Rat Adrenocortical Cells Research Articles

Exendin-4, a GLP-1 Receptor Agonist, Stimulates Rat Pituitary-Adrenocortical Axis

Preproglucagon-derived peptides, among which glucagon and glucagon-like peptide-1 (GLP-1), are known to modulate the activity of the hypothalamo-pituitary-adrenal (HPA) axis. Many effects of GPL-1, however, are preventedby its very short half-life. Hence we investigated in vivo and in vitro the effects on the rat HPA axis of the GLP-1-receptor agonist exendin-4 (EX4), which possesses a longer biological half-life than GLP-1. The bolus systemic administration of EX4 (12 nmol/kg) evoked significant rises in ACTH, aldosterone and corticosterone concentrations. EX4 (10 - 7 M) induced a weak but significant increase in corticosterone secretion from dispersed rat adrenocortical cells, and the effect was abrogated by the GLP-1-receptor antagonist exendin (9-39). Collectively, our findings indicate that EX4, acting via GLP-1 receptors, stimulates HPA axis in rats, acting on both its central (hypothalamo-pituitary) and peripheral (adrenocortical) branch.

Specificity of a new lipid mediator produced by testicular and peritoneal macrophages on steroidogenesis.

Macrophage-derived factor (MDF) is a lipophilic factor produced by rat testicular and peritoneal macrophages that maximally stimulates testosterone production by rat Leydig cells through a steroidogenic acute regulatory protein independent mechanism. The purpose of the present study was to determine whether MDF is also produced by human macrophages, and/or if it acts on human steroidogenic cells. We also studied the tissue-specific functions of MDF by determining if it also acts on steroidogenic cells of the ovary and adrenal glands and, if so, does it require new protein synthesis. It was found that MDF was produced by human peritoneal macrophages, and was capable of stimulating human steroidogenic cells. In terms of tissue specificity, it was found that primary cultures of rat adrenocortical cells respond to MDF with increased secretion of aldosterone and corticosterone, as did rat granulosa cells by producing progesterone. MDF acted in the presence of cycloheximide, indicating that it does not require new protein synthesis. These results indicate that MDF may have significant therapeutic potential and provide a basis for future studies concerning its physiological role in humans. These results further suggest that MDF is not only involved in paracrine regulation of Leydig cells, but also has the potential for the local regulation of steroidogenesis in both granulosa and adrenal cortical cells.

Tyrphostin-23 Enhances Steroid-Hormone Secretion from Dispersed Human and Rat Adrenocortical Cells

Tyrphostin-23 is commonly used as inhibitor of tyrosine kinase (TK). We found that tyrphostin-23 concentration-dependently increased basal steroid-hormone secretion from dispersed human and rat adrenocortical cells, the maximal effective concentration being 10-5 M Tyrphostin-23 (10-5 M) enhanced 10-9 Mangiotensin-II- and endothelin-1-stimulated secretion of human and rat adrenocortical cells, but not the secretory response to 10-9 MACTH. However, it increased the response to lower concentrations (10-12 or 10-11 M) of ACTH The secretagogue effect of tyrphostin-23 on dispersed rat adrenocortical cells was abolished by either the adenylate cyclase inhibitor SQ-22536 (10-4 M) or the protein kinase A (PKA) inhibitor H-89 (10-5 M). Tyrphostin-23 (10-5 M) raised basal cyclic-AMP release by dispersed rat adrenocortical cells, but in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 10-3 M) it was ineffective Both tyrphostin-23 and BMX increased cyclic-AMP release by rat adrenocortical cells in response to 10-10 M ACTH, and their effects were not additive Taken together, our findings suggest that tyrphostin-23, acting as an inhibitor of phosphodiesterases in adrenocortical cells, increases the intracellular concentration of cyclic-AMP available for PKA activation thereby stimulating steroid-hormone secretion They also stress that caution must be used in interpreting the results of studies aimed at investigating the possible cross-talk between adenylate cyclase- and TK-dependent signaling cascades.

P-589 - The sources of steroidogenic cholesterol on steroidogenesis in rat adrenocortical cells

P-589 - The sources of steroidogenic cholesterol on steroidogenesis in rat adrenocortical cells

Daily Regeneration of Rat Adrenocortical Cells: Circadian and Zonal Variations in Cytogenesis

Daily regeneration of rat adrenocortical cells were investigated in terms of circadian and zonal variations by following the cells at the DNA-synthesizing stage. An S-phase was assessed by 5-bromo-2′-deoxyuridine (BrdU) incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The BrdU-positive cells were observed throughout the day mainly in two regions of the adrenal cortex, i.e. the innermost portion of the zona glomerulosa and the outermost portion of the zona fasciculata. Cells only in a latter region showed a distinct circadian rhythm of cell proliferation with a peak at 3–4 a.m. A remarkable rise in the plasma adrenocorticotropin (ACTH) concentration preceded such an increase in the cell proliferation by about 4 hours. This phenomenon could be mimicked by raising the plasma ACTH concentration by the administration of Cortrosyn Z or metyrapone. Angiotensin II-stimuli induced by Na-deficiency increased the proliferation of zona glomerulosa cells in the former region at 6–7p.m without significant effects on that of the zona fasciculata cells in the latter region. Thus at least two sites, which respond differentially to the day/night cycle and circulating hormone levels, exist in rat adrenal cortex being responsible for the cytogenesis in this endocrine organ.

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10 −8 M. In contrast, orexins did not affect either maximally ACTH (10 −9 M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10 −6 M) annulled corticosterone response of ZF/R cells to ACTH (10 −9 M), but not to orexins (10 −8 M). Orexins (10 −8 M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10 −5 M) abolished corticosterone responses to both ACTH (10 −9 M) and orexins (10 −8 M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.

Characterization of serotonergic receptors in frog, rat and human adrenocortical cells

Characterization of serotonergic receptors in frog, rat and human adrenocortical cells

Effects of glucagon and glucagon-like peptide-1 on glucocorticoid secretion of dispersed rat adrenocortical cells

The effects of glucagon and glucagon-like peptide-1 (GLP-1) on the secretory activity of rat adrenocortical cells have been investigated in vitro Neither hormones affected basal or agonist-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells or basal corticosterone production of zona fasciculata-reticularis (inner) cells In contrast, glucagon and GLP-I partially (40%) inhibited ACTH (10 −9 M)-enhanced corticosterone secretion of inner cells, maximal effective concentration being 10 −7 M. The effect of 10 −7 M glucagon or GPL-1 was suppressed by 10 −6 M Des-His 1-[Glu 9]-glucagon amide (glucagon-A) and exendin-4(3-39) (GPL-1-A), which are selective antagonists of glucagon and GLP-1 receptors, respectively Glucagon and GLP-1 (10 −7 M) decreased by about 45–50% cyclic-AMP production by dispersed inner adrenocortical cells in response to ACTH (10 −9 M), but not to the adenylate cyclase activator forskolin (10 −5 M). Again this effect was blocked by 10 −6 M glucagon-A or GLP-1-A. The exposure of dispersed inner cells to 10 −7 M glucagon plus GLP-1 completely suppressed corticosterone response to ACTH (10 −9 M) However, they only partially inhibited (by about 65–70%) both corticosterone response to forskolin (10 −5 M) or dibutyryl-cyclic-AMP (10 −7 M) and ACTH (10 −9 M)-enhanced cyclic-AMP production. Quantitative HPLC showed that 10 −7 M glucagon or GLP-1 did not affect ACTH-stimulated pregnenolone production, evoked a slight rise in progesterone and 11-deoxycorticosterone release, and markedly reduced (by about 55%) corticosterone secretion of dispersed inner adrenocortical cells. In light of these findings the following conclusion are drawn: (i) glucagon and GLP-1, via the activation of specific receptors, inhibit glucocorticoid response of rat adrenal cortex to ACTH; and (ii) the mechanism underlying the effect of glucagon and GLP-1 is probably two-fold, and involves both the inhibition of the ACTH-induced activation of adenylate cyclase and the impairment of the late steps of glucocorticoid synthesis.

The inhibitor of phospholipase-A2, AACOCF3, stimulates steroid secretion by dispersed human and rat adrenocortical cells

AACOF3 is a trifluomethylketone analog of arachidonic acid, which inhibits phospholipase-A2 (PLA2). AACOCF3 was found to concentration-dependently increase basal aldosterone and corticosterone secretion by dispersed rat zona glomerulosa and zona fasciculata/reticularis cells, respectively, as well as aldosterone and cortisol production by dispersed human adrenocortical cells Maximal effective concentration was 10 −5M, and elicited about 2.5–3.0-fold rises in steroid output. 10 −5 M AACOCF3 also enhanced submaximally ( 10 −15 10 −12M ), but not maximally (10 −9 M) ACTH-stimulated hormonal secretion. Quantitative HPLC showed that 10 −5 M AACOCF3 evokes similar increases (from 2.0- to 3.0-fold) in the basal release of the entire spectrum of adrenocortical steroids ( i.e. both intermediate and definitive products of steroid synthesis), thereby suggesting that AACOCF3 acts on the early steps of steroid synthesis. Accordingly, when pregnenolone metabolism is prevented by cyanoketone, 10 −5 M AACOCF3 increased by about 8–10-fold the production of this steroid. In conclusion, we have demonstrated a side-effect of AACOCF3, which may become relevant in studies where this chemical is used to inhibit PLA2 in tissues able to convert cholesterol to pregnenolone.

P–644 - Effect of dichlorvos on the steroidogenesis in dispersed hamster, bovine and rat adrenocortical cells.

P–644 - Effect of dichlorvos on the steroidogenesis in dispersed hamster, bovine and rat adrenocortical cells.

Comparison of the Effects of VIP and PACAP on Steroid Secretion of Dispersed Rat Adrenocortical Cells

Comparison of the Effects of VIP and PACAP on Steroid Secretion of Dispersed Rat Adrenocortical Cells

Guanylin: a novel regulatory peptide possibly involved in the control of Ca2+-dependent agonist-stimulated aldosterone secretion in rats.

Guanylin is a 15-amino acid peptide, which activates guanylate cyclase (GC) and plays a major role in the regulation of water and electrolyte secretion by intestinal mucosa. The expression of guanylin prohormone has been recently demonstrated in the rat adrenal gland, and this prompted us to investigate whether guanylin, like other peptides secreted by adrenal medulla, affects the function of the adrenal cortex. Autoradiography demonstrated the presence of [125I]guanylin binding sites in the zona glomerulosa (ZG), but not zona fasciculata-reticularis. Guanylin did not change either basal or ACTH-stimulated steroid secretion of dispersed rat adrenocortical cells, but concentration-dependently (from 10(-10) M to 10(-8) M) inhibited aldosterone response of ZG (capsular) cells to both angiotensin-II (ANG-II) and K+. Guanylin (10(-8) M) blocked the aldosterone secretagogue effect of the Ca2+-channel activator BAYK-8644, and the Ca2+-ionophore ionomycin counteracted the inhibitory action of this peptide on the secretory responses of capsular cells to ANG-II and K+. As expected, guanylin did not affect cyclic-AMP release by capsular cells, but evoked a sizeable increase in cyclic-GMP production. Both the inhibitor of GMP synthase decoyinine and the GC-inhibitor LY-83583, although suppressing cyclic-GMP release, did not affect guanylin-evoked inhibition of K+-stimulated aldosterone secretion. Collectively, these findings allow us to conclude that guanylin: i) inhibits aldosterone secretion of rat ZG cells by interfering with the agonist-induced activation of voltage-gated Ca2+-channels, the stimulation of guanylate cyclase conceivably playing a negligible role; and ii) could be included in that group of regulatory peptides, secreted by medullary chromaffin cells, which are able to counteract an exceedingly high aldosterone secretion.

Corticotropin-inhibiting peptide enhances aldosterone secretion by dispersed rat zona glomerulosa cells

Corticotropin-inhibiting peptide (CIP), the 7–38 fragment of human ACTH(1–39), is known to act as an antagonist of ACTH receptors. Accordingly, CIP has been found to inhibit ACTH-stimulated glucocorticoid secretion of dispersed rat adrenocortical cells, without per se affecting the basal production. We confirmed these findings, but unexpectedly observed that CIP concentration-dependently raised basal aldosterone secretion from fresh suspensions of rat zona glomerulosa (ZG) cells, maximal effective concentration being 10 −6 M. CIP (10 −6 M) partially reversed the ZG-cell response to ACTH, but not to the Ca 2+-dependent agonists angiotensin-II (ANG-II) and K +. The aspecific ANG-II-receptor antagonist saralasin (10 −6 M) blocked the aldosterone response of ZG cells to 10 −6 M CIP, and in the presence of the Ca 2+-channel blocker verapamil CIP was ineffective. Collectively, these findings suggest that CIP enhances aldosterone secretion of rat ZG through a mechanism involving the activation of ANG-II receptors and the consequent rise in the cytosolic Ca 2+ concentration. They also stress that this side-effect of CIP must be taken into account in interpreting the results of investigations on the adrenal cortex, where CIP has been employed as an ACTH-receptor antagonist.

Effects of a water-soluble extract of Cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells.

Cordyceps sinensis contains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus-pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water-soluble extract of Cordyceps sinensis (CS) and the signaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose-dependent manner by CS, reaching a maximum at 25 microg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS-induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS-treated cells. However, combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS-induced steroidogenesis.

Selective Uptake of Low Density Lipoprotein-Cholesteryl Ester Is Enhanced by Inducible Apolipoprotein E Expression in Cultured Mouse Adrenocortical Cells

Apolipoprotein (apo) E is expressed at high levels by steroidogenic cells of the adrenal gland, ovary, and testis. The cell surface location of apoE in adrenocortical cells suggests that apoE may facilitate the uptake of lipoprotein cholesterol by either the endocytic or the selective uptake pathways, or both. To examine these possibilities, the human apoE gene was expressed in murine Y1 adrenocortical cells under control of an inducible tetracycline-regulated promoter. The results show that induction of apoE yielded a 2-2.5-fold increase in the uptake of low density lipoprotein-cholesteryl ester (LDL-CE) but had little effect on high density lipoprotein-CE uptake. Analysis of lipoprotein uptake pathways showed that apoE increased LDL-CE uptake by both endocytic and selective uptake pathways. In terms of cholesterol delivery to the adrenal cell, the apoE-mediated enhancement of LDL-CE selective uptake was quantitatively more important. Furthermore, the predominant effect of apoE expression was on the low affinity component of LDL-CE selective uptake. LDL particles incubated with apoE-expressing cells contained 0.92 +/- 0.11 apoE molecules/apoB after gel filtration chromatography, indicating stable complex formation between apoE and LDL. ApoE expression by Y1 cells was necessary for enhanced LDL-CE selective uptake. This result may indicate an interaction between apoE-containing LDL and cell surface apoE. These data suggest that apoE produced locally by steroidogenic cells facilitates cholesterol acquisition by the LDL selective uptake pathway.

ACTH-mediated glucocorticoid and mineralocorticoid production is inhibited by an inositolphosphoglycan and a glycosylphosphatidylinositol-phospholipase C is activated by the hormone in mammalian adrenocortical cells

In the present paper, we report that an inositolphosphoglycan (IPG), derived from a Trypanosoma cruzi glycoinositolphosphoceramide (LPPG), is able to inhibit ACTH-mediated accumulation of a glucocorticoid, cortisol, in calf adrenocortical cells. This IPG is also able to inhibit the stimulation by ACTH of the production of the main glucocorticoid, corticosterone and the main mineralocorticoid, aldosterone, in rat adrenocortical cells. Nitrous acid deamination confirmed that IPG is responsible for this inhibition. In order to study the involvement of glycosylphosphatidylinositol (GPI) in ACTH response in rat adrenal cortex, the activation of a phospholipase that hydrolyzes GPI (GPI-PLC) was evaluated. It was found that the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium is increased in rat adrenocortical cells by ACTH treatment. In addition, ACTH stimulates the release of ceramide from the glycoinositolphosphoceramide purified from T. cruzi. These data suggest that ACTH activates a GPI-PLC in rat adrenal cortex, which is in agreement with our previous data in calf adrenocortical cells; thus, the hydrolysis of GPI provoked by ACTH takes place in different mammals and the IPG released could inhibit ACTH-mediated synthesis of aldosterone, corticosterone and cortisol.

Acth regulates ldl receptor and CLA-1 mRNA in the rat adrenal cortex

Rat adrenocortical cells utilize both low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol for steroid hormone production. In addition to exogenous lipoprotein-derived cholesterol, cells produce cholesterol de novo. Adrenocorticotropin (ACTH) increases both steroid hormone secretion and uptake of LDL and HDL. We studied the expression of LDL receptor mRNA and CLA- I (a putative HDL receptor) mRNA in cultured rat adrenocortical cells. ACTH increased the amounts of LDL receptor mRNA during 2 to 48 h of stimulation, the highest levels being detected after 2–4 h. Similar results were obtained with cyclic AMP (cAMP) derivatives, 8-bromo cAMP (8-Br cAMP) or dibutyryl cAMP. ACTH increased CLA-1 mRNA during 2 to 24 h of stimulation, the highest levels being detected after 4 h. In conclusion, ACTH up regulates both LDL and HDL receptor mRNA in rat adrenocortical cells.

Regulation of 11β-hydroxysteroid dehydrogenase type II in rat adrenocortical cells

The regulation of 11β-hydroxysteroid dehydrogenase type II (11β-HSD2) gene expression was studied in primary cultures of rat adrenocortical cells. The protein kinase A (PKA) pathway agonists forskolin, dibutyrylcAMP and ACTH caused a 5-10 fold increase in 11β-HSD2 mRNA as determined by semiquantitative PCR. The effect of forskolin could be partially inhibited by the addition of the phorbol ester TPA, an activator of the protein kinase C (PKC) pathway. The increase in mRNA encoding 11β-HSD2 was accompanied by increased synthesis of 11β-HSD2 as measured by immunoprecipitation of labeled protein. It is concluded that both the PKA and PKC pathways are involved in the regulation of rat adrenal 1β-HSD2 gene expression.

The possible involvement of pancreatic polypeptide in the paracrine regulation of human and rat adrenal cortex

Pancreatic polypeptide (PP) is a member of a family of 36-amino acid braingut peptides, including neuropeptide Y (NPY) and polypeptide YY (PYY) and acting through many subtypes of Y receptors belonging to the superfamily of the G protein-coupled receptors. PP was found to increase both glucocorticoid and cyclic-AMP production by dispersed rat and human adrenocortical cells in a concentration-dependent manner. Minimal and maximal effective concentrations were 10-10 and 10-8 M, respectively. The glucocorticoid secretagogue effect of 10-8 M PP was blocked by the protein kinase A (PKA) unhibitor H-89, but not by the ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP) Autoradiography showed the presence of [125I]PP binding sites in the inner zones of rat and human adrenal cortex, which were not displaced by NPY, PYY, ACTH or CIP. Sizable amounts of PP-immunoreactivity were detected in the medulla of both rat and human adrenals (about 50–100 fmol/mg); this content may give rise, upon submaximal stimulation of PP release, to local intraadrenal concentrations of about 10-8/10-7 M. Collectively, these findings allow us to draw the following conclusions: (i) PP stimulates glucocorticoid secretion, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway, and (ii) PP could be included in that group of regulatory peptides, contained in adrenal medulla, which are able to control the secretory function of the cortex acting in a paracrine manner.

Steroidogenic effect of peptide histidine-isoleucine (PHI) in vitro: comparison with vip

VIP and PHI originate from the same precursor, and the two peptides share considerable sequence homology. Present studies, however, revealed notable differences in their steroidogenic effect on isolated rat adrenocortical cells. VIP seems to act through the VIP and/or PACAP receptor while PHI acts, directly or by sensitivity increase, through the ACTH receptor.