Stimulatory Effect of Food Restriction on the Steroidogenesis of Aldosterone in Ovariectomized Rats.

Food or calorie restriction (FR or CR) induces several physiological changes including weight loss, metabolic adaptations, mineral and hormonal changes. However, the effects of FR on aldosterone steroidogenesis in zona glomerulosa (ZG) cells have not been elucidated. Therefore, the present study was designed to investigate the effects of FR on aldosterone secretion and the involved mechanisms in ovariectomized (Ovx) rats. Ovx rats were divided into ad libitum fed (control) and FR groups. The FR rats exhibited decreased body weight, water intake, urine flow, sodium excretion and increased plasma aldosterone in comparison with control rats. FR elevated the basal and angiotensin II-stimulated aldosterone secretion from ZG cells. The conversions of 25-hydroxy-cholesterol to pregnenolone or corticosterone to aldosterone in ZG cells of FR group were greater than that in control group. FR group had a higher protein expression of steroidogenic acute regulatory (StAR) protein in ZG cells. However, there was no different protein expression of cytochrome P450 sidechain cleavage enzyme (P450scc) in ZG cells between control and FR groups. In summary, the increased activities of P450scc and aldosterone synthase as well as the protein expression of StAR protein in ZG cells are involved in the effects of FR on aldosterone steroidogenesis in Ovx rats. We also suggest that the increase of aldosterone might be associated with anti-diuresis and antinatriuresis in FR group. These results are helpful for understanding the role of aldosterone in physiological adaptation and renal sodium conservation during FR.

Inhibitory effect of bufalin and cinobufagin on steroidogenesis via the activation of ERK in human adrenocortical cells.

BACKGROUND AND PURPOSE: Bufalin and cinobufagin exhibit cardiotonic and natriuretic activities. The aim of this study was to evaluate the effects of bufalin and cinobufagin on aldosterone and cortisol secretion and their mechanisms of action in human adrenocortical cells (NCI-H295). EXPERIMENTAL APPROACH: H295 cells were incubated with bufalin or cinobufagin in the presence or absence of angiotensin II (Ang II), forskolin, 8-Br-cAMP, corticosterone or deoxycortisol. The role of ERK1/2 was studied by use of the inhibitor of MEK (U0126). The binding of transcription factor steroidogenic factor 1 (SF-1) to steroidogenic acute regulatory (StAR) gene promoter was analysed by EMSA. KEY RESULTS: Bufalin and cinobufagin markedly inhibited basal, Ang II-, forskolin- or 8-Br-cAMP-stimulated aldosterone and cortisol secretion, and the conversions of corticosterone to aldosterone and deoxycortisol to cortisol. Bufalin and cinobufagin also inhibited StAR protein expression and SF-1 binding to StAR gene promoter. They both increased phosphorylation of ERK1/2, and U0126 fully abolished these effects on ERK1/2 in H295 cells. Furthermore, U0126 reversed the inhibitory effects of bufalin and cinobufagin on StAR protein expression and the binding of SF-1 to StAR gene promoter. However, U0126 did not completely reverse their inhibitory effects on aldosterone and cortisol release. CONCLUSIONS AND IMPLICATIONS: The inhibitory effects of bufalin and cinobufagin on steroidogenesis of aldosterone and cortisol were associated with inhibition of aldosterone synthase and 11ß-hydroxylase, as well as the suppression of StAR protein expression and SF-1 binding to StAR promoter via the phosphorylation of ERK1/2 in H295 cells.

Involvement of ERK phosphorylation in the prevention of ischemia-induced ovarian follicular depletion by stem cells.

Ovarian failure is commonly caused by aging, autoimmune disease, menopause and cancer therapy. We used an ischemic model in the ovary to test the hypothesis that stem cells are helpful for ovarian regeneration after injury. Three treatment regimes were employed: sham-operated control, ligation plus PBS, and ligation plus immortalized human bone marrow stromal cells (stem cells) groups. After ligation-induced ischemia, stem cells or PBS were injected into rat ovaries. Then, pregnant mare serum gonadotropin was given intra-peritoneally to initiate folliculogenesis. The animals were then sacrificed. The ovary gland was weighed, and ovarian folliculogenesis, stem cell differentiation and vascular neogenesis were evaluated. In order to study improvement of folliculogenesis after ovarian ischemia, steroidogenic acute regulatory protein (StAR), p44/p42 MAPK (T-ERK1/2), and phospho-p44/ p42 MAPK (P-ERK1/2) expression were specifically evaluated. Results indicated that ovarian size was smaller and that the rate of folliculogenesis was lower in ovarian ischemic-reperfusion animals, but both recovered after stem cell treatment. The stem cells migrated into the ovary and differentiated into theca cells, granulosa cells, corona radiata cells and vascular endothelial cells. In addition, von Willebrand factor (vWF) expression was increased; 17beta-estradiol (E2), progesterone (P4), P-ERK1/2 and StAR protein expression was recovered by stem cells treatment in the ischemic ovaries. The serum LH was significantly increased in ovaries of ischemia-reperfusion animals, but the stem cell treatment restored the effects. These results suggest that stem cells might be helpful for ovarian regeneration after injuries by promoting vascular neogenesis and steroidogenesis through the MAPK pathway.

Acute effects of digoxin on plasma aldosterone and cortisol in monkeys.

Digoxin, a cardiac glycoside, is used to increase cardiac contractility via inhibition of Na(+)/K(+)-adenosinetriphosphatase (ATPase) and increase intracellular calcium in congestive heart failure. Inhibitory effects of digoxin have been demonstrated on the biosynthesis of gonadal hormones and adrenal glucocorticoids in rats. However, acute effects of digoxin on levels of adrenal corticosteroid hormones in the primates in vivo are uncertain. Therefore, we test the hypothesis that a single injection of digoxin decreases the secretion of aldosterone and cortisol in monkeys. An intravenous injection of digoxin (1 microg/kg) inhibited basal and adrenocorticotropin (ACTH)- or KCl-stimulated aldosterone release in monkeys. Furthermore, digoxin induced a decrease in ACTH- and KCl-stimulated cortisol release. Administration of digoxin did not alter plasma concentrations of Na(+) and K(+). Ouabain, a selective inhibitor of Na(+)/K(+)-ATPase, did not affect ACTH- or KCl-stimulated aldosterone and cortisol release. These results revealed that injection of digoxin induced an inhibitory effect on aldosterone and cortisol secretion in monkeys. Because ouabain did not affect levels of plasma aldosterone or cortisol, we suggest that (1) the Na(+)/K(+)-ATPase pathway may not be involved in the mechanism of action of digoxin on aldosterone or cortisol secretion in monkeys and/or (2) the Na(+)/K(+)-ATPase is more sensitive to digoxin than to ouabain in monkeys.

Effect of aging on corticosterone secretion in diestrous rats.

The roles of age and prolactin (PRL) in regulating glucocorticoid secretion in diestrous rats were investigated. Adrenal zona fasciculata-reticularis (ZFR) cells from young, adult, middle (mid)-aged, and old female rats were isolated. Estrous cycle stage was determined by light microscopy after vaginal smears. Blood samples were collected from right jugular vein at 0, 30, 60, and 120 min after challenge with adrenocorticotropin (ACTH). During the diestrous phase, plasma levels of estradiol and progesterone were lower in mid-aged and old rats than in either young or adult rats. Age-dependent increases of the basal levels of plasma PRL and corticosterone were observed. No difference of ACTH-increased plasma concentrations of corticosterone was observed among young, adult, mid-aged, and old rats. Aging increased the basal, ACTH-, PRL-, forskolin (an adenylate cyclase activator)-, and 3-isobutyl-l-methylxanthine (IBMX, a non-selective phosphodiesterase inhibitor)-stimulated release of corticosterone and production of adenosine 3', 5'-cyclic monophosphate (cAMP) in ZFR cells. However, the 8-Br-cAMP (a membrane-permeable cAMP)-stimulated release of corticosterone was not affected by age. Taken together, these data indicated that aging increased corticosterone secretion in female rats during diestrous phase, which is in part due to an increase in cAMP accumulation. In conclusion, aging and PRL play a stimulatory role in the co-regulation of corticosterone secretion.

Inhibitory effects of digoxin and ouabain on aldosterone synthesis in human adrenocortical NCI-H295 cells.

The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI-H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)-stimulated release of aldosterone by NCI-H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11beta-hydroxylase, P450c11beta) to cortisol were reduced by digoxin or ouabain. The basal and 22-hydroxy-cholesterol (a membrane-permeable cholesterol, substrate of cytochrome P450 side-chain cleavage enzyme, P450scc)-stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II-stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI-H295 cells. Digoxin or ouabain (10(-6) M, 24 h)-treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10(-6) M digoxin or ouabain, the digoxin- or ouabain-induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11beta and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI-H295 cells.

Effects of chronic hypogonadism on corticosterone secretion and cyclic AMP production in male rat adrenocortical cells.

AIM: To determine the secretion of corticosterone (CCS) both in vivo and in vitro during different intervals after orchidectomy in male rats. METHODS: Three- and 12-month-old rats had been orchidectomized 0, 3, 6, or 9 months before decapitation. RESULTS: Orchidectomy increased the concentrations of plasma CCS, the basal release of CCS, and the adenosine 3', 5'-cyclic monophosphate (cAMP) production in rat zona fasciculata reticularis (ZFR) cells. The forskolin/3-isobutyl-l-methylxanthine-stimulated releases of CCS and cAMP production by ZFR cells were higher in rats with chronic hypogonadism. The CCS release from ZFR cells of orchidectomized rat was not altered by 8-bromo-cAMP treatment. Orchidectomy enhanced the stimulatory effect of deoxycorticosterone on CCS release in ZFR cells. CONCLUSION: These results suggest that orchidectomy-related increases of CCS secretion in rats are associated with an increase of adenylate cyclase activity, cAMP generation, and 11-beta-hydroxylase activity in ZFR cells.

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.

Stimulatory effects of hyperprolactinemia on aldosterone secretion in ovariectomized rats.

BACKGROUND: To evaluate the effects of hyperprolactinemia on aldosterone secretion and its mechanisms of action in ovariectomized (OVX) rats. METHODS: Hyperprolactinemia was induced by the transplantation of rat anterior pituitary (AP) glands under the kidney capsule for 6 weeks in female rats. Control rats underwent cerebral cortex (CX) transplantation. Four weeks after transplantation, the rats were OVX 2 weeks before decapitation. After decapitation, the trunk blood was collected, and the adrenal glands of CX- and AP-grafted rats were prepared as zona glomerulosa (ZG) cells for in vitro study. RESULTS: Plasma prolactin and aldosterone in the rats were increased by AP gland transplantation. In the in vitro study, the basal aldosterone secretion by the adrenal ZG cells was higher in AP-grafted rats than in CX-grafted rats. The AP-grafted group showed increased responsiveness to angiotensin II (10(-8) M), KCl (8 x 10(-3) M), or 8-bromo-adenosine 3',5'-cyclic monophosphate (8-br-cAMP; 10(-4) M, a membrane-permeable analogue of cAMP) with regard to aldosterone secretion as compared with the CX-grafted group. N-(2-[p-Bromocinnamylamine]ethyl)-5-isoquinolinesulfonamide (H89; 10(-6), 10(-5) M, a protein kinase A inhibitor) or tetrandrine (10(-5) M, a blocker for both L-type and T-type Ca2+ channels) induced a greater suppression of aldosterone secretion in the AP-grafted group than in the CX-grafted group. No significant differences between the CX- and AP-grafted groups were observed, however, with regard to the adrenocorticotropichormone (10(-9) M)-, forskolin (10(-5) M, an adenylyl cyclase activator)-, or nifedipine (10(-5) M, an L-type Ca2+ channel blocker)-induced responsiveness of aldosterone secretion. In addition, there was no difference in the expression of desmolase (i.e., cytochrome P450 side-chain cleavage enzyme) in ZG cells between AP- and CX-grafted rats. The conversions of 25-OH-cholesterol into pregnenolone in the presence of trilostane (an inhibitor of 3beta-hydroxysteroid dehydrogenase) and corticosterone into aldosterone, as well as the expression of the steroidogenic acute regulatory protein in ZG cells, were greater in AP-grafted rats than in CX-grafted rats. CONCLUSIONS: These results suggest that hyperprolactinemia increases basal, angiotensin II- and KCl-stimulated aldosterone secretion by ZG cells in OVX rats through activation of T-type Ca2+ channels, the post-cAMP and protein kinase A pathway, cytochrome P450 side-chain cleavage enzyme, and aldosterone synthase, as well as by causing increased expression of steroidogenic acute regulatory protein in ZG cells.

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.