Rat Zona Fasciculata-reticularis Cells Research Articles

Down Regulation of Acrolein on Corticosterone Secretion in Male Rats

Acrolein is a small unsaturated aldehyde and can be found in a wide range of resources including all types of smoke and exhaust gases from gasoline engines. Although the toxicity and damage of acrolein have been recognized, the action mechanisms of acrolein, especially that of acrolein on the response of stresshormones are still unclear. The present study hypothesized that administration of acrolein altered the secretion of both adrenocorticotropin (ACTH) and corticosterone via the regulation of steroid biosynthetic pathway in rat zona fasciculata-reticularis (ZFR) cells. Both in vivo and in vitro approaches were uased. In the in vivo study, intraperitonal injection of acrolein (2 mg/ml/kg) once daily for 1 or 3 days resulted in a reduction of plasma levels of ACTH and corticosterone as well as the intracellular cAMP and ACTH-induced secretion of corticosterone. The protein expression of ACTH receptor (ACTHR) in rat ZFR cells was also reduced by 40-60% after treatment of acrolein for 1 day and 3 days, respectively. In the in vitro study, rat ZFR cells were prepared and chanllenged with ACTH (10 -9 M), forskolin (an adenylyl cyclase activitior, 10 -5 M), 8-Br-cAMP (a permeable synthetic cAMP, 5x10 -5 M), 25-OH-cholesterol (10 -5 M) ± trilostane (an inhibitor of 3β-hydroxysteroid dehydrogenase, 3β-HSD, 10 -5 M). The evoked release of corticosterone by ACTH, forskolin, 8-Br-cAMP and the induced release of pregnenolone in response to 25-OH-cholesterol plus triolostane were decreased. Since the accumulation of pregnenolone after blocking 3β-HSD by trilostane represents the activity of P450 scc, therate-limiting step of steroid biosynthesis, we suggest that not only the cAMP pathway was inhibited, but also the enzyme activity of P450 scc was attenuated following administration of acrolein. Although insignificant, the protein expression of steroidogenic acute regulatory protein (StAR) was decreased by 40% in ZFR cells after treatment of acrolein in vivo . Incubation of ZFR cells with acrolein (10 -9 ~10 -7 M) also decreased the in vitro release of corticosterone. These results suggest that administration of acrolein inhibited corticosterone production via the attenuation of cAMP pathway, StAR protein expression, and the enzyme activity of P450 scc. The attenuation of protein expression of ACTHR (also named melanocortin 2 receptor, MC2R) and reduced secrection of ACTH indicated that the hypothalamus-pituitary-adrenal (H-P-A) axis was also down- regulated by the administration of acrolein.

Effects of acrolein on the production of corticosterone in male rats

Acrolein, an α, β-unsaturated aldehyde, exists in a wide range of sources. Acrolein can be not only generated from all types of smoke but also produced endogenously from the metabolism by lipid peroxidation. The cellular influence of acrolein is due to its electrophilic character via binding to and depleting cellular nucleophiles. Although the toxicity of acrolein has been extensively studied, there is relatively little information about its impact on hormone release. This study aimed at the effect of acrolein on hypothalamic–pituitary–adrenal (H–P–A) axis. In an in vivo study, male rats were administrated with acrolein for 1 or 3days. The plasma corticosterone in response to a single injection of adrenocorticotropic hormone (ACTH) increased slowly in acrolein-pretreated rats than in control rats. Further investigating the steroidogenic pathway, the protein expressions of steroidogenic acute regulatory protein (StAR) and the upper receptor-melanocortin 2 receptor (MC2R) were attenuated in acrolein-treated groups. Another experiment using trilostane showed less activity of P450scc in zona fasciculata-reticularis (ZFR) cells in acrolein-treated groups. In addition to the suppressed ability of corticosterone production in ZFR cells, acrolein even had extended influence at higher concentrations. The lower ACTH was observed in the plasma from acrolein-pretreated rats. In an in vitro study, ZFR cells were incubated with acrolein and the results showed that corticosterone concentrations in media were decreased in a dose-dependent manner. Acrolein also desensitized the response of the ZFR cells to ACTH. These results suggested that acrolein decreased the releasing ability of corticosterone via an inhibition on the response of ZFR cells to ACTH and the reduction of protein expressions of StAR and MC2R as well as the activity of P450scc in rat ZFR cells. The present evidences showed that the H–P–A axis was affected by the administration of acrolein.

Effects of Propylthiouracil on the Production of Aldosterone in Rat Zona Glomerulosa Cells

Propylthiouracil (PTU), an anti-thyroid drug, is widely used for the treatment in hyperthyroid patients. Previous studies indicate that thyroxine inhibit the spontaneous and adrenocorticotropin (ACTH)-stimulated corticosterone secretion by acting directly at adrenal glands via decrease cAMP production. It has also been shown that PTU suppresses corticosterone release by male rat zona fasciculata-reticularis cells. However, the direct effects and mechanisms of PTU on the aldosterone production in adrenal zona glomerulosa (ZG) cells are still unclear. ZG cells were prepared from adrenocortical tissues of male rats, and then challenged with or without angiotensin II (10^(-7), 10^(-6) M), ACTH (10^(-9) M), A23187 (10^(-5) M), cyclopiazonic acid (CPA, 10^(-5) M), forskolin (10^(-5) M), 8-Br-cAMP (10^(-5) M), trilostane (10^(-6) M, 3β-HSD inhibitor) or steroidogenic precursors (e.g. 10^(-6) M 25-OH-cholesterol and 10^(-7), 10^(-5) M corticosterone) at 37°C for 1 h. Our results showed that PTU (0-3 mM) dose-dependently decreased the aldosterone release in response to the above hormones, drugs and the steroidogenic precursors in vitro. PTU also decreased steroidogenic acute regulatory (StAR) protein expression, but did not alter the protein expression of P450 side chain cleavage enzyme (P450scc) during steroidogenesis of aldosterone. These results suggested that PTU has a direct inhibitory effect on aldosterone production via cAMP, and Ca^2+ down stream pathway and the steroidogenic enzymes activities including P450scc and aldosterone synthase, as well as StAR protein expression.

Effects of Propylthiouracil on the Production of Aldosterone in Rat Zona Glomerulosa Cells

Propylthiouracil (PTU), an anti-thyroid drug, is widely used for the treatment in hyperthyroid patients. Previous studies indicate that thyroxine inhibit the spontaneous and adrenocorticotropin (ACTH)-stimulated corticosterone secretion by acting directly at adrenal glands via decrease cAMP production. It has also been shown that PTU suppresses corticosterone release by male rat zona fasciculata-reticularis cells. However, the direct effects and mechanisms of PTU on the aldosterone production in adrenal zona glomerulosa (ZG) cells are still unclear. ZG cells were prepared from adrenocortical tissues of male rats, and then challenged with or without angiotensin II (10^(-7), 10^(-6) M), ACTH (10^(-9) M), A23187 (10^(-5) M), cyclopiazonic acid (CPA, 10^(-5) M), forskolin (10^(-5) M), 8-Br-cAMP (10^(-5) M), trilostane (10^(-6) M, 3β-HSD inhibitor) or steroidogenic precursors (e.g. 10^(-6) M 25-OH-cholesterol and 10^(-7), 10^(-5) M corticosterone) at 37°C for 1 h. Our results showed that PTU (0-3 mM) dose-dependently decreased the aldosterone release in response to the above hormones, drugs and the steroidogenic precursors in vitro. PTU also decreased steroidogenic acute regulatory (StAR) protein expression, but did not alter the protein expression of P450 side chain cleavage enzyme (P450scc) during steroidogenesis of aldosterone. These results suggested that PTU has a direct inhibitory effect on aldosterone production via cAMP, and Ca^2+ down stream pathway and the steroidogenic enzymes activities including P450scc and aldosterone synthase, as well as StAR protein expression.

Effect of 17a-Ethinylestradiol on Corticosterone Release from Rat Zona Fasciculata-Reticularis Cells.

Effect of 17a-Ethinylestradiol on Corticosterone Release from Rat Zona Fasciculata-Reticularis Cells.

Effects and Mechanisms of Nonylphenol on Corticosterone Release in Rat Zona Fasciculata-Reticularis Cells

Alkylphenol ethoxylate, consisting of ∼80% nonylphenol ethoxylate (NPEO), is a major group of nonionic surfactant. The primary degradation product of NPEO, nonylphenol (NP), interferes with reproduction, induces cell death in gonads, and leads to changes in other reproductive parameters. With such apparent stress, NP is believed to induce stress response mechanism, i.e., adrenal cortical hormone. However, the effects and action mechanisms of NP on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of NP on corticosterone release. ZFR cells were incubated with NP in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3',5'-adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxyl cholesterol (25-OH-cholesterol), pregnenolone, progesterone, or deoxycorticosterone at 37°C for 1 h. The concentrations of corticosterone or pregnenolone in the spent media were measured by radioimmunoassay. The expressions of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc) protein, and 11β-hydroxylase in the cells were measured by Western blot. The data demonstrated that (1) NP stimulated corticosterone release induced by ACTH, 8-Br-cAMP, FSK, 25-OH-cholesterol, pregnenolone, progesterone, or deoxycorticosterone; (2) NP significantly increased pregnenolone release in the control, 25-OH-cholesterol, trilostane, and 25-OH-cholesterol + trilostane groups; (3) NP-stimulated corticosterone release was estrogen receptor dependent, but mediated by nitric oxide and p38 mitogen-activated protein kinase pathway independent; and (4) NP did not affect StAR, 11β-hydroxylase, or P450scc protein expression. These results suggest that NP acts directly on rat ZFR cells to stimulate corticosterone release and that the stimulation mechanism of NP mediates through post-cAMP corticosterone manufacture enzymes, i.e., P450scc and 11β-hydroxylase.

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.

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.

Direct effects of IL‐15 on corticosterone secretion by rat adrenocortical cells

Cytokines might regulate the function of the hypothalamic-pituitary-adrenal axis. IL-15 is a potent non-T-cell-derived cytokine with IL-2-like activities. It has been shown that IL-15 can reverse the inhibition of glucocorticoids on PBMC. In vitro experiments were designed to assess the direct effect of IL-15 on corticosterone (CORT) secretion in the adrenal zona fasciculata-reticularis (ZFR) cells of male rats. Administration of IL-15 dose dependently decreased the basal and adrenocorticotropin-stimulated release of CORT and production of cAMP in ZFR cells. The stimulatory effect of forskolin (an adenylate cyclase activator) on CORT secretion and accumulation of cAMP in ZFR cells was attenuated by the administration of IL-15. However, 8-Br-cAMP (a cAMP analogue)-stimulated release of CORT was not affected by IL-15. Exogenous administration of IL-15 (10(-7) mol/L) significantly attenuated the pregnenolone (the substrate of 3beta-hydroxysteroid dehydrogenase)- or deoxycorticosterone (the substrate of 11beta-hydroxylase)-induced release of CORT. The results indicate that decrease of CORT secretion by IL-15 is in part because of (i) the decrease of adenylate cyclase activity and cAMP production and (ii) the inhibition of 3beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase activities in rat ZFR cells.

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), 11β-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°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 11β-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 11β-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.

Inhibitory effects of bromocriptine on corticosterone secretion in male rats

Bromocriptine, a dopamine D2 receptor agonist, is widely used for treating prolactinoma, Parkinson's disease and galactorrhea. However, the influence of bromocriptine on the endocrine system, especially adrenal function, is not clear. The present study was aimed to investigate the effects of bromocriptine on corticosterone production in rats. Male rats were treated or not treated by bromocriptine (5 mg/kg, s.c.) twice per day for 2 days before decapitation. The adrenal zona fasciculata–reticularis cells were prepared and incubated with adrenocorticotropic hormone (ACTH), forskolin (an adenylyl cyclase activator), 8-bromo-adenosine 3′:5′ cyclic monophosphate (8-Br-cAMP, a membrane-permeable analogue of cAMP), and steroidogenic precursors including 25-OH-cholesterol and pregnenolone. The concentrations of prolactin, corticosterone and pregnenolone in the plasma and/or medium were measured by radioimmunoassay (RIA). The protein expression of cytochrome P450 side-chain cleavage (P450scc) enzyme and steroidogenic acute regulatory protein (StAR) was analyzed by Western blotting. Administration of bromocriptine in vivo resulted in a decrease in the levels of plasma prolactin and corticosterone. Basal—and ACTH—as well as forskolin-stimulated corticosterone secretion by zona fasciculata–reticularis cells was also lower in bromocriptine-treated rats than in control animals. The decreased production of corticosterone in zona fasciculata–reticularis cells could be reversed by administration of 8-Br-cAMP. The corticosterone and pregnenolone release induced by 25-OH-cholesterol in zona fasciculata–reticularis cells was reduced by administration of bromocriptine. The protein expression of both StAR protein and P450scc in zona fasciculata–reticularis cells was inhibited in the bromocriptine-treated group. Administration of bromocriptine in vitro reduced the release of corticosterone stimulated by ACTH and forskolin in rat zona fasciculata–reticularis cells. These results suggested that bromocriptine caused adrenal dysfunction through inhibition of ACTH action and of the activity of adenylyl cyclase, and impaired the early steps of corticosterone biosynthesis.

Involvement of cAMP but not PKA in the increase of corticosterone secretion in rat zona fasciculata-reticularis cells by aging

The effects and mechanisms of aging on corticosterone secretion in zona fasciculata-reticularis (ZFR) cells of ovariectomized (Ovx) rats were studied. Young (3-month) and old (24-month) female rats were Ovx for 4 days before decapitation. ZFR cells were isolated and incubated with different hormones or reagents at 37°C for 30 min. Aging increased the basal secretion of corticosterone both in vivo and in vitro. The adrenocorticotropin (ACTH)-, forskolin-, 3-isobutyl-l-methylxanthine (IBMX)-, 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP)-, and ovine prolactin (oPRL)-stimulated release of corticosterone by ZFR cells was greater in old than in young Ovx rats. H89, an inhibitor of protein kinase A (PKA), decreased the production of corticosterone in ZFR cells from young but not old Ovx rats. Forskolin-, or IBMX-induced production of cAMP was greater in old than in young Ovx animals, which correlated with the increase of corticosterone production by aging. The activity of 11β-hydroxylase that converts deoxycorticosterone (DOC, 10−9 or 10−8 M) to corticosterone in rat ZFR cells was decreased by age. However, the corticosterone production in response to high dose of DOC (10−7 M) was indifferent between young and old groups. These results suggest that aging increases corticosterone production in Ovx rats via a mechanism in part associated with an increase of adenylyl cyclase activity and a decrease of phosphodiesterase activity, and then an increase of the generation of cAMP, but not related to either PKA activity or 11β-hydroxylase. J. Cell. Biochem. 85: 35–41, 2002. © 2002 Wiley-Liss, Inc.

Involvement of cAMP but not PKA in the increase of corticosterone secretion in rat zona fasciculata‐reticularis cells by aging

The effects and mechanisms of aging on corticosterone secretion in zona fasciculata-reticularis (ZFR) cells of ovariectomized (Ovx) rats were studied. Young (3-month) and old (24-month) female rats were Ovx for 4 days before decapitation. ZFR cells were isolated and incubated with different hormones or reagents at 37 degrees C for 30 min. Aging increased the basal secretion of corticosterone both in vivo and in vitro. The adrenocorticotropin (ACTH)-, forskolin-, 3-isobutyl-l-methylxanthine (IBMX)-, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP)-, and ovine prolactin (oPRL)-stimulated release of corticosterone by ZFR cells was greater in old than in young Ovx rats. H89, an inhibitor of protein kinase A (PKA), decreased the production of corticosterone in ZFR cells from young but not old Ovx rats. Forskolin-, or IBMX-induced production of cAMP was greater in old than in young Ovx animals, which correlated with the increase of corticosterone production by aging. The activity of 11 beta-hydroxylase that converts deoxycorticosterone (DOC, 10(-9) or 10(-8) M) to corticosterone in rat ZFR cells was decreased by age. However, the corticosterone production in response to high dose of DOC (10(-7) M) was indifferent between young and old groups. These results suggest that aging increases corticosterone production in Ovx rats via a mechanism in part associated with an increase of adenylyl cyclase activity and a decrease of phosphodiesterase activity, and then an increase of the generation of cAMP, but not related to either PKA activity or 11 beta-hydroxylase.

Age-related differences in corticosterone secretion in female rats

The effects of age on steroidogenesis in rat zona fasciculata-reticularis (ZFR) cells were studied. Young, adult, and middle-aged rats were ovariectomized (Ovx) and received replacement therapy with oil or estradiol benzoate ([EB] 25 μg/mL/kg). Rat ZFR cells were incubated with corticotropin (ACTH), prolactin (PRL), or forskolin at 37°C for 1 hour. The effects of age on the activity of steroidogenic enzymes of ZFR cells were measured by the amount of intermediate steroidal products separated by thin-layer chromatography. Plasma levels were higher for PRL (54% to 254%) and corticosterone (179% to 257%) in middle-aged versus young rats. In oil-treated Ovx rats, basal and ACTH-stimulated corticosterone release by ZFR cells were also greater in middle-aged compared with young rats. Replacement with EB in Ovx rats increased the ACTH-stimulated release of corticosterone. Administration of ovine PRL in vitro resulted in a dose-dependent increase of corticosterone production. In oil-treated middle-aged rats, ovine PRL-stimulated corticosterone release was higher than in young rats. Forskolin-induced production of cyclic adenosine 3′,5′-monophosphate (cAMP) was greater in middle-aged versus young rats and correlated with the increase of corticosterone production. The activity of steroidogenic enzymes in rat ZFR cells was unchanged by age. These results suggest that the age-related increase of corticosterone production in female rats is associated with the stimulatory effect of PRL on ZFR cells and is due in part to an increase of cAMP generation.