POD-1/Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells

During gonad and adrenal development, the POD-1/capsulin/TCF21transcription factor negatively regulates SF-1/NR5A1expression, with higher SF-1 levels being associated with increased adrenal cell proliferation and tumorigenesis. In adrenocortical tumor cells, POD-1 binds to the SF-1 E-box promoter region, decreasing SF-1 expression. However, the modulation of SF-1 expression by POD-1 has not previously been described in normal adrenal cells. Here, we analyzed the basal expression of Pod-1 and Sf-1 in primary cultures of glomerulosa (G) and fasciculata/reticularis (F/R) cells isolated from male Sprague-Dawley rats, and investigated whether POD-1 overexpression modulates the expression of endogenous Sf-1 and its target genes in these cells. POD-1 overexpression, following the transfection of pCMVMycPod-1, significantly decreased the endogenous levels of Sf-1 mRNA and protein in F/R cells, but not in G cells, and also decreased the expression of the SF-1 target StAR in F/R cells. In G cells overexpressing POD-1, no modulation of the expression of SF-1 targets, StAR and CYP11B2, was observed. Our data showing that G and F/R cells respond differently to ectopic POD-1 expression emphasize the functional differences between the outer and inner zones of the adrenal cortex, and support the hypothesis that SF-1 is regulated by POD-1/Tcf21 in normal adrenocortical cells lacking the alterations in cellular physiology found in tumor cells.

POD-1/Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells.

During gonad and adrenal development, the POD-1/capsulin/TCF21transcription factor negatively regulates SF-1/NR5A1expression, with higher SF-1 levels being associated with increased adrenal cell proliferation and tumorigenesis. In adrenocortical tumor cells, POD-1 binds to the SF-1 E-box promoter region, decreasing SF-1 expression. However, the modulation of SF-1 expression by POD-1 has not previously been described in normal adrenal cells. Here, we analyzed the basal expression of Pod-1 and Sf-1 in primary cultures of glomerulosa (G) and fasciculata/reticularis (F/R) cells isolated from male Sprague-Dawley rats, and investigated whether POD-1 overexpression modulates the expression of endogenous Sf-1 and its target genes in these cells. POD-1 overexpression, following the transfection of pCMVMycPod-1, significantly decreased the endogenous levels of Sf-1 mRNA and protein in F/R cells, but not in G cells, and also decreased the expression of the SF-1 target StAR in F/R cells. In G cells overexpressing POD-1, no modulation of the expression of SF-1 targets, StAR and CYP11B2, was observed. Our data showing that G and F/R cells respond differently to ectopic POD-1 expression emphasize the functional differences between the outer and inner zones of the adrenal cortex, and support the hypothesis that SF-1 is regulated by POD-1/Tcf21 in normal adrenocortical cells lacking the alterations in cellular physiology found in tumor cells.

Interleukin-6 inhibits adrenal androgen release from bovine adrenal zona reticularis cells by inhibiting the expression of steroidogenic proteins.

Interleukin-6 (IL-6) is secreted by adrenocortical cells and modifies cortisol secretion. In this study, the effects of IL-6 on adrenal androgen release were investigated. The zona reticularis (ZR) was generally isolated from bovine adrenal glands by dissection. In select experiments, the intact adrenal cortex (ie, all 3 adrenocortical zones) was dissected from the adrenal glands. For androgen release experiments, ZR and intact adrenocortical cubes were dispersed into isolated cells, the cells cultured and exposed to IL-6 and/or adrenocorticotropic hormone (ACTH), and androgen release determined by radioimmunoassay. Basal and ACTH-stimulated androgen release from the ZR was inhibited by IL-6 in a concentration-dependent (10-1000 pg/mL) and time-dependent (4-24 h) manner (P < 0.01 by 1-way analysis of variance and the Bonferroni test). In contrast, IL-6 increased basal and ACTH-stimulated androgen release from mixed adrenocortical cells (P < 0.01). The mechanism of IL-6 inhibition of androgen release was investigated by exposing ZR strips to IL-6 and measuring the expression of the messenger RNA (mRNA) and protein of steroidogenic factors. Basal and ACTH-stimulated expression of the mRNA and protein for steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 3-ß-hydroxysteroid dehydrogenase type 2, steroid 17-α-hydroxylase/17,20 lyase/17,20 desmolase, and the nuclear factor steroidogenic factor 1 (SF-1), that stimulates steroidogenesis, were decreased by IL-6 (P < 0.01). In contrast IL-6 increased the mRNA and protein for dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1), a nuclear factor that inhibits steroidogenesis (P < 0.01). In summary, IL-6 decreased androgen release and the expression of steroidogenic factors in the ZR, and this decrease may be mediated in part through increasing DAX-1 and decreasing SF-1.

The proliferative effect of synthetic N-POMC(1-28) peptides in rat adrenal cortex: a possible role for cyclin E.

Modified synthetic N-POMC(1-28) without disulfide bridges has been shown to act as an adrenal mitogen. Cyclins and their inhibitors are the major cell cycle controls, but in the adrenal cortex the effect of ACTH and N-POMC on the expression of these proteins remains unclear. In this work, we evaluate the effect of different synthetic N-POMC peptides on the S-phase of the cell cycle. In addition, we examine the cyclin E expression in rat adrenal cortex. Rats treated with dexamethasone were injected with ACTH and/or synthetic modified N-POMC and/or synthetic N-POMC with disulfide bridges. DNA synthesis was determined by BrdU incorporation and protein expression was analyzed by immunoblotting and immunohistochemistry. The results showed that similarly to modified N-POMC without disulfide bridges, administration of synthetic N-POMC with disulfide bridges and the combination of ACTH and N-POMC promoted an increase of BrdU-positive nuclei in adrenal cortex. However, the proliferative effect of N-POMC was comparable to that of ACTH only in the zona glomerulosa. An increase in cyclin E expression was observed 6 h after N-POMC treatment in the outer fraction of the adrenal cortex, in agreement with immunohistochemical findings in the zona glomerulosa. In summary, the effect of synthetic N-POMC with disulfide bridges was similar to modified synthetic N-POMC, increasing proliferation in the adrenal cortex, confirming previous evidence that disulfide bridges are not essential to the N-POMC mitogenic effect. Moreover, cyclin E appears to be involved in the N-POMC- and ACTH-stimulated proliferation in the zona glomerulosa of the adrenal cortex.

Development of the human adrenal zona reticularis: morphometric and immunohistochemical studies from birth to adolescence.

Age-related morphologic development of human adrenal zona reticularis (ZR) has not been well examined. Therefore, in this study, 44 human young adrenal autopsy specimens retrieved from large archival files (n=252) were examined for immunohistochemical and morphometric analyses. Results demonstrated that ZR became discernible around 4 years of age, and both thickness and ratio per total cortex of ZR increased in an age-dependent fashion thereafter, although there was no significant increment in total thickness of developing adrenal cortex. We further evaluated immunoreactivity of both KI67 and BCL2 in order to clarify the equilibrium between cell proliferation and apoptosis in the homeostasis of developing human adrenals. Results demonstrated that proliferative adrenocortical cells were predominantly detected in the zona glomerulosa and partly in outer zona fasciculata (ZF) before 4 years of age and in ZR after 4 years of age, but the number of these cells markedly decreased around 20 years of age. The number of BCL2-positive cells increased in ZR and decreased in ZF during development. Adrenal androgen synthesizing type 5 17beta-hydroxysteroid dehydrogenase (HSD17B5 or AKR1C3 as listed in the Hugo Database) was almost confined to ZR of human adrenals throughout development. HSD17B5 immunoreactivity in ZR became discernible and increased from around 9 years of age. Results of our present study support the theory of age-dependent adrenocortical cell migration and also indicated that ZR development is not only associated with adrenarche, but may play important roles in an initiation of puberty.

Effects of evodiamine and rutaecarpine on the secretion of corticosterone by zona fasciculata-reticularis cells in male rats.

Evodiamine (EVO) and rutaecarpine (RUT) are two bioactive alkaloid isolated from Chinese herb named Wu-Chu-Yu. Previous studies have shown that EVO and RUT possess thermoregulation, vascular regulation, anti-allergic, anti-nociceptive and anti-inflammatory activities. The mechanisms of EVO and RUT effect on steroidogenesis are not clear. The goal of this study was to characterize the mechanism by which EVO and RUT affect corticosterone production in rat zona fasciculata-reticularis (ZFR) cells. ZFR cells were isolated from adrenal glands of male rats and incubated with adrenalcorticotropin (ACTH, 10(-9) M), forskolin (an adenylyl cyclase activator, 10(-5) M), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP, a permeable cAMP analog, 10(-4) M), or steroidogenic precursors including 25-hydroxycholesterol, pregnenolone, progesterone, and deoxycorticosterone, 10(-5) M each, in the presence or absence of EVO and RUT respectively (0-10(-3) M) at 37 degrees C for 1 h. The concentrations of corticosterone, pregnenolone and progesterone in the media were measured by radioimmunoassay. After administration of ZFR cells with EVO or RUT (10(-4) M) for 60 and 120 min, Western blot analysis was employed to explore the influence of EVO and RUT on the expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). EVO and RUT reduced both basal and ACTH-, forskolin-, as well as 8-Br-cAMP-stimulated corticosterone production in rat ZFR cells. The enhanced corticosterone production caused by the administration of four steroidogenic precursors was decreased following EVO or RUT challenge. These results suggest that EVO and RUT inhibit corticosterone production in rat ZFR cells via a mechanism involving: (1) a decreased activity of cAMP-related pathways; (2) a decreased activity of the steroidogenic enzymes, that is, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 11beta-hydroxylase (P450c11), during steroidogenesis; and (3) an inhibition of StAR protein expression.

Mechanisms of inhibition of dehydroepiandrosterone upon corticosterone release from rat zona fasciculata-reticularis cells.

We have demonstrated that dehydroepiandrosterone (DHEA) acts directly on rat zona fasciculata-reticularis (ZFR) cells to diminish corticosterone secretion by an inhibition of post-cAMP pathway, and decreases functions of steroidogenic enzymes after P450(scc) as well as steroidogenic acute regulatory (StAR) protein expression. However, the mechanisms by which DHEA engages with environmental messenger signals which translate into interfering StAR protein expression are still unclear. This study explored the effects of DHEA on the phosphorylation/activation of extracellular signal-regulated kinases (ERKs). ERK activation resulted in enhancing phosphorylation of steroidogenic factor-1 (SF-1) and increased StAR protein expression. ZFR cells were incubated in the presence or absence of adrenocorticotropin (ACTH), forskolin (FSK), 25-OH-cholesterol, U0126, and H89 at 37 degrees C. The concentration of corticosterone released into the media was measured by radioimmunoassay (RIA). The cells were used to extract protein for Western blot analysis of ERKs or StAR protein expression or immunoprecipitation of SF-1 analysis. The results suggested that (1) ERK pathway of rat ZFR cells might be PKA dependent, (2) ERK activity was required for SF-1 phosphorylation to upregulate steroidogenesis in rat ZFR cells, and (3) DHEA did not affect ERK phosphorylation, however, it attenuated forskolin-stimulated SF-1 phosphorylation to affect StAR protein expression.

Distribution of cells expressing Jun and Fos proteins and synthesizing DNA in the adrenal cortex of hypophysectomized rats: regulation by ACTH and FGF2.

Protein expression of the early response genes, jun and fos, has been suggested to play an important role in the in vitro and in vivo proliferation of adrenal cells. To elucidate the immunolocalization of proliferative cells and the patterns of adrenal gland expression of members of the activating protein-1 (AP-1) family of oncogenes, we used hypophysectomized rats. The effects of adrenocorticotropic hormone (ACTH) and fibroblast growth factor 2 (FGF2) on Fos and Jun protein expression were investigated, and DNA synthesis was assessed by using bromodeoxyuridine (BrdU) incorporation. No change was detectable in the adrenal cortex at 2 days after hypophysectomy, although a reduction occurred in the number of BrdU-positive cells in the zona fasciculata. This hypophysectomy-induced early phase of adrenal cortex atrophy in the zona fasciculata was correlated with JunB protein induction, suggesting the formation of an inhibitory AP-1 complex. Accumulation of c-Jun/JunD and c-Fos/FosB, but not of JunB, in the zona fasciculata and zona reticularis implied that, after ACTH stimulation, these proteins were the principal AP-1 components in these zones. In these same zones, ACTH increased BrdU-positive cell counts, indicating that the composition of the AP-1 complex in these zones was proliferation-related. However, FGF2 induced an antagonistic modulation of the response to ACTH, by reducing the numbers of Jun-/Fos-positive cells and inhibiting DNA synthesis. Our results implicate the AP-1 family of transcription factors (in particular, the dynamics within the Jun protein family) in the regulation of cell control during ACTH-induced proliferation of the adrenal cortex.

Immunohistochemical Jun/Fos protein localization and DNA synthesis in rat adrenal cortex after treatment with ACTH or FGF2.

In vitro and in vivo studies have suggested that the expression of the early response genes for Jun and Fos proteins plays an important role in adrenal cell proliferation. In order to study the expression pattern of the activating protein-1 (AP-1) family of oncogenes in the adrenal gland, we have used immunohistochemistry to localize Jun and Fos protein expression in rat adrenal cortex infused in situ with adrenocorticotropic hormone (ACTH), fibroblast growth factor 2 (FGF2), or both. The expression of AP-1 factors has been found to be correlated with in vivo ACTH and FGF2 proliferation in rats treated with dexamethasone and bromodeoxyuridine (BrdU). Induction of c-Jun and c-Fos in the zona fasciculata and of FosB in the zona reticularis suggests that, after ACTH stimulation, these proteins are the main AP-1 components in these zones. In vivo, ACTH increases BrdU-positive cells in the zona fasciculata and zona reticularis suggesting that the composition of AP-1 complexes in these zones is correlated with proliferation. Patterns of Fos and Jun induction by FGF2 do not resemble those after ACTH induction. However, in isolation, neither affects the zona glomerulosa. In the zona fasciculata, and more so in the zona reticularis, FGF2 modulates responses to ACTH, reducing the numbers of Jun-positive cells, Fos-positive cells, and DNA synthesis. This indicates that FGF2 antagonizes ACTH, and that ACTH thus controls the trophic effect independently of exogenous FGF2. Our results implicate the AP-1 family of transcription factors in the regulation of cell progression and the control of ACTH-induced proliferation in the zona fasciculata and zona reticularis.

Effects of crude adlay hull acetone extract on corticosterone release from rat zona fasciculata-reticularis cells.

Adlay is a grass crop which has been used in traditional Chinese medicine and also as a nourishing food. It has been shown to posses anti-allergic, antimutagenic and hypolipemic effects. However, the effects and action mechanisms of crude adlay hull acetone extract (AHA) on adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of AHA on corticosterone release. ZFR cells were incubated with AHA in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3': 5'- adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxy cholesterol (25-OH-cholesterol), pregnenolone, progesterone or deoxycorticosterone. The concentrations of corticosterone or pregnenolone in the media were measured by radioimmunoassay (RIA). The cells were used to measure the expression of steroidogenic acute regulatory (StAR) protein by Western blot. The present data demonstrated that: (1) AHA inhibited ACTH-, 8-Br-cAMP-, forskolin-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) AHA (800 microg/ml) caused more pregnenolone release in control group, but not in 25-OH-cholesterol, trilostane or 25-OH-cholesterol+trilostane group; (3) kinetic study showed an uncompetitive inhibition model of AHA to P450 side chain cleavage enzyme (P450scc); (4) kinetic study showed a noncompetitive inhibition model of AHA to 11beta-hydroxylase; and (5) AHA inhibited the expression of StAR protein. These results suggest that AHA acts directly upon rat ZFR cells to diminish corticosterone release. These results indicate the inhibitory mechanism of AHA mediates through an inhibition of the activities of the post-cAMP corticosterone synthesis enzymes, i.e. 3beta-HSD, 21-hydroxylase, 11beta-hydroxylase, and inhibition of StAR protein expression.

Differences between the growth regulatory pathways in primary rat adrenal cells and mouse tumor cell line.

In this study, DNA synthesis, phosphorylation of ERK1/2 and CREB proteins, as well as induction of c-Fos protein, were examined in rat adrenocortical, glomerulosa and fasciculata/reticularis cells, as well as in the Y1 cell line. We found that FGF2 was mitogenic only in glomerulosa cells and although ACTH did not activate ERK1/2, it did activate CREB protein, indicating efficient transduction of signals initiated in the ACTH receptors of rat adrenocortical cells. The FGF2 activated ERK1/2 in rat adrenal cells by a mechanism that might be modulated by upstream PKA pathway phosphorylation of MEK and despite the nonmitogenic effect of ACTH on rat adrenal cells it effectively induces c-Fos protein. The results presented herein describe distinct differences between the ACTH and FGF2 signal transduction mechanisms seen in adrenocortical cells and those observed in the Y1 cell line, indicating that, in vitro, ACTH blockage of the mitogenic effect occurs in normal adrenal cells after induction of c-Fos protein.

Response of rat fasciculata-reticularis cells in primary culture to bacterial lipopolysaccharide.

The aim of this study was to determine the direct effect of a wide range of concentrations of lipopolysaccharide (LPS) of Escherichia coli O111:B4 on fasciculata-reticularis cells in primary cultures. In short-term cultures of fasciculata-reticularis cells, the presence of low (1-10 microg/ml) doses of LPS in the medium produced a decrease in ACTH-induced corticosterone secretion, in a dose-dependent manner and independent of the culture medium. The corticosterone production stimulated by db-cAMP was slightly decreased by the presence of LPS in culture medium, while the pregnenolone induced corticosterone biosynthesis was not modified. LPS modified the binding of [125I]-Tyr23-ACTH to the fasciculata-reticularis cell membrane and the signal transduction pathway, as LPS reduced ACTH-induced cAMP production. In long-term cultures, the presence of LPS in the medium produces a decrease in the specific binding of [125I]-Tyr23-ACTH, while the presence of ACTH in the culture medium produced an increase in its specific binding. The use of high doses of LPS (100-250 microg/ml) has helped to clarify some aspects of the LPS action. These doses of LPS severely inhibited ACTH-induced corticosterone production, and clearly reduced the corticosterone production stimulated by db-cAMP and the binding of ACTH to its receptors. In long-term cultures, LPS decreased the number of ACTH receptors, an effect that was reversed by subsequent exposure to ACTH. These results indicate that LPS exerts a direct action on fasciculata-reticularis cells and a model of the mechanism of LPS action is proposed.

Inhibitory effects of digoxin and digitoxin on corticosterone production in rat zona fasciculata-reticularis cells.

The aim of the present study was to investigate the direct effects and action mechanisms of digitalis on the production of corticosterone in rat adrenocortical cells. Male rats were challenged with digoxin (1 microg ml(-1) kg(-1)) in the presence or absence of adrenocorticotropin (ACTH, 5 microg ml(-1) kg(-1)) administered by intravenous injection to the right jugular vein. Blood samples were collected at 0, 30, 60, and 120 min following the challenge. The concentration of corticosterone in the rat plasma samples was measured by radioimmunoassay. Zona fasciculata-reticularis (ZFR) cells in male rats were prepared and then incubated with or without digoxin or digitoxin in the presence or absence of ACTH (10(-9) m), forskolin (10(-7) m), 8-bromo-cyclic 3' : 5'-adenosine monophosphate (10(-4) m), cyclopiazonic acid (CPA, 10(-5) m), trilostane (10(-6) m), 25-OH-cholesterol (10(-5) m), pregnenolone (10(-5) m), progesterone (10(-5) m), or deoxycorticosterone (10(-5) m) at 37 degrees C for 1 h before collection of the media. Corticosterone or pregnenolone levels were measured by radioimmunoassay. A single injection of digoxin did not alter the basal level of plasma corticosterone, but did inhibit the level of plasma corticosterone released in response to ACTH in vivo. Administration of digoxin or digitoxin decreased both spontaneous and ACTH-stimulated release of corticosterone in vitro. Digoxin (10(-7)-10(-5) m) and digitoxin (10(-7)-10(-5) m), but not ouabain (10(-7)-10(-5) m), dose-dependently inhibited corticosterone production in response to forskolin and 8-Br-cyclic AMP in rat ZFR cells. Both digoxin (10(-6)-10(-5) m) and digitoxin (10(-6)-10(-5) m) attenuated corticosterone production in response to CPA. Digoxin (10(-5) m) or digitoxin (10(-5) m) inhibited cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc) activity (catalyses conversion of cholesterol to pregnenolone in the presence of trilostane) in rat ZFR cells. The enzyme activity of 11 beta-hydroxylase (catalyses conversion of deoxycorticosterone to corticosterone) in ZFR cells was also inhibited by the administration of digoxin (10(-5) m) or digitoxin (10(-5) m).10 These results together suggest that digoxin and digitoxin decrease the release of corticosterone by acting directly on ZFR cells via a Na+, K+-ATPase-independent mechanism involving the inhibition of the activities of adenylyl cyclase, cytochrome P450scc and 11 beta-hydroxylase, as well as the functioning of cyclic AMP and intracellular calcium.

Comparison of c-Fos immunoreactivity in pancreatic beta cells and cells with neural crest, endoderm and mesoderm origin in rats.

AIMS: Pancreatic beta cells are known to share many similarities with neuronal cells, but their origin remains controversial. It has been hypothesized that pancreatic beta cells are derived from neural crest cells. The aim of this study was to investigate the similarities between pancreatic beta cells and cells with neural crest, endoderm and mesoderm origin with respect to c-Fos immunoreactivity (c-Fos-ir), which has a role in important cellular processes including cellular proliferation, growth, differentiation and apoptosis. METHODS: c-Fos-ir was analyzed by immunohistochemical methods in formaline-fixed, paraffin-embedded sections of rat pancreatic beta cells (BCs), in adrenal medullary chromaffin cells (CCs) that are derived from neural crest, in exocrine pancreatic acinar cells (ACs) that are derived from endoderm, and in adrenal cortex zona reticularis cells (RCs) that are derived from mesoderm. RESULTS: The statistical comparisons revealed no significant differences between BCs and CCs with respect to c-Fos-ir (p>0.05). However, a highly significant difference (p<0.001) with respect to c-Fos-ir both between ACs and RCs, and between these two cell types and each of the two other cell types was noted. CONCLUSIONS: As opposed to findings in cells without neural crest origin, the observed similarity between BCs and CCs with respect to c-Fos-ir, provides additional evidence for the similarity of these cells with cells derived from neural crest.

Effects of S-petasin on cyclic AMP production and enzyme activity of P450scc in rat zona fasciculata-reticularis cells.

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of P. hybridus, which has been used to relieve gastrointestinal pain, lung disease, and spasms of urogenital tract. We have demonstrated that S-petasin inhibited corticosterone release from rat zona fasiculata-reticularis cells. However, the mechanism and molecular effects of S-petasin on zona fasiculata-reticularis cells are still unclear. This study explored the effects of S-petasin on cellular adenosine 3':5'-cyclic monophosphate (cAMP) production, the functions of steroidogenic enzymes including cytochrome P450 side-chain cleavage enzyme (P450scc), 11beta-hydroxylase, and the expression levels of steroidogenic acute regulatory protein or P450scc. In this experiment, zona fasciculata-reticularis cells were incubated with S-petasin in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), forskolin, 25-OH-cholesterol, deoxycorticosterone at 37 degrees C for 0.5, 1 or 3 h. The media were used to measure the concentration of corticosterone or pregnenolone by radioimmunoassay. The cells were used to measure the content of cAMP by radioimmunoassay and extracted protein for Western blot or messenger RNA (mRNA) for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Our data demonstrated that (1) S-petasin inhibits ACTH- or forskolin-stimulated cellular cAMP production, (2) S-petasin increased the Michaelis constants of P450scc and 11beta-hydroxylase and (3) S-petasin decreased the expression levels and mRNA of steroidogenic acute regulatory protein. In summary, the actions of S-petasin mediate the inhibition of cAMP formation, decrease the activities of key enzymes P450scc and 11beta-hydroxylase, and reduce mRNA of steroidogenic acute regulatory protein and expression of steroidogenic acute regulatory protein.

Effects of dehydroepiandrosterone on corticosterone release in rat zona fasciculata-reticularis cells.

The decline of plasma dehydroepiandrosterone (DHEA) and maintenance of glucocorticoid levels with increasing age contribute to excess body fat accumulation, hyperglycaemia, hyperlipidaemia, hyperinsulinaemia and cancer. Although opposing actions of DHEA and corticosterone have been proposed in a rat model, the effects and action mechanisms of DHEA on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study addressed the effects of DHEA on corticosterone release, cellular cAMP production, the functions of steroidogenic enzymes and the expression levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc). ZFR cells were incubated with DHEA in the presence or absence of adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone or deoxycorticosterone at 37 degrees C for 30 min, 1 h or 5 h and the concentration of corticosterone or pregnenolone measured subsequently in the media by RIA. The cells were used to measure the content of cAMP by RIA and to extract protein for Western blot or mRNA for RT-PCR analysis. The data demonstrated that (1) DHEA inhibited ACTH-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA increased the K(m) of 11beta-hydroxylase but not P450scc; (4) DHEA affected the expression levels of StAR protein but not of P450scc. These results suggest that DHEA acts directly on rat ZFR cells to diminish corticosterone secretion by inhibition within the post-cAMP pathway, by inhibiting steroidogenic enzymes downstream from P450scc and by inhibiting StAR expression.

Effects of fasting on corticosterone production by zona fasciculata-reticularis cells in ovariectomized rats.

BACKGROUND: To investigate the function and mechanism of fasting on the production of corticosterone in vitro by zona fasciculata-reticularis (ZFR) cells from ovariectomized (OVX) rats. METHODS: Female rats were OVX for 4 days before decapitation. Rats were fed or fasted for 1 day before experiment. ZFR cells from fed and fasted rats were incubated with adrenocorticotropic hormone (ACTH), forskolin, 8-bromo-3',5'-cyclic adenosine monophosphate, SQ22536, nifedipine, chelerythrine chloride, trilostane or steroidogenic precursors at 37 degrees C for either 60 or 30 minutes. Corticosterone, pregnenolone concentrations in spent media, and the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration were determined by radioimmunoassay. The effects of fasting in response to ACTH on the protein expressions of steroidogenic acute regulatory protein (StAR) or cytochrome P450 side-chain cleavage enzyme (P450scc) in ZFR cells were determined by Western blot analysis. RESULTS: The concentration of plasma corticosterone in fasted rats was significantly higher than that in fed rats (P<0.01). One-day fasting significantly increased the responsiveness of ZFR cells to ACTH, forskolin, and precursor-stimulated corticosterone productions and to forskolin-stimulated cAMP accumulation. The corticosterone production was reduced in fasted group when adenylyl cyclase was inhibited by SQ22536. The fasting-enhanced level of corticosterone production in ZFR cells was decreased by the administration of nifedipine but not altered by that of chelerythrine chloride. Fasting significantly increased trilostane-stimulated production of pregnenolone in ZFR cells. The activities of enzymes which converting cholesterol, pregnenolone, progesterone, and deoxycorticosterone to corticosterone and the expressions of StAR in ZFR cells were greater in fasted rats than in fed rats. CONCLUSIONS: This study demonstrated that fasting increased the release of corticosterone and the accumulation of cAMP by rat ZFR cells. The action mediated through enhancing the responsiveness to ACTH stimulation, cAMP cascades and the activity of L-type calcium channels. The activities of steroidogenic enzymes including P450scc, 3beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11beta-hydroxylase were all enhanced by the fasting treatment.

Signaling pathways involved in the A and B receptor-mediated cortisol secretagogue effect of endothelins in the human adrenal cortex.

Endothelins (ETs) are a family of 21-amino acid hypertensive peptides, which together with their receptors ETA and ETB are expressed in human adrenal cortex. Evidence has been provided that ETs exert a potent secretagogue effect on human adrenocortical cells, acting through both ETA and ETB receptors. Therefore, it seemed worthwhile to study the signaling cascades mediating the cortisol secretagogue effect of the two receptor subtypes. Normal adrenal glands were obtained from consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Dispersed zona fasciculata-reticularis (ZF/R) cells were obtained by collagenase digestion and mechanical disaggregation. The selective activation of ETA and ETB receptors was obtained by exposing dispersed cells to ET-1 plus the ETB receptor antagonist BQ-788 and to the selective ETB receptor agonist BQ-3020, respectively. ETA and ETB receptors about equally contributed to the cortisol response of dispersed ZF/R cells to ETs. The phospholipase (PL) C inhibitor U-73122 abolished ETA-mediated secretory response, but only partially prevented the ETB-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes, while the Ca(2+)-channel blocker nifedipine was ineffective. The ETB receptor-, but not the ETA receptor-mediated cortisol response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. The inhibitors of adenylate cyclase, PKA, tyrosine kinase and lipoxygenase did not affect the secretory response to the activation of either receptor subtype. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZF/R cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate cortisol secretion from human ZF/R cells, acting through ETA receptors exclusively coupled with PLC/PKC-dependent pathway and ETB receptors coupled with both PLC/PKC- and COX-dependent cascades.

Characterisation of the muscarinic receptor subtype M3 in the bovine zona fasciculata-reticularis cells by receptor binding, mRNA and functional studies.

In the present work we have investigated which muscarinic (M) receptor subtype is responsible for the steroidogenic effect of muscarinic agonists in bovine zona fasciculata-reticularis (ZFR) cells in culture. Radioligand binding studies using the muscarinic antagonist [(3)H]quinuclidinyl benzilate ([(3)H]QNB) demonstrated binding sites of high affinity (K(d)=0.45 nM) and low capacity ( approximately 8000 sites/cell). Pharmacological characterisation of muscarinic receptors was assessed by evaluating the effects of the M(3)>M(1)>>M(2) antagonist 4-DAMP (4-diphenylacetyl-N-methylpiperidine) and the M(1)=M(4)> M(3)>>M(2) antagonist pirenzepine on the binding of [(3)H]QNB and carbachol-induced cortisol production. For both parameters, the potency of 4-DAMP was about two orders of magnitude higher than that of pirenzepine. Reverse transcriptase (RT)-PCR analysis of bovine ZFR mRNAs using specific primers for M(2), M(3) and M(4) receptors revealed the expression of only M(3) mRNA. Moreover, carbachol significantly stimulated inositol phosphate accumulation, but had no inhibitory effect on basal or ACTH-induced cAMP production. Indeed, carbachol potentiated ACTH-induced cAMP production and this effect was, in part, mediated through protein kinase C. Lastly, neomycin, an inhibitor of phosphoinositide turnover, significantly attenuated carbachol-evoked cortisol production. Thus, pharmacological, biochemical and mRNA studies indicate that the M(3) receptor subtype is responsible for the biological effects of muscarinic agonists in bovine ZFR cells.

Direct effects of prolactin on corticosterone release by zona fasciculata-reticularis cells from male rats.

The role of prolactin (PRL) in the male is not fully defined. The aim of this study was to investigate the function and mechanism of PRL on the production of corticosterone by zona fasciculata-reticularis (ZFR) cells in vitro. The ZFR cells were obtained from male rats under normal, hyperprolactinemic, or hypoprolactinemic situation. PRL stimulated the corticosterone release in a dose-dependent pattern in the ZFR cells from normal male rats. The cellular adenosine 3'-5'-cyclic monophosphate (cAMP) concentration positively correlated with PRL concentration in the presence of forskolin or 3-isobutyl-1-methylxanthine (IBMX). PRL enhanced the stimulatory effects of cAMP mimetic reagents, i.e., forskolin, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and IBMX on the release of corticosterone. The adenylate cyclase inhibitor (SQ22536) inhibited the corticosterone release in spite of presence of PRL. Nifedipine (L-type calcium channel blocker) did not inhibit corticosterone release. The hyperprolactinemic condition was actualized by transplantation of donor rat anterior pituitary glands (APs) under kidney capsule. By comparison with the cerebral cortex (CX)-grafted group, AP-graft resulted in an increased release of corticosterone, 3beta-hydroxysteriod dehydrogenase (HSD) activity and cAMP production by ZFR cells. Acute hypoprolactinemic status was induced by bromocriptine for 2 days. The results showed the productions of corticosterone were lower in hypoprolactinemic group than in control group, which were persistent along with different ACTH concentrations. These results suggest that PRL increase the release of corticosterone by ZFR cells via cAMP cascades and 3beta-HSD activity.