Marcin Trejter

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 [125I]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.

Adrenomedullin stimulates proliferation and inhibits apoptosis of immature rat thymocytes cultured in vitro

Adrenomedullin (AM) is a hypotensive peptide, which derives from the proteolytic cleavage of pro(p)AM, and acts through two subtypes of receptors, named L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR functions as either a calcitonin gene-related peptide (CGRP) receptor or a selective AM receptor depending on which member of a family of receptor-activity-modifying proteins (RAMPs) is expressed: RAMP1 generates CGRP receptors, while RAMP2 and RAMP3 produce AM receptors. Reverse transcription (RT)-polymerase chain reaction (PCR) consistently allowed the detection of pAM and peptidyl-glycine alpha-amidating monooxygenase (the enzyme converting immature AM to the mature peptide) mRNAs in the thymus cortex of immature (10-day-old) rats. Accordingly, radioimmune assay (RIA) measured low but sizeable AM concentrations in this tissue. RT-PCR also demonstrated the presence of the specific mRNAs of L1-R, CRLR and RAMPs. AM (from 10(-9) to 10(-7)M) increased proliferation index and lowered apoptotic index of cultured immature rat thymocytes, and the effects were annulled by the AM receptor antagonist AM(22-52). In conclusion, our study demonstrated that (1) immature rat thymus cortex expresses AM and the AM receptors L1-R and CRLR/RAMP; and (2) AM, acting via AM(22-52)-sensitive receptors, exerts a potent growth promoting effect on immature rat thymus, by enhancing proliferation and lowering apoptotic death of thymocytes. Taken together, these findings could suggest that AM may play a role in the development of immunity.

Sex-related gene expression profiles in the adrenal cortex in the mature rat: microarray analysis with emphasis on genes involved in steroidogenesis.

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.

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats

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 ERα, ERβ and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERβ mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERRα and ERRβ were similar, and only in the ZF/R of female rats ERRγ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5α-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 ERRβ receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

Visinin-like peptide 1 in adrenal gland of the rat. Gene expression and its hormonal control

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.

Enucleation-Induced Rat Adrenal Gland Regeneration: Expression Profile of Selected Genes Involved in Control of Adrenocortical Cell Proliferation

Enucleation-induced adrenal regeneration is a highly controlled process; however, only some elements involved in this process have been recognized. Therefore, we performed studies on regenerating rat adrenals. Microarray RNA analysis and QPCR revealed that enucleation resulted in a rapid elevation of expression of genes involved in response to wounding, defense response, and in immunological processes. Factors encoded by these genes obscure possible priming effects of various cytokines on initiation of regeneration. In regenerating adrenals we identified over 100 up- or downregulated genes involved in adrenocortical cell proliferation. The changes were most significant at days 2-3 after enucleation and their number decreased during regeneration. For example, expression analysis revealed a notable upregulation of the growth arrest gene, Gadd45, only 24 hours after surgery while expression of cyclin B1 and Cdk1 genes was notably elevated between days 1–8 of regeneration. These changes were accompanied by changes in expression levels of numerous growth factors and immediate-early transcription factors genes. Despite notable differences in mechanisms of adrenal and liver regeneration, in regenerating adrenals we identified genes, the expression of which is well recognized in regenerating liver. Thus, it seems legitimate to suggest that, in the rat, the general model of liver and adrenal regeneration demonstrate some degree of similarity.

Expression of selected genes involved in steroidogenesis in the course of enucleation-induced rat adrenal regeneration

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.

Angiogenesis in the course of enucleation-induced adrenal regeneration--expression of selected genes and proteins involved in development of capillaries.

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.

Effects of galanin on the secretion and proliferative activity of the immature and regenerating adrenal glands of rats.

The effects of galanin and the galanin-receptor antagonist (galanin-A) [D-Thr(6),D-Trp(8,9),15-ol]-galanin(1-15) on the immature and regenerating rat adrenal glands have been investigated in vivo. Adult female rats with adrenal regeneration and their offpring (20-day-old) were given three subcutaneous injections (28, 16, and 4 h before being killed) of 2 nmol/100 g galanin and/or galanin-A, and 0.1 mg/100 g vincristin 3 h before being killed. Plasma corticosterone concentration was measured by radioimmunoassay, and the mitotic index ( per thousand of metaphase-arrested cells) was evaluated. In immature rats, galanin increased plasma corticosterone concentration, without affecting mitotic index; the secretagogue effect was reversed by galanin-A, which alone was ineffective. In rats with regenerating adrenal, galanin-A increased both blood level of corticosterone and mitotic index; galanin was ineffective, but blocked the effects of galanin-A. These findings allowed us to draw the following conclusions: 1) galanin exerts a moderate glucocorticoid secretagogue action on immature rat adrenals, but endogenous galanin does not play a major physiological role in the functional control of the gland; and 2) endogenous galanin exerts a maximal tonic inhibitory control on both glucocorticoid secretion and proliferative activity of regenerating rat adrenals, whose physiological relevance remains to be investigated.

Neuromedin-U inhibits unilateral adrenalectomy-induced compensatory adrenal growth 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 and to control the growth of the rat adrenal cortex. The present study aimed to investigate the possible involvement of NMU in the regulation of unilateral adrenalectomy-induced compensatory adrenal growth, a phenomenon known to be neurally mediated. The right adrenal gland of mature female rats was removed, the contralateral gland was then analyzed at 24 and 72h following surgery. Groups of rats were given 3 subcutaneous injections (24, 16 and 8h before decapitation) of NMU8 (1.5 or 3.0 nmol/100g/per injection). Three hours before sacrifice all rats received an intraperitoneal injection of 0.1mg/100g body weight of vincristin. By means of RT-PCR the presence of NMUR1 mRNA was detected in adrenal cortex of both intact and hemiadrenalectomized rats. As expected, unilateral adrenalectomy-induced an increase in adrenal weight, associated with increased plasma ACTH, aldosterone and corticosterone levels. The administration of NMU to hemiadrenalectomized rats did not significantly affect these parameters. NMU administration, however, notably inhibited the unilateral adrenalectomy-induced adrenocortical cell proliferation in both zona glomerulosa and zona fasciculata, as assessed by the metaphase index and the number of parenchymal cell nuclei per unit area of the tissue. When compared to hemiadrenalectomized animals receiving saline, a significant decrease of blood corticosterone levels was observed after 24h in rats treated with the highest dose of NMU. Since these effects were independent on changes in blood ACTH, they could reflect an interaction of NMU with the neural system innervating the adrenal gland.