Mechanisms of digoxin and digitoxin on the production of corticosterone in zona fasciculata-reticularis cells of ovariectomized rats.

Previous studies have indicated that digoxin (DG) inhibits testosterone production by rat testicular interstitial cells through both in vivo and in vitro experiments. DG and digitoxin (DT), but not ouabain, inhibit the progesterone, pregnenolone, and corticosterone secretion by rat granulosa cells, luteal cells, and zona fasciculata-reticularis (ZFR) cells, respectively. However, the effect of DG and DT on the enzyme kinetics of cytochrome P450 side chain cleavage enzyme (P450scc), the protein expression of P450scc and steroidogenic acute regulatory protein (StAR), and mRNA expression of StAR are unclear. ZFR cells were prepared from adrenocortical tissues of ovariectomized rats, and then challenged with adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, A23187, cyclopiazonic acid (CPA), nicotinic acid adenine dinucleotide phosphate (NAADP), trilostane, 25-OH-Cholesterol, progesterone, or deoxycorticosterone in the presence of DG, DT, or ouabain for 1 h. Enzyme kinetics of P450scc, protein expression of acute regulatory protein (StAR) and P450scc, and mRNA expression of StAR were investigated. DG and DT but not ouabain suppressed basal and other evoked-corticosterone release significantly. DG and DT also inhibited pregnenolone production. The Vmax of the DG and DT group was the same as the control group, but the Km was higher in DG- and DT-treated group than in control group. DT and ouabain significant suppressed mRNA expression of StAR. DG and DT had no effect on the P450scc and StAR protein expression at basal state, but diminished ACTH-induced StAR protein expression to basal level. These results indicated that DG and DT have an inhibitory effect on corticosterone production via a Na+, K+-ATPase-independent mechanism by diminishing actions on cAMP-, Ca2+-pathway, competitive inhibition of P450scc enzyme and reduction of StAR mRNA expression.

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.