating basal cell functions.

VSNL1 encodes the calcium-sensor protein visinin-like 1 and was identified previously as an upregulated gene in a sample set of aldosterone-producing adenomas. Recently, by means of microarray studies we demonstrated high expression of Vsnl1 gene in rat adrenal zona glomerulosa (ZG). Only scanty data are available on the role of this gene in adrenal function as well as on regulation of its expression by factors affecting adrenal cortex structure and function. Therefore we performed relevant studies aimed at clarifying some of the above issues. By Affymetrix ® Rat Gene 1.1 ST Array Strip, QPCR and immunohistochemistry we demonstrated that expression levels of Vsnl1 in the rat adrenal ZG are notably higher than in the fasciculata/reticularis zone. In QPCR assay this difference was approximately 10 times higher. Expression of this gene in the rat adrenal gland or adrenocortical cells was acutely down regulated by ACTH, while chronic administration of corticotrophin or dexamethasone did not change Vsnl1 mRNA levels. In enucleation-induced adrenocortical regeneration expression levels of both Vsnl1 and Cyp11b2 were notably lowered and positively correlated. Despite these findings, the physiological significance of adrenal Vsnl1 remains unclear, and requires further investigation

Nicotinamide phosphoribosyltransferase (Nampt), also termed visfatin, catalyses the rate‑limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. In addition to its intracellular function (iNampt), extracellular Nampt (eNampt) also affects numerous intracellular signalling pathways. The current study investigated the role of Nampt in the regulation of the hypothalamic‑pituitary‑adrenal (HPA) axis in rats. At 1 h after intraperitoneal administration of eNampt (4 µg/100 g) in adult male rats, serum adrenocorticotropic hormone(ACTH) and aldosterone levels remained unchanged, while corticosterone levels were notably elevated compared with the control group, as determined by ELISA. The results of reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) demonstrated that, in the hypothalami of eNampt‑treated rats, the mRNA expression levels of Fos proto‑oncogene, which is also termed c‑Fos, were not significantly different compared with the control group; however, the mRNA expression levels of proopiomelanocortin (POMC) were markedly increased in the pituitary gland of eNampt‑treated rats compared with the control group. Furthermore, in hypothalamic explants, ELISA results demonstrated that the addition of the eNampt protein exhibited no effect on corticotropin‑releasing hormone (CRH) release into the incubation medium and prevented potassium ion‑induced CRH release. Additionally, the eNampt‑induced increase in ACTH output by pituitary gland explants was not statistically significant, compared with the control group. However, RT‑qPCR indicated that exposure of pituitary gland explants to eNampt and CRH increased the levels of POMC mRNA expression; the effect of eNampt, but not CRH, was inhibited by FK866, which is a specific Nampt inhibitor. In primary rat adrenocortical cell cultures, eNampt exhibited no effect on basal aldosterone or corticosterone secretion, while increases in aldosterone and corticosterone levels in response to ACTH were retained. To assess the potential role of iNampt in the regulation of adrenal steroidogenesis, experiments involving a specific Nampt inhibitor, FK866, were performed. Exposure of cultured cells to FK866 notably lowered basal aldosterone and corticosterone output compared with the control group, and completely eliminated the response of cultured cells to ACTH. The results of the present study indicated that the injected eNampt may have increased the corticosterone serum levels by acting at the pituitary level. In addition, iNampt may exert a tonic stimulating effect on the secretion of aldosterone and corticosterone from rat adrenocortical cells, as normal iNampt levels were required to retain the response of cultured rat adrenocortical cells to ACTH. Thus, these data suggest an important physiological role of both iNampt and eNampt in the regulation of the HPA axis activity in the rat.

Introduction. Sirtuins are NAD dependent class III histone deacetylases. In adrenal cortex mitochondria are able to transform — via nicotinamide nucleotide transhydrogenase (NNT) — NAD into NADPH, which is required for steroidogenesis. These findings suggest that sirtuins expressed in mitochondria, Sirt3, Sirt4 and Sirt5, may be associated with adrenal steroidogenesis. Therefore, the purpose of this study was to characterize the expression of mitochondrial sirtuins (Sirt3–5) in individual compartments of rat adrenal cortex, their developmental regulation and to demonstrate whether their expression is dependent on adrenocorticotrophic hormone (ACTH) and Nampt (nicotinamide phosphoribosyltransferase also known as visfatin/PBEF), the rate-limiting enzyme in the regulation of mammalian NAD synthesis. Material and methods. Studies were performed on rat adrenal glands or on primary culture of rat adrenocortical cells. Expression of mitochondrial sirtuins (Sirt3–5) was evaluated by Affymetrix microarray system or QPCR. The bulk of data were extracted from our earlier experiments which have been reanalyzed in regard to Sirt3–5 mRNAs expression levels and — if necessary — validated by QPCR. Results. Sirt3–5 were expressed throughout the rat adrenal, with the highest expression level of Sirt5. The level of expression of all sirtuins is higher in the zona glomerulosa (ZG) and zona fasciculata/reticularis (ZF/R) than in the adrenal medulla. Sirt3 and Sirt5 expression levels were similar in adult male and female rats, while Sirt4 expression level was higher in females. As revealed by analysis of the available open database, no significant changes in Sirt3–5 expression levels in whole adrenal glands were observed up to week 104 of life of both male and female rats. Moreover, 60 min after intraperitoneal ACTH injection the expression level of Sirt3 in the en­tire gland was elevated while Sirt5 expression level lowered. On the other hand, chronic ACTH infusion (48 h) did not change expression of studied sirtuins. In cultured cells, ACTH greatly increased the expression levels of the Sirt4 and Sirt5. In cultured cells, Fk866 — a highly specific competitive inhibitor of Nampt — reduced expression level of Sirt5 only. In enucleation-induced regenerating rat adrenal, the expression levels of all studied sirtuins were significantly reduced in relation to the control group. Finally, in primary rat adrenal culture the FCS depletion elevates the Sirt3 and Sirt4 expression levels and downregulates Sirt5 expression. Conclusions. Sirt3–5 are expressed throughout the rat adrenal, with the highest expression levels in adrenal cortex. Performed experiments (ACTH stimulation, FCS depletion, regeneration) suggest that in the adrenal cortex, the mitochondrial Sirt5 is the primary mitochondrial sirtuin involved in regulating the biological activity of adrenocortical cells. Our results also suggest that normal levels of intracellular Nampt (iNampt) enzymatic activity are required to maintain normal (control) levels of Sirt5 mRNA in cultured cells.

The enucleation-induced (EI) rapid proliferation of adrenocortical cells is followed by their differentiation, the degree of which may be characterized by the expression of genes directly and indirectly involved in steroid hormone biosynthesis. In this study, out of 30,000 transcripts of genes identified by means of Affymetrix Rat Gene 1.1 ST Array, we aimed to select genes (either up- or downregulated) involved in steroidogenesis in the course of enucleation-induced adrenal regeneration. On day 1, we found 32 genes with altered expression levels, 15 were upregulated and 17 were downregulated [i.e., 3β-hydroxysteroid dehydrogenase (Hsd3β), nuclear receptor subfamily 0, group B, member 1 (Nr0b1), cytochrome P450 aldosterone synthase (Cyp11b2) and sterol O-acyltransferase 1 (Soat1)]. On day 15, the expression of only 2 genes was increased and that of 3 was decreased. The investigated genes were clustered according to an hierarchical clustering algorithm and 4 clusters were obtained. Quantitative PCR (qPCR) confirmed the much lower mRNA expression levels of steroidogenic acute regulatory protein (StAR) during the regeneration process compared to the control, while the cholesterol side-chain cleavage enzyme (cholesterol desmolase; Cyp11a1) and Hsd3β genes presented similar expression profiles throughout the entire regeneration process. Cyp11b2 mRNA levels remained very low during the whole regeneration period. Fatty acid binding protein 6 (Fabp6) was markedly upregulated, whereas hormone-sensitive lipase (Lipe) was downregulated. The expression of Soat1 was lowest on regeneration day 1 and, subsequently, its expression increased from there on, reaching levels higher than the control. Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (Dax-1) mRNA levels were lowest on day 1 of the experiment; however, throughout the entire experimental period, there were no statistically significant differences observed. After the initial decrease in steroidogenic factor 1 (Sf-1) mRNA levels observed on the 1st day of the experiment, a marked upregulation in its expression was observed from there on. Data from the current study strongly suggest the role of Fabp6, Lipe and Soat1 in supplying substrates of regenerating adrenocortical cells for steroid synthesis. Our results indicate that during the first days of adrenal regeneration, intense synthesis of cholesterol may occur, which is then followed by its conversion into cholesteryl esters. Moreover, our data demonstrated that in enucleation-induced regeneration, the restoration of genes involved in glucocorticoid synthesis is notably shorter than that of those involved in aldosterone synthesis.

Neuropeptide B (NPB) and W (NPW) regulate food intake and energy homeostasis in humans via two G-protein-coupled receptor subtypes, termed as GPR7 and GPR8. Rodents express GPR7 only. In animals, NPW decreases insulin and leptin levels, whereas the deletion of either NPB or GPR7 leads to obesity and hyperphagia. Metabolic and endocrine in vitro activities of NPW/NPB in adipocytes are unknown. We therefore characterize the effects of NPB and NPW on the secretion and expression of leptin and resistin, and on lipolysis, using rat adipocytes. Isolated rat adipocytes express GPR7 mRNA. NPB and NPW are expressed in macrophages and preadipocytes but are absent in mature adipocytes. Both, NPB and NPW reduce the secretion and expression of leptin from isolated rat adipocytes. NPB stimulates the secretion and expression of resistin, whereas both, NPB and NPW increase lipolysis. Our study demonstrates for the first time that NPB and NPW regulate the expression and secretion of leptin and resistin, and increase lipolysis in isolated rat adipocytes. These effects are presumably mediated via GPR7. The increase of resistin secretion, stimulation of lipolysis and the decrease of leptin secretion may represent mechanisms, through which NPB and NPW can affect glucose and lipid homeostasis, and food intake in rodents.

Adiponectin (ADN) is a regulatory peptide secreted mostly by adipose tissue and acting via two receptors: AdipoR1 and AdipoR2. Our aim was to investigate expression of adiponectin system genes in the rat adrenal gland as well as its ontogenetic and physiological control. Furthermore, we examined the effects of acute and prolonged activation of HPA axis on ADN system in adipose tissue. By means of QPCR, ADN and AdipoR1 expression was demonstrated in rat adrenal cortex both at mRNA and protein levels, while AdipoR2 could only be detected at mRNA levels. ADN expression level was significantly upregulated in a developing and regenerating adrenal cortex. Globular domain of adiponectin at 10−9 M stimulated corticosterone output and BrdU incorporation by cultured rat adrenocortical cells. Moreover, both acute (ACTH and ether stress) and prolonged (ACTH) adrenal stimulation resulted in lowered ADN levels, while expression of AdipoR1 and AdipoR2 was upregulated by the acute treatment. Depending on its site of origin, visceral (VAT) or subcutaneous (SAT) adipose tissue responded differently to alterations in HPA axis. VAT expression of ADN and its receptors remained almost unchanged by experimental manipulations. In SAT, on the other hand, expression of ADN and AdipoR2 was markedly increased by ACTH treatment and stress, while dexamethasone suppressed ADN and AdipoR1 mRNA levels. The results of this study provide new evidence for direct and indirect interactions between adipokines and HPA axis.

Gonadotropin-inducible ovarian transcription factor-1 (Giot1) belongs to a family of fast-responsive genes, and gonadotropins rapidly induce its expression in steroidogenic cells of ovaries and testes of rats. Gonadal Giot1 gene expression is regulated by cyclic adenosine monophosphate (cAMP) -dependent protein kinase A pathway, with essential role of orphan nuclear receptor NR4A1 transcription factor (nuclear receptor subfamily 4, group A, member 1). A recent study reports that Giot1 is also expressed in adrenals, however, the mechanism of its regulation in adrenal gland is yet to be identified. Therefore, the aim of this study was to characterise the changes in Giot1 gene expression in male and female rat adrenals using wide range of in vivo and in vitro experimental models. Special emphasis was directed at the Giot1 gene regulation by ACTH and gonadotropin. In our study, we found that ACTH rapidly stimulates Giot1 expression both in vivo and in vitro. However, gonadotropin does not affect the adrenal Giot1 gene expression, presumably due to the low expression of gonadotropin receptor in adrenals. Both testosterone and estradiol administered in vivo had inhibitory effect on Giot1 gene expression in the adrenals of post-gonadectomized adult rats. Further, our studies revealed that the intracellular mechanism of Giot1 gene regulation in rat adrenals is similar to that of gonads. As in the case of gonads, the expression of Giot1 in adrenal gland is regulated by cAMP-dependent signaling pathway with essential role of the NR4A1 transcription factor. The results of our studies suggest that Giot1 may be involved in the regulation of rat adrenocortical steroidogenesis.

The orexigenic peptide ghrelin (GHREL) and obestatin (OBS) originate from the same peptide precursor, preproghrelin (ppGHREL). Apart from orexigenic effect, GHREL also regulates neuroendocrine function. We investigated GHREL and OBS effects on corticosterone secretion by freshly isolated and cultured rat adrenocortical cells. Classic RT-PCR revealed the presence of ppGHREL, GHS-R1a, GPR39v1 and GPR39v2 and GOAT4 (ghrelin O-acyl transferase) mRNAs in rat adrenals and cultured for 4 days rat adrenocortical cells. Expression of ppGHREL, GHS-R1a, and GOAT genes was notably higher in the cortex than in medulla. High expression level of GOAT gene was found in the zona glomerulosa, while expression level of both GPR39v1 and GPR39v2 genes was similar in adrenal cortical zones and in medulla. In freshly isolated cells neither GHREL nor OBS had an effect on corticosteroid output. Prolonged exposure of cultured cells to GHREL resulted in a potent, comparable to ACTH, stimulating effect of GHREL on corticosterone secretion. Prolonged exposure to OBS was ineffective. Neither GHREL nor OBS had any effect on proliferation of studied cells, while ACTH notably lowered it. GHREL down regulated GHS-R1a gene expression while both ACTH and GHREL stimulated expression level of GPR39v1 gene. Expression of CYP11A1 gene was notably stimulated and that of StAR gene remained unaffected by ACTH or GHREL. Thus, our study is the first to demonstrate direct stimulating effect of GHREL on corticosterone output by cultured rat adrenocortical cells. This stimulating action differs from that evoked by ACTH and is not dependent on the presence of functional ACTH receptor.

OBJECTIVES Neuromedin U (NmU) is a neuropeptide with anorexigenic activity. Two receptor subtypes (NmUR1 and NmUR2) confer the effects of NmU on target cells. We have recently demonstrated that NmU reduces insulin secretion from isolated pancreatic islets. Aim of our current study is to investigate the role of somatostatin at mediating the effects of NmU on insulin secretion. METHODS Expression of NmU in the pancreas was detected by immunohistochemistry. Insulin and somatostatin secretion from in situ perfused rat pancreas and isolated pancreatic islets was measured by radioimmunoassay. The paracrine effects of somatostatin within pancreatic islets were blocked by cyclosomatostatin, a somatostatin receptor antagonist. RESULTS Receptor subtype NmUR1, but not NmUR2, was expressed in the endocrine pancreas, predominantly in the periphery. Neuromedin U reduced insulin secretion from in situ perfused rat pancreas and stimulated somatostatin secretion from isolated pancreatic islets. Neuromedin U stimulated somatostatin secretion at both physiological and supraphysiological glucose concentrations. Cyclosomatostatin increased insulin secretion and reduced NmU-induced inhibition of insulin secretion. CONCLUSIONS Neuromedin U reduces insulin and increases somatostatin secretion. Blockade of somatostatin action abolishes the inhibition of insulin secretion by NmU. The results of the study suggest that somatostatin mediates the inhibitory action of NmU on insulin secretion.

N-homocysteinylation of epsilon-amino group of protein lysine residues by homocysteine (Hcy) thiolactone has been implicated in vascular disease in humans. We have previously generated polyclonal rabbit anti-N-Hcy-protein IgG antibodies that specifically recognize the Nepsilon-Hcy-Lys epitope on N-homocysteinylated proteins. The present work was undertaken to examine the utility of these antibodies for the immunohistochemical detection of N-homocysteinylated proteins in biological samples. We found that the rabbit antibody specifically detected N-Hcy-protein in a dot-blot assay, that the signal resulting from the reaction of the antibody with N-Hcy-protein depended on the amount of the antigen, and that the sensitivity of the assay was protein-dependent. The rabbit anti-N-Hcy-protein IgG also specifically detected Nepsilon-Hcy-Lys epitopes in human tissues, as shown by positive immunohistochemical staining of myocardium and aorta samples from cardiac surgery patients, and a lack of staining when the antibody was pre-adsorbed with N-Hcy-albumin. We also observed increased immunohistochemical staining for N-Hcy-proteins in aortic lesions from ApoE-/- mice with hyperhomocysteinemia induced by a high methionine diet, relative to ApoE-/- mice fed a control chow diet. In conclusion, polyclonal rabbit anti-N-Hcy-protein antibody can detect and monitor N-homocysteinylated proteins in human and mouse tissues with good sensitivity and specificity.

Endocrine-disrupting chemicals (EDCs) became the focus of both public and scientific interest when defects in sexual behavior and reproductive ability of wild-living animals were ascribed to their steroid-like or anti-steroid androgenic properties (Colborn et al. 1993). In the aftermath, these observations were reproduced in many laboratory animal models. Whether human reproduction is also affected by the action of EDCs is currently a topic of controversial discussion (Safe 2004; Toppari 2002; Waring and Harris 2005). Furthermore, there is growing evidence that, in addition to the reproductive, other endocrine systems such as the hypothalamus–pituitary–thyroid (HPT) axis may be targets of endocrine disruption. In particular, poly-halogenated phenolic compounds such as polychlorinated biphenyls (PCBs) and poly-brominated diphenyl ethers (PBDEs), probably because of their structural resemblance to thyroid hormones (Cody et al. 1986), may cause disturbance of thyroid hormone homeostasis, hypothyroidism, thyroid hyperplasia, and neoplasia (Hagmar 2003; Siddiqi et al. 2003), and developmental defects of the central nervous system (CNS) in experimental animals and humans (Koopman-Esseboom et al. 1994; Meerts et al. 2004). It now has been shown that there may be multiple targets for interference by various EDCs with the complex regulatory network of thyroid hormone synthesis, metabolism, distribution, and action on the various levels of endocrine regulation and feedback control. These targets include thyrotropin receptor (Auf’mkolk et al. 1985a, 1985b; Santini et al. 2003); iodide uptake by the sodium iodide symporter (NIS; Schroder-van der Elst et al. 2004); type I 5′-deiodinase (5′DI) (Auf’mkolk et al. 1986; Ferreira et al. 2002; Schmutzler et al. 2004); transthyretin (TTR) (Kohrle et al. 1988; van den Berg 1990; Yamauchi et al. 2003); thyroid hormone receptor (TR) (Bogazzi et al. 2003; Moriyama et al. 2002); and thyroid hormone-dependent growth of pituitary cells (Ghisari and Bonefeld-Jorgensen 2005). In the brain, PCBs may disrupt normal differentiation regulated by thyroid hormones, although they do not act as thyroid hormone-like ligands (Zoeller 2005). Moreover, complex biological developmental programs controlled by thyroid hormones may be disturbed by EDC action such as metamorphosis in the amphibian Xenopus laevis (Kloas 2002). Yet, data on EDCs affecting the HPT axis are comparatively scarce. Within the CREDO (cluster of research into endocrine disruption in Europe) Cluster, which is part of the Sixth European Union framework program, the EURISKED (multi-organic risk assessment of selected endocrine disruptors) consortium worked on a group of projects assessing multiorganic effects of selected endocrine disruptors. These projects included a focus on the thyroid gland and its target organs. In this article we summarize the first results. Chosen for analysis was an environmentally and nutritionally relevant collection of substances suspected to have endocrine-disrupting activity because of their steroid-like or anti-steroid effects. These substances included genistein as well as glycitein and daidzein (isoflavones from soybean); resveratrol (phytoalexin from grapes); silymarin (flavonol–lignane mixture from the milk thistle); xanthohumol (XN; prenylated chalcone from hops, Humulus lupulus L.); 8-prenylnaringenin (8-PN; prenylated flavonone from hops); benzophenone-2, ben-zophenone-3, 4-methylbenzylidene camphor, and octyl-methoxycinnamate [BP2, BP3, OMC, 4-MBC, respectively; ultraviolet (UV) filters]; F21388 (synthetic, halogenated flavonoid); 4-nonylphenol (4-NP; e.g., emulgator); bisphenol A (BPA; building block for, e.g., polycarbonate plastics); dibutylphthalate (e.g., plasticizer); linuron and procymidon (pesticides); 5α-androstane-3β,17β-diol [adiol; proposed to be an endogenous ligand of estrogen receptor β (ER-β)] and 17β-estradiol benzoate (E2). We show that several of these substances also interfere with multiple targets at various levels of the HPT axis in a tissue-specific manner. These targets included weight and morphology of the thyroid gland, iodide uptake by the NIS, iodide organification by thyroid peroxidase (TPO), binding of the thyroid hormone thyroxine (T4) to TTR, the metabolism of thyroid hormones by deiodinases, and the action of the biologically active thyroid hormone triiodothyronine (T3) mediated by TRs functioning as ligand-dependent transcription factors.

Structural alterations in thymuses of female rats during the first 2 years of life were evaluated by morphometric analysis and, then, correlated with organization of epithelial cells in various thymic compartments, examined for their cytokeratin immunoreactivity. With an advancing age, the thymuses demonstrated morphological modifications related to maturation and senescence, the dynamics of which varied between particular thymic compartments, and involved subpopulations of thymic epithelial cells. In the entire period of life the most dynamic changes were found in the cortex while the medulla was demonstrated to be a rather "stable" region. Morphometric studies revealed a negative correlation between the volume of thymic cortex and medulla and age of rats and a linear, positive relationship between the volume of connective tissue compartment and age. Changes in organization of epithelial network in the medulla preceded those observed in the cortex. Decreased proliferative activity of subset of medullary cells, which probably represented a self-renewable population, was accompanied by alterations in the immunocytochemically characterized (cytokeratines) differentiation process. At the same period of life, hypertrophy and hyperplasia of superficial epithelial cells seems to functionally replace medullary cells. This process begins around 3rd month of life and expands on all thymic compartments. The first changes in the cortex appeared around 8th month and were connected with reduced cytokeratin immunoreactivity. The involution observed in older animals was preceded by age-related alterations in epithelial network pattern which, in the course of stable morphometric parameters (between 5th and 12th month), showed character of a structural and functional adaptation.

The Esophageal Cancer Related Gene 4 (ECRG4) is a highly conserved tumour suppressor gene encoding various peptides (augurin, CΔ16 augurin, ecilin, argilin, CΔ16 argilin) which can be processed and secreted. In the present work, we examined ECRG4 expression and location in a wide range of rat organs and reviewed the available literature. ECRG4 mRNA was identified in all examined tissues by quantitative PCR (qPCR). ECRG4 immunoreaction was mainly cytoplasmic, and was detected in heart and skeletal muscles, smooth muscle cells showing only weak reactions. In the digestive system, ECRG4 immunostaining was stronger in the esophageal epithelium, bases of gastric glands, hepatocytes and pancreatic acinar epithelium. In the lymphatic system, immunoreactive cells were detectable in the thymus cortex, lymph node medulla and splenic red pulp. In the central and peripheral nervous systems, different neuronal groups showed different reaction intensities. In the endocrine system, ECRG4 immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei, hypophysis, thyroid and parathyroid glands, adrenal zona glomerularis and medulla and Leydig cells, as well as in follicular and luteal cells of the ovary. In the literature, ECRG4 has been reported to inhibit cell proliferation and increase apoptosis in various cell types. It is down-regulated, frequently due to hypermethylation, in esophageal, prostate, breast and colon cancers, together with glioma (oncosuppressor function), although it is up-regulated in papillary thyroid cancer (oncogenic role). ECRG4 expression is also higher in non-proliferating cells of the lymphatic system. In conclusion, our identification of ECRG4 in many structures suggests the involvement of ECRG4 in the tumorigenesis of other organs and also the need for further research. In addition, on the basis of the location of ECRG4 in neurons and endocrine cells and the fact that it can be secreted, its role as a neurotransmitter/neuromodulator and endocrine factor must be examined in depth in the future.

Beacon, a hypothalamic peptide involved in the regulation of food intake, has been recently shown to be expressed in the adrenal cortex, and to inhibit its secretion and growth. To further characterize the role of beacon in the control of adrenal growth, we investigated the level of beacon gene expression in the regenerating rat adrenal cortex. Conventional reverse transcription-polymerase chain reaction (PCR) and immunocytochemistry demonstrated the expression of beacon mRNA and protein in the adrenals at both days 5 and 8 of regeneration after enucleation and contralateral adrenalectomy. Semiquantitative real time-PCR revealed a net down-regulation of beacon mRNA in the regenerating glands, as compared to the intact adrenal cortex of sham-operated animals. Beacon gene expression was higher at day 8 than at day 5 of regeneration. Mitotic index, as assayed by the stachmokinetic method with vincristin, was negligible in the intact adrenal, but greatly elevated in regenerating gland, with a higher index found at day 5 than at day 8 after surgery. Taken together our findings indicate that the level of beacon gene expression is inversely correlated with the proliferative activity of adrenocortical cells, and suggest that beacon might act as an endogenous inhibitor of adrenocortical growth in the rat.

Orexins A and B are hypothalamic peptides, that act via two subtypes of receptors, named OX1-R and OX2-R. Rat and human adrenal cortexes are provided with both OX1-R and OX2-R, and we have previously shown that orexin-A, but not orexin-B, enhances glucocorticoid secretion from dispersed adrenocortical cells. Since OX1-Rs preferentially bind orexin-A and OX2-Rs are non-selective for both orexins, the hypothesis has been advanced that the secretagogue effect of orexin-A is exclusively mediated by the OX1-R. Here, we aimed to verify this contention and to gain insight into the signaling mechanism(s) underlying the secretagogue effect of orexins using primary cultures of rat and human adrenocortical cells. Reverse transcription-polymerase chain reaction showed that cultured cells, as freshly dispersed cells, expressed both OX1-R and OX2-R mRNAs. Orexin-A, but not orexin-B, concentration-dependently increased corticosterone and cortisol secretion from cultured rat and human adrenocortical cells, respectively. The blockade of OX1-Rs by selective antibodies abrogated the secretagogue effect of orexin-A, while the immuno-blockade of OX2-Rs was ineffective. The glucocorticoid response of cultured cells to orexin-A was annulled by the adenylate cyclase and protein kinase (PK) A inhibitors SQ-22536 and H-89, and unaffected by the phospholipase C and PKC inhibitors U-73122 and calphostin-C. Orexin-A, but not orexin-B, enhanced cyclic-AMP production from cultured cells, and did not alter inositol-3-phosphate release. Collectively, our present results allow us to conclude that orexins stimulate glucocorticoid secretion from rat and human adrenocortical cells, exclusively acting through OX1-Rs coupled to the adenylate cyclase/PKA-dependent signaling cascade.

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) × 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 11β- and 18-hydroxylation remains to be investigated.

Pneumadin (PNM) is a decapeptide, originally isolated from mammalian lungs, which exerts a potent stimulating effect on arginine-vasopressin (AVP) release, thereby evoking an antidiuretic effect. We have established a specific radioimmunoassay (RIA) method for rat PNM determination, the sensitivity of which is sufficient for measuring tissue content of the peptide. Moreover, raised antibodies have been used for the immunocytochemical detection of PNM in several rat organs. As expected, high concentrations of PNM were detected by RIA in newborn and adult rat lungs and immunocytochemistry (ICC) localized PNM immunoreactivity (IR) in the bronchial and bronchiolar epithelium. Very high concentrations of PNM were measured by RIA in the prostate, and ICC showed that PNM-IR is contained in the epithelial cells. RIA and ICC demonstrated the presence of low amounts of PNM in the thymus. The highest content of radioimmunoassayable PNM was found in the kidneys and intestinal tract, but dilution test suggested the presence of some interfering substances in these tissues. Accordingly, ICC-detectable PNM-IR was absent in the kidneys and present only in the duodenal criptae and Brunner's glands of the intestinal tract. RIA did not measure sizeable PNM concentrations in the thyroid gland, but ICC showed PNM-IR in C-cells. RIA and ICC did not detected PNM in testes, seminal vesicles, ovaries, uterus, pancreas, liver, spleen, adrenal glands, and heart. Taken together, our findings suggest that PNM, in addition to its role as hypothalamo-pituitary AVP secretagogue, may be involved in the autocrine-paracrine functional regulation of other peripheral organs, like lungs and prostate and perhaps duodenum, thymus and thyroid gland.

Orexin A and B are recently identified as peptides that are derived from the same precursor and their expression is highly specifically localized in neurons located in the lateral hypothalamic area, a region implicated in the feeding behaviour. These peptides appear to be a part of a complex circuit that integrates the aspects of energy metabolism, cardiovascular function, hormone homeostasis and sleep/wake behaviours. The functional linking of orexins with leptin and insulin suggests the possibility of its involvement in the regulation of the adipoinsular axis, and the present investigation was designed to examine the potential role of orexins in this axis regulation. In all the tested doses (8, 16 and 40 nmol/kg body weight (b.w.)), subcutaneous (s.c.) injections of orexin A caused the significant increase in insulin and leptin blood levels. These elevations were observed 60 and 120 min after peptide administration. On the other hand, after the orexin B administration, elevated insulin and leptin blood concentrations were found only at 60 min of the experiment, and in that time point, the increases were comparable to that evoked by orexin A. In comparison with the control animals, the administration of orexins for 7 days resulted in a significant gain in body weight. Prolonged administration of either orexin A or orexin B significantly elevated insulin and leptin blood concentrations. Under these conditions, the orexin A effect on the leptin secretion was more marked than on the insulin secretion, and this difference is reflected by the lowered insulin/leptin molar ratio. These results suggest that orexins play an important role in the adipoinsular axis function and may be a significant regulator of both insulin and leptin secretion. In this regard, we suggest the updated functional model of Kieffer and Habener [Am. J. Physiol.: Endocrinol. Metab. 27 (2000) E1] that proposed the adipoinsular axis. Our model is extended by the probable humoral links between orexins and leptin and orexins and insulin and points on the dependence of the effects evoked by orexins, leptin and insulin on the blood glucose levels.

Cholecystokinin is a regulatory peptide, that acts through two subtypes of receptors, 1 and 2. RT-PCR demonstrated the expression of both cholecystokinin receptors 1 and 2 genes in the zona glomerulosa, but not the zona fasciculata-reticularis, of rat adrenals. Autoradiography demonstrated the presence of abundant [(125)I]cholecystokinin-binding sites in the zona glomerulosa, but not the zona fasciculata-reticularis, which were displaced by both cholecystokinin receptor 1- and 2-selective antagonists (cholecystokinin 1-A and 2-A). Cholecystokinin increased basal aldosterone secretion from dispersed zona glomerulosa cells without affecting corticosterone secretion from zona fasciculata-reticularis cells. The aldosterone response to cholecystokinin was blunted by cholecystokinin 1-A and 2-A, which when added together abolished it. ACTH-stimulated aldosterone production was not affected by cholecystokinin; in contrast, cholecystokinin potentiated aldosterone response to both angiotensin II and K(+). Cholecystokinin enhanced cAMP, but not IP(3), release by dispersed zona glomerulosa cells. The aldosterone response to cholecystokinin was abolished by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89, but not by either the PLC inhibitor U-73122 or the PKC inhibitor calphostin C. In conclusion, our study provides evidence that cholecystokinin, acting through cholecystokinin receptors 1 and 2 coupled with the adenylate cyclase/PKA cascade, exerts a sizeable secretagogue action on rat zona glomerulosa cells.

The effect of adrenomedullin (ADM) on the proliferative activity of the rat adrenal cortex has been investigated in vivo, using an in situ perfusion technique of the intact left gland. ADM and other chemicals were dissolved in the perfusion medium, and the perfusion was continued for 180 min. ADM infusion concentration dependently increased the mitotic index and [3H]thymidine incorporation into DNA in the zona glomerulosa (ZG; the maximal effective concentration was 10(-8) M), but not in inner adrenocortical layers, where basal proliferative activity was negligible. The effect of 10(-8) M ADM was equipotently counteracted by both the calcitonin gene-related peptide (CGRP) type 1 receptor antagonist CGRP-(8-37) and ADM-(22-52). The adenylate cyclase inhibitor SQ-22536 (10(-4) M), the cAMP blocker Rp-cAMP-S (10(-3) M), and the protein kinase A inhibitor H-89 (10(-5) M), although counteracting the ZG proliferogenic action of 10(-9) M ACTH, did not affect the 10(-8) M ADM-elicited increase in ZG DNA synthesis. Similar results were obtained using the phospholipase C inhibitor U-73122 (10(-5) M), the inositol-1,4,5-trisphosphate antagonist D,L-myo-inositol-1,4,5-trisphosphothiate (10(-4) M), and the protein kinase C inhibitor calphostin C (10(-5) M), which, however, significantly inhibited the ZG proliferogenic effect of 10(-9) M angiotensin II. The growth-promoting action of 10(-8) M ADM was not affected by the phospholipase A2 inhibitor AACOCF3 (10(-5) M), the cyclooxygenase (COX) inhibitor indomethacin (10(-5) M), or the mixed COX/lipoxygenase inhibitor phenidone (10(-5) M). In contrast, the ZG proliferogenic effect of 10(-8) M ADM was abolished by either the tyrosine kinase (TK) inhibitor tyrphostin-23 (10(-5) M) or the mitogen-activated protein kinase (MAPK) antagonists PD-98059 and U0216 (10(-4) M). ADM (10(-8) M) stimulated TK and p42/p44 MAPK activity in dispersed ZG, but not ZF, cells, and the effect was reversed by either 10(-6) M CGRP-(8-37) and ADM-(22-52) or preincubation with 10(-5) M tyrphostin-23. Collectively, our findings indicate that 1) ADM stimulates cell proliferation in the rat ZG, through CGRP-(8-37)- and ADM-(22-52)-sensitive receptors, probably of the CGRP1 subtype; and 2) the mitogenic effect of ADM is mediated by activation of the TK-MAPK cascade, without any involvement of the adenylate cyclase/protein kinase A-, phospholipase C/protein kinase C-, and COX- or lipoxygenase-dependent signaling pathways.

Secretin. Glucagon, gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH) belong, together with vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase (AC)-activating polypeptide, to a Family of peptides (the VIP-secretin-glucagon family), which also includes growth hormone-releasing hormone and exendins. All the members of this peptide family possess a remarkable amino-acid sequence homology, and bind to G-protein-coupled receptors, whose signaling mechanism primarily involves AC/protein kinase A and phospholipase C/protein kinase C cascades. VIP and pituitary AC-activating polypeptide play a role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and in this: review we survey findings that also other members of the VIP-secretin-glucagon Family may have the same function. Secretin and secretin receptors are expressed in the hypothalamus and pituitary gland, and secretin inhibits adrenocorticotropic hormone (ACTH) release. No evidence is available for the presence of secretin receptors in adrenal glands, but secretin selectively depresses the glucocorticoid response to ACTH of dispersed zona fasciculata-reticularis (ZF/R) cells. Glucagon and glucagon-like peptide-1 are contained in the hypothalamus, and all the components of the HPA axis are provided with glucagon and glucagons-like-1 receptors. These peptides exert a short-term inhibitory effect on stress-induced pituitary ACTH release and depress the ZF/R cell response to ACTH by inhibiting the AC/protein kinase A cascade; they also stimulate hypothalamic arginine-vasopressin release. GIP receptors are present in the ZF/R of the normal adrenals, and are particularly abundant in some types of adrenocortical adenomas and hyperplasias. GIP, through the activation of the AC/protein kinase A cascade, evokes a sizeable glucocorticoid secretagogue effect, leading to the identification of a food/GIP-dependent Cushing's syndrome. PTH and PTH-related protein are expressed in the hypothalamus and pituitary gland, and PTH and PTH-related protein receptors in all the components of the HPA axis. Both peptides enhance ACTH and arginine-vasopressin release, as well as stimulate aldosterone and glucocorticoid secretion of dispersed zona glomerulosa and ZF/R cells, respectively. The involvement of growth hormone-releasing hormone and exendins in the functional regulation of the HPA axis has not yet been extensively investigated. (C) 2000 Elsevier Science Inc. All rights reserved.

Leptin is an adipose-tissue secreted hormone, that acts to decrease caloric intake and to increase energy expenditure. Some of the leptin effects on the energy balance are known to be mediated by the hypothalamo-pituitary-adrenal (HPA) axis, but the role of this cytokine in the regulation of the growth and steroidogenic capacity of adrenal cortex is still controversial. Therefore, the present study was designed to explore the long-term effects of native leptin[1-147] and its biologically active fragment leptin[116-130] (6 daily subcutaneous injection of 20 nmol/kg) on the rat HPA axis. Leptin[1-147] and leptin[116-130] caused a significant adrenal atrophy, which was mainly due to the decrease in the volume of zona fasciculata (ZF) and in the number of its parenchymal cells. Both leptins provoked a marked drop in the plasma concentrations of ACTH and corticosterone, the main hormone produced by ZF cells. The effects of leptin[116-130] were more intense than those of leptin[1-147]. Leptin[1-147], but not its fragment, evoked a clear-cut rise in the plasma concentration of aldosterone. Collectively, these findings indicate that prolonged leptin administration, by inhibiting pituitary ACTH release, exerts a potent suppressive action on the growth and glucocorticoid secretory capacity of the adrenal cortex in the rat. The mechanism(s) underlying the aldosterone secretagogue action of native leptin remain(s) to be investigated.

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.

Galanin, a 29-amino acid peptide widely distributed in the central and peripheral nervous systems, was found to induce a concentration-dependent increase in corticosterone secretion and cyclic-AMP release by dispersed rat inner adrenocortical cells (maximal effective concentration, 10 −7 M). The effect of 10 −7 M galanin was blocked by 10 −6 M galantide, a specific antagonist of galanin receptors. Galanin (10 −7 M) also enhanced corticosterone and cyclic-AMP responses of dispersed cells to submaximal but not maximal (10 −9 M) effective concentrations of ACTH, and again this effect was reversed by galantide. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10 −6 M) blocked corticosterone response of dispersed cells to 10 −9 M ACTH but not to 10 −7 M galanin; conversely, the specific protein kinase A inhibitor H-89 (10 −5 M) annulled the secretory response to both ACTH and galanin. In light of these findings, we conclude that galanin stimulates adrenal glucocorticoid secretion in rats, acting through specific receptors, coupled, like those of ACTH, with the adenylate cyclase/protein kinase A-dependent signaling pathway.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are members of a family of regulatory peptides that are widely distributed in the body and share numerous biologic actions. The two peptides display a remarkable amino acid-sequence homology, and bind to a class of G protein-coupled receptors, named PACAP/VIP receptors (PVRs), whose signaling mechanism mainly involves the activation of adenylate-cyclase and phospholipase-C cascades. A large body of evidence suggests that VIP and PACAP play a role in the control of the hypothalamo--pituitary-adrenal (HPA) axis, almost exclusively acting in a paracrine manner, since their blood concentration is very low. VIP and PACAP are contained in both nerve fibers and neurons of the hypothalamus, and VIP, but not PACAP, is also synthesized in the pituitary gland. Both peptides are expressed in the adrenal gland, and especially in medullary chromaffin cells. All the components of the HPA axis are provided with PVRs. VIP and PACAP enhance pituitary ACTH secretion, VIP by eliciting the hypothalamic release of CRH and potentiating its secretagogue action, and PACAP by directly stimulating pituitary corticotropes. Through this central mechanism, VIP and PACAP may increase mineralo- and glucocorticoid secretion of the adrenal cortex. VIP but not PACAP also exerts a weak direct secretagogue action on adrenocortical cells by activating both PVRs and probably a subtype of ACTH receptors. VIP and PACAP raise aldosterone production via a paracrine indirect mechanism involving the stimulation of medullary chromaffin cells to release catecholamines, which in turn enhance the secretion of zona glomerulosa cells via a beta-adrenoceptor-mediated mechanism. PACAP appears to be able to evoke a glucocorticoid response through the activation, at least in the rat, of the intramedullary CRH/ACTH system. The relevance of these effects of VIP and PACAP under basal conditions is questionable, although there are indications that endogenous VIP is involved in the maintenance of the normal growth and steroidogenic capacity of rat adrenal cortex. However, indirect evidence suggests that these peptides might play a relevant role under paraphysiological conditions (e.g., in the mediation of HPA axis responses to cold and inflammatory stresses) or may be somehow involved in the pathogenesis of Cushing disease or some case of hyperaldosteronism associated with secreting pheochromocytomas.

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

The effects of endothelins (ET) on the proliferative activity of the rat adrenal cortex have been investigated in vivo, using an in situ perfusion technique of the intact left gland. The chemicals were dissolved in the perfusion medium, and the perfusion continued for 120 min. ET-1 concentration dependently increased the mitotic index and [3H]thymidine incorporation into DNA in the zona glomerulosa (ZG; 6- and 3-fold increases, respectively, at a 10(-8) M concentration), but not in the inner adrenocortical layers, where the basal proliferative activity was negligible. The effect of 10(-8) M ET-1 was blocked by the ETA receptor antagonist BQ-123, whereas the ETB receptor antagonist BQ-788 was ineffective. ET-2 and ET-3 (10(-8) M) enhanced DNA synthesis in the ZG, but their effects were less intense than that of 10(-8) M ET-1 and were directly related to their binding potency for the ETA receptor subtype (ET-1 > ET-2 >> ET-3). The selective ETB receptor agonists BQ-3020, IRL-1620, and sarafotoxin-6B were ineffective. The ZG proliferogenic action of 10(-8) M ET-1 was reversed by both the protein kinase C inhibitor Ro31-8220 and the tyrosine kinase inhibitor tyrphostin-23; a complete blockade was obtained at a 10(-6)-M concentration of each inhibitor. In contrast, neither the protein kinase A inhibitor H-89 (10(-5) M) nor the cyclooxygenase and lipoxygenase inhibitors indomethacin and phenidone (10(-5) M) affected ET-1 action. Collectively, our findings indicate that ETs stimulate the proliferation of rat adrenal ZG cells, acting through ETA receptors coupled with protein kinase C- and tyrosine kinase-dependent signaling pathways. The results of the present study are in keeping with the view that in mammals, ZG is the proliferative layer involved in the maintenance of growth of the entire adrenal cortex and with the previous autoradiographic demonstration that ZG is the only adrenocortical layer provided with ETA receptors.

Using an in situ perfusion technique of isolated left rat adrenal gland, it has been demonstrated that angiotensin-II (ANG-II) increases DNA synthesis in the zona glomerulosa (ZG), but not fasciculata-reticularis cells. The AT1 receptor antagonist DuP753 abolished the effect of ANG-II, while the AT2 receptor antagonist PD 123319 potentiated it. Both Ro31-8220, an inhibitor of protein kinase C (PKC), and tyrphostin-23, an inhibitor of tyrosine kinase (TK), evoked a partial reversal of ANG-II effect, and when added together to the perfusion medium abolished it. In contrast, the phospholipase C inhibitor U-73122 alone was able to induce a complete blockade of ANG-II effect. Neither the phospholipase A2 inhibitor AACOCF3 nor the cyclooxygenase inhibitor indomethacin and the lipoxygenase inhibitor phenidone affected ANG-II-induced stimulation of DNA synthesis, thereby making unlikely the involvement of the arachidonic acid signaling pathways. Our findings suggest that (i) ANG-II stimulates rat ZG cell proliferation acting via AT1 receptors coupled with phospholipase C, which activates both PKC and TK signaling systems; and (ii) the proliferogenic effect of ANG-II is partially counteracted by the activation of the AT2 receptor subtype.

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.

Hormonal regulation of adrenal function occurs primarily through activation of GPCRs. GPCRs are central to many of the body's endocrine and neurotransmitter pathways. Recently, it was shown that activation of GPR103 by its ligand QRFP induced feeding, locomotor activity, and metabolic rate, and QRFP is bioactive in adipose tissue of obese individuals. Given that the adrenal gland is a pivotal organ for energy balance and homeostasis, we hypothesized that GPR103 and QRFP are involved in steroidogenic responses. Using qRT-PCR and immunohistochemistry, we mapped both GPR103 and QRFP in human fetal and adult adrenal gland as well as rat adrenals. Both were primarily localized in the adrenal cortex but not in the medulla. Activation of GPR103 in human adrenocortical H295R cells led to a decrease in forskolin-increased cAMP and an increase of intracellular Ca2+ levels. In addition, treatment of H295R cells with QRFP induced aldosterone and cortisol secretion as measured by ELISA. These increases were accompanied by increased expression and activity of StAR, CYB11B1, and CYP11B2 as assessed by qRT-PCR and luciferase reporter assay, respectively. Using specific inhibitors, we also demonstrated that aldosterone induction involves MAPK, PKC, and/or T-type Ca2+ channel-dependent pathways. These novel data demonstrate that QRFP induces adrenal steroidogenesis in vitro by regulating key steroidogenic enzymes involving MAPK/PKC and Ca2+ signaling pathways.

The effect of 17 beta-estradiol and testosterone on glucocorticoid secretion were studied in vitro by using dispersed inner adrenocortical cells obtained from gonadectomized female and male rats. Independently of the sex of animals, estradiol enhanced basal, but not ACTH-stimulated corticosterone (B) secretion; conversely, testosterone inhibited ACTH-stimulated, but not basal B output. HPLC analysis of steroid secreted demonstrated that estradiol induced comparable rises (53-62%) in basal pregnenolone (PREG) and total post-PREG secretion (progesterone, 11-deoxycorticosterone and B). Testosterone inhibited by about 30% ACTH-stimulated PREG production and by about 54% total post-PREG secretion (B was decreased to 56% of the control value, and other steroid hormones were below the limit of sensitivity of our assay system). These findings indicate that sex hormones directly affect rat adrenocortical secretion, mainly by acting on the rate-limiting step of steroidogenesis (i.e. the conversion of cholesterol to PREG); moreover, they suggest that testosterone is also able depress the activity of the enzymes operating distally to cholesterol side-chain cleavage.

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 occured 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.

The effect of an i.p. bolus injection of 200 micrograms.kg-1 bacterial lipopolysaccharide (LPS) on the plasma concentrations of ACTH and corticosterone (B) were studied in intact and hypophysectomized/ACTH replaced (Hx) rats. Hormonal blood levels were measured by RIA, 30, 60, 120, 180 and 240 min after the injection. The stress evoked by the vehicle i.p. injection provoked significant rises in ACTH and B blood levels at 30 and 60 min in intact rats, but not in Hx animals. In intact rats, LPS enhanced (over the respective control value) ACTH plasma level at 60, 120 and 180 min, and B plasma concentration at 120, 180 and 240 min. In Hx rats, LPS did not affect ACTH blood level, but raised B plasma concentration at 60, 120 and 180 min. B response to LPS at 120 min was completely annulled, in both intact and Hx rats, by the simultaneous administration of 25 nmol.kg-1 alpha-helical-CRH and corticotropin-inhibiting peptide that are competitive inhibitors of CRH and ACTH, respectively. The hypothesis is advanced that LPS may activate hypothalamo-pituitary adrenal axis in rats, by stimulating not only the central (hypothalamo-pituitary), but also the peripheral (intra-adrenal) branch of the CRH/ACTH system.

Enucleation-induced rapid proliferation of adrenocortical cells and restoration of adrenals structure requires formation of new blood vessels. The performed studies aimed to select from around 30,000 transcripts, identified by means of Affymetrix ® Rat Gene 1.1 ST Array, the genes involved in angiogenesis in the course of enucleation-induced adrenal regeneration and to characterize their expression levels in regenerating gland between days 1 and 15 after surgery. At day 1 of regeneration almost 2000 genes showed more than 2-fold up/down-regulation. At days 1–3 after surgery the highest expression demonstrated genes involved in the development of inflammation and blood clot formation. From around 2000 genes we selected genes involved in angiogenesis. During the regeneration 62 genes involved in angiogenesis were identified as up- or down-regulated. Some data were also validated by QPCR. Levels of Vegfa and Kdr (Vegfr-2) mRNAs were very low at day 1 of regeneration and remained unchanged thereafter. The highest expression of Figf gene was found at day 5 while that of Vwf gene at days 1 and 2 after surgery. Levels of Thy1 mRNA increased notably between days 2 and 5 of the experiment. In comparison to control rats, Mc2r (ACTH receptor) expression was lowered at day 1 of the experiment and remained unchanged thereafter. This suggests that enucleation-induced adrenal neoangiogenesis does not require elevated expression of ACTH receptor. Results of our studies strongly suggest that enucleation-induced adrenal regeneration is an angiogenesis-dependent process. Moreover, immunohistochemistry suggests that regenerating adrenal parenchymal cells release numerous angiogenic factors which paracrinally may regulate formation of new vessels.

Production of n-octanoyl-modified ghrelin (GHREL), an active form of the peptide requires prohormone processing protease and GHREL O-acyltransferase (GOAT), as well as n-octanoic acid. Recently a selective GOAT antagonist (GO-CoA-Tat) was invented and this tool was used to study the possible role of endogenous GHREL in regulating HPA axis function in the rat. Administration of GOAT inhibitor (GOATi) resulted in a notable decrease in plasma ACTH, aldosterone and corticosterone concentrations at min 60 of experiment. Octanoic acid (OA) administration had no effect on levels of studied hormones. Plasma levels of unacylated and acylated GHREL remained unchanged for 60min after either GOATi or OA administration. Under experimental conditions applied, no significant changes were observed in the levels of GOAT mRNA in hypothalamus, pituitary, adrenal and stomach fundus. After GOATi injection hypothalamic CRH mRNA levels were elevated at 30 min and pituitary POMC mRNA levels at 60 min. Both GOATi and OA lowered basal, but not K(+)-stimulated CRH release by hypothalamic explants and had no effect on basal or CRH-stimulated ACTH release by pituitary slices. Neither GOATi nor OA affected corticosterone secretion by freshly isolated or cultured rat adrenocortical cells. Thus, results of our study suggest that in the rat endogenous GHREL exerts tonic stimulating effect on hypothalamic CRH release. This effect could be demonstrated by administering rats with selected inhibitor of ghrelin O-acyltransferase, the enzyme responsible for GHREL acylation, a process which is absolutely required for both GHSR-1a binding and its central endocrine activities.

VIP dose-dependently increased basal, but not submaximally ACTH (10 −10 M)-stimulated, aldosterone (ALDO) and corticosterone (B) secretion of dispersed rat capsular and inner adrenocortical cells, respectively. The maximal stimulatory effect (60–70% rise) was obtained with a VIP concentration of 10 −8 M. [4-Cl-D-Phe 6 ,Leu 17 ]-VIP, a VIP-receptor antagonist (VIP-A), and corticotropin inhibiting peptide (CIP), an ACTH receptor antagonist (both 10 −6 M), completely annulled VIP (10 −8 M)-evoked rises in basal ALDO and corticosterone secretions. The ACTH (10 −10 M)-enhanced (about 5-fold) production of both hormones was completely reversed by CIP (10 −6 M) and only partially reduced (about −30%) by VIP-A (10 −6 M). The hypothesis is advanced that the weak secretagogue effect of VIP on dispersed rat capsular and inner adrenocortical cells may be due to its positive interaction with ACTH receptors.

The effect of a s.c. bolus injection of 2 micrograms galanin on the hypothalamo-pituitary-adrenocortical (HPA) axis were investigated in both normal and ether-stressed (2 min ether-vapor inhalation) or cold-stressed (20 min at 4 degrees C) rats. Blood concentrations of ACTH, aldosterone (ALDO) and corticosterone (B) were measured by specific RIA, 1, 2 or 4 h after galanin injection. Galanin administration to normal rats resulted in a marked rise in the blood levels of ACTH, ALDO and B at 1h and 2 h, the values returned to the baseline after 4 h. Ether and cold stresses notably raised the blood levels of ACTH, ALDO and B, and these rises lasted unchanged until 4 h. Galanin markedly potentiated ACTH and ALDO responses to ether stress at 1 and 2 h, but B response remained unchanged. ACTH response to cold stress was not affected by galanin; however, galanin magnified ALDO response to cold stress at 4 h, and enhanced at 1 h and depressed at 2 h that of B. In light of these findings the following conclusions can be drawn: (i) galanin exerts a stimulatory effect on HPA axis of rats under basal conditions; (ii) under our experimental conditions, ether stress exerts a stronger stimulation of HPA axis than cold stress; (iii) the galaninergic mechanisms involved in the stimulation of ACTH release do not interfere with ether stress-activated ones controlling ACTH secretion, and are probably similar to those underlying the effect of cold stress; (iv) steroidogenic capacity of adrenal cortex, at least in term of glucocorticoid hormones, is a rate-limiting step in the response of rat HPA axis to severe stresses; and (v) galanin exerts a direct secretory action of the rat adrenal gland, that can manifest itself only in the case of submaximally cold stress-stimulated HPA axis.

Adult female rats were sc injected with 0.6 or 1.2 nmol/100 g body wt of neurotensin (NT); control animals received a sc injection of saline vehicle. The plasma concentrations of ACTH, aldosterone and corticosterone were measured 1, 2 or 4 after the injection. Our RIA estimates revealed that the lower dose of NT exerted a net stimulatory effect on the rat pituitary-adrenocortical function, that can be ascribed to the well-known NT-evoked enhancement of CRH release. Conversely, the higher dose of NT appeared to have a marked inhibitory effect on both pituitary ACTH release and adrenocortical secretion. The mechanism underlying this last action of NT remains to be elucidated.

Leptin is known to play a role in the modulation of metabolism and control of breathing acting mainly on central nervous structures, although additional actions on peripheral arterial chemoreceptors have also been suggested in the literature. We therefore examined by means of immunohistochemistry the expression of leptin and leptin receptors in the carotid bodies of rats and humans. Leptin expression and relative expression of leptin receptor isoforms were also studied in rats by real-time PCR. No leptin or leptin receptor immunoreactivities were visible in the type II cells of either series. In rat carotid bodies, diffuse positive stainings for leptin and leptin receptors (both with antibody recognizing all receptor isoforms and antibody specific for Ob-Rb) were observed in type I cells. In human carotid bodies, the mean percentage (±standard error) of leptin immunoreactive type I cells was 39.4%±5.1% and the percentages of leptin receptor immunoreactive type I cells were 57.3%±3.9% with antibody recognizing all receptor isoforms and 33.3%±4.2% with antibody specific for isoform Ob-Rb. Double immunofluorescences with anti-tyrosine hydroxylase (type I cell marker) and anti-glial fibrillary acidic protein (type II cell markers) confirmed the selective location of leptin and Ob-Rb in type I cells. Real-time PCR showed the expression of leptin and Ob-Ra, Ob-Rb, Ob-Rc and Ob-Rf isoform mRNA in the rat carotid body, levels of expression being Ob-Rf>Ob-Rc>>Ob-Ra>>Ob-Rb. Ob-Re mRNA was not detected. The above findings suggest a role of circulating or locally produced leptin in the regulation of chemoreceptor discharge and/or metabolic sensing function, by means of direct action on type I cells.

A 7-day subcutaneous infusion with the AVP antagonist [Deamino-Pen 1 , Val 4 , d -Arg 8 ]-vasopressin (AVP-A; 3 nmol·kg −1 ·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 ·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.

Summary Adult female rats were treated with prazosin, a selective α 1 -adrenoceptor inhibitor, for 60 days in doses of 0.5 mg/kg/day or 0.5 mg/kg/8 h. Prolonged prazosin administration lowered the gain of body weight and significantly decreased systolic blood pressure and heart weight. Prazosin administered every 8 h also lowered serum triglycerides but did not change the total cholesterol level. Selective α 1 -inhibitor treatment increased the adrenal gland weight and decreased the number of adrenocortical cells; in every 8 h treated group this effect being connected with a marked cell hypertrophy (in all adrenocortical zones). Our results suggest that prazosin induced changes in the structure of the rat adrenal cortex depend on activation of the hypothalamo-pituitary-adrenocortical axis and possibly on the small inhibition of the proliferative activity of rat adrenocortical cells.

Atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides act by binding to three main subtypes of receptors, named NPR-A, -B, and -C. NPR-A and NPR-B are coupled with guanylate cyclase. Not only NPR-C is involved in removing natriuretic peptides from the circulation but it also acts through inhibition of adenylyl cyclase. NPR-A binds ANP and BNP; NPR-B preferentially binds CNP; and NPR-C binds all natriuretic peptides with similar affinities. All natriuretic peptides and their receptors are widely present in the hypothalamus, pituitary, adrenal cortex, and medulla. In the hypothalamus, they reduce norepinephrine release, inhibit oxytocin, vasopressin, corticotropin-releasing factor, and luteinizing hormone-releasing hormone release. In the hypophysis, natriuretic peptides inhibit basal and induced ACTH release. Conversely, the effects of natriuretic peptides on secretion of growth, luteinizing, and follicle-stimulating hormones are not clear. Natriuretic peptides are known to inhibit basal and stimulated aldosterone secretion, through an increase of intracellular cGMP, and to inhibit the growth of zona glomerulosa. Inhibition or stimulation of glucocorticoid secretion by adrenocortical cells has been reported on the basis of the species involved, and an indirect effect mediated by adrenalmedullary cells has been hypothesized. In the adrenal medulla, natriuretic peptides inhibit catecholamine release and increase catecholamine uptake. It appears that natriuretic peptides may play a role in the pathophysiology of adrenocortical neoplasias and pheochromocytomas.

Spexin is a highly conserved peptide which was recently identified through the bioinformatics approach. Immunohistochemical analysis of its expression has not yet been performed. Thus, in this study, we examined spexin location in a wide range of rat organs by both RT-PCR and IHC. RT-PCR identified spexin mRNA in all tissues examined. Spexin immunoreaction was mainly cytoplasmic. Spexin was immunohistochemically detected, although with different staining intensities, in epithelia and glands of skin and respiratory, digestive, urinary, and reproductive systems. Smooth muscle cells showed weak immunostaining, and connective tissue was negative. In the central nervous system, neuronal groups showed different intensities for reaction product. Immunoreaction was also found in ganglionic cells of both trigeminal and superior cervical ganglia and in photoreceptor, inner nuclear, and ganglionic layers of the retina. In the endocrine system, spexin immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei; adenohypophysis, thyroid, and parathyroid glands; adrenal cortex and medulla (mainly ganglionic cells); Leydig cells; and thecal, luteal, and interstitial cells of the ovary. Because of its widespread expression, spexin is probably involved in many different physiological functions; in particular, location of spexin in neurons and endocrine cells suggests its roles as neurotransmitter/neuromodulator and endocrine factor. (J Histochem Cytochem 58:825–837, 2010)