Mei-Mei Kau

Effects of estradiol on aldosterone secretion in ovariectomized rats.

The effects of estradiol benzoate (EB) on steroidogenesis in rat zona fasciculata‐reticularis (ZFR) cells were studied. Female rats were ovariectomized (Ovx) for 2 weeks and then injected subcutaneously with oil or EB for 3 days before decapitation. ZFR cells were isolated and incubated with adrenocorticotropin (ACTH) or prolactin (PRL) for 1 h. Corticosterone concentrations in plasma and cell media, and adenosine 3′,5′‐cyclic monophosphate (cAMP) production in ZFR cells were determined by radioimmunoassay. The effects of EB replacement in vivo on the activities of steroidogenic enzymes in ZFR cells were measured by the amounts of intermediate steroidal products separated by thin‐layer chromatography. Replacement of EB in vivo resulted in a dose‐dependent increase of plasma PRL and corticosterone in Ovx rats. The basal, ACTH‐, and PRL‐stimulated release of corticosterone by ZFR cells was greater in EB‐ than in oil‐treated animals. Forskolin‐induced production of cAMP was greater in the EB‐replaced rats than in oil‐treated animals, which correlated with the increase of corticosterone production. The 3‐isobutyl‐l‐methylxanthine (IBMX) plus ACTH‐, IBMX plus PRL‐, and forskolin plus PRL‐stimulated productions of cAMP were higher in EB‐ than in oil‐treated rats. The enzyme activities of postpregnenolone were not affected by EB replacement in Ovx rats. These results suggest that the EB‐related increase of corticosterone production in Ovx rats is associated with an increase of cAMP generation and the stimulatory effect of PRL on ZFR cells. J. Cell. Biochem. 77:560‐568, 2000.

The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine

1 The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats. 2 Administration of amphetamine (10−12–10−6 M) in vitro resulted in a dose‐dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml−1)‐stimulated release of testosterone. 3 Amphetamine (10−9 M) enhanced the basal and hCG‐increased levels of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells. 4 Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine. 5 Nifedipine (10−6 M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10−10–10−6 M). 6 Amphetamine (10−10–10−6 M) significantly (P<0.05 or P<0.01) decreased the activities of 3β‐hydroxysteroid dehydrogenase (3β‐HSD), P450c17, and 17‐ketosteroid reductase (17‐KSR) as indicated by thin‐layer chromatography (t.l.c.). 7 These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3β‐HSD, P450c17, and 17‐KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

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, 3β‐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 3β‐HSD activity. J. Cell. Biochem. 73:563–572, 1999.

Effects of prolactin on aldosterone secretion in rat zona glomerulosa cells

Acute effects and action mechanisms of prolactin (PRL) on aldosterone secretion in zona glomerulosa (ZG) cells were investigated in ovariectomized rats. Administration of ovine PRL (oPRL) increased aldosterone secretion in a dose‐dependent manner. Incubation of [3H]‐pregnenolone combined with oPRL increased the production of [3H]‐aldosterone and [3H]‐deoxycorticosterone but decreased the accumulation of [3H]‐corticosterone. Administration of oPRL produced a marked increase of adenosine 3′,5′‐cyclic monophosphate (cAMP) accumulation in ZG cells. The stimulatory effect of oPRL on aldosterone secretion was attenuated by the administration of angiotensin II (Ang II) and high potassium. The Ca2+ chelator, ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA, 10−2 M), inhibited the basal release of aldosterone and completely suppressed the stimulatory effects of oPRL on aldosterone secretion. The stimulatory effects of oPRL on aldosterone secretion were attenuated by the administration of nifedipine (L‐type Ca2+ channel blocker) and tetrandrine (T‐type Ca2+ channel blocker). These data suggest that the increase of aldosterone secretion by oPRL is in part due to (1) the increase of cAMP production, (2) the activation of both L‐ and T‐type Ca2+ channels, and (3) the activation of 21‐hydroxylase and aldosterone synthase in rat ZG cells. J. Cell. Biochem. 72:286–293, 1999.

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. J. Cell. Biochem.

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 11β‐hydroxylase, P450c11β) 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 P450c11β and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI‐H295 cells.

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 (8x10-3 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 adrenocorticotropic hormone (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 3β-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.

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.

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-β-hydroxylase activity in ZFR cells.

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.