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.

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.

1 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. 2 Male rats were challenged with digoxin (1 mg ml À1 kg À1 ) in the presence or absence of adrenocorticotropin (ACTH, 5 mg ml À1 kg À1 ) 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. 3 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 À9 M), forskolin (10 À7 M), 8-bromo-cyclic 3 0 : 5 0 -adenosine monophosphate (10 À4 M), cyclopiazonic acid (CPA, 10 À5 M), trilostane (10 À6 M), 25-OH-cholesterol (10 À5 M), pregnenolone (10 À5 M), progesterone (10 À5 M), or deoxycorticosterone (10 À5 M) at 371C for 1 h before collection of the media. Corticosterone or pregnenolone levels were measured by radioimmunoassay. 4 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. 5 Administration of digoxin or digitoxin decreased both spontaneous and ACTH-stimulated release of corticosterone in vitro. 6 Digoxin (10 À7 -10 À5 M) and digitoxin (10 À7 -10 À5 M), but not ouabain (10 À7 -10 À5 M), dosedependently inhibited corticosterone production in response to forskolin and 8-Br-cyclic AMP in rat ZFR cells. 7 Both digoxin (10 À6 -10 À5 M) and digitoxin (10 À6 -10 À5 M) attenuated corticosterone production in response to CPA. 8 Digoxin (10 À5 M) or digitoxin (10 À5 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. 9 The enzyme activity of 11 b-hydroxylase (catalyses conversion of deoxycorticosterone to corticosterone) in ZFR cells was also inhibited by the administration of digoxin (10 À5 M) or digitoxin (10 À5 M). 10 These results together suggest that digoxin and digitoxin decrease the release of corticosterone by acting directly on ZFR cells via a Na þ , K þ -ATPase-independent mechanism involving the inhibition of the activities of adenylyl cyclase, cytochrome P450scc and 11 b-hydroxylase, as well as the functioning of cyclic AMP and intracellular calcium.

No abstract

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(beta-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.