Ethanol induction of steroidogenesis in rat adrenal and brain is dependent upon pituitary ACTH release and de novo adrenal StAR synthesis

Abstract

The mechanisms of ethanol actions that produce its behavioral sequelae involve the synthesis of potent GABAergic neuroactive steroids, specifically the GABAergic metabolites of progesterone, (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP), and deoxycorticosterone, (3alpha,5alpha)-3,21-dihydroxypregnan-20-one. We investigated the mechanisms that underlie the effect of ethanol on adrenal steroidogenesis. We found that ethanol effects on plasma pregnenolone, progesterone, 3alpha,5alpha-THP and cortical 3alpha,5alpha-THP are highly correlated, exhibit a threshold of 1.5 g/kg, but show no dose dependence. Ethanol increases plasma adrenocorticotropic hormone (ACTH), adrenal steroidogenic acute regulatory protein (StAR), and adrenal StAR phosphorylation, but does not alter levels of other adrenal cholesterol transporters. The inhibition of ACTH release, de novo adrenal StAR synthesis or cytochrome P450 side chain cleavage activity prevents ethanol-induced increases in GABAergic steroids in plasma and brain. ACTH release and de novo StAR synthesis are independently regulated following ethanol administration and both are necessary, but not sufficient, for ethanol-induced elevation of plasma and brain neuroactive steroids. As GABAergic steroids contribute to ethanol actions and ethanol sensitivity, the mechanisms of this effect of ethanol may be important factors that contribute to the behavioral actions of ethanol and risk for alcohol abuse disorders.

Fig. 1

Threshold for ethanol-induced increases in plasma steroids. Ethanol was administered at varying doses and plasma and cerebral cortex were collected after 60 min to measure steroid levels. (a) Plasma pregnenolone, (b) plasma progesterone, (c) plasma 3α,5α-THP and (d) cerebral cortical 3α,5α-THP. *p < 0.001 and **p < 0.05 compared to saline control (one-way ANOVA followed by Newman-Keuls test), n = 4–5 for each group in duplicate.

Fig. 2

Hypophysectomy abolishes ethanol-induced increases in ACTH release as well as plasma and brain steroid levels. (a) Plasma ACTH, (b) plasma progesterone and (c) cerebral cortical 3α,5α-THP levels following hypophysectomy compared to sham-operated controls. *p < 0.001 compared to all groups (two-way ANOVA followed by Bonferroni post-tests), n = 8–9 in duplicate.

Fig. 3

Dexamethasone inhibits ACTH release as well as ethanol-induced increases in steroid levels. Effect of dexamethasone (0.1 mg/kg, i.p.) for 90 min on the ethanol-induced increase in (a) plasma ACTH, (b) plasma progesterone, (c) 3α,5α-THP in the cerebral cortex and hippocampus and (d) adrenal StAR protein expression. Representative blot shown. StAR protein was normalized to β-actin and presented as % control. *p < 0.001 compared to control, **p < 0.01 compared to control, #p < 0.001 compared to ethanol (EtOH) (ANOVA, followed by Newman–Keuls test), n = 6 in duplicate.

Fig. 4

Ethanol exhibits a threshold for plasma ACTH and adrenal StAR induction, and StAR expression and plasma progesterone levels are rapidly increased. Rats were administered various doses of ethanol and (a) plasma ACTH and (b) adrenal StAR protein expression were measured. A separate group of rats was administered ethanol and blood was collected at varying time points. (c) Time course of StAR protein induction. (d) Time course of plasma progesterone induction. *p < 0.01 compared to control, #p < 0.01 compared to 1.5 g/kg ethanol, and @p < 0.05 compared to 2.5 g/ kg ethanol (Student's t-test or ANOVA followed by Newman–Keuls test where appropriate), n = 4–6 for each group in duplicate.

Fig. 5

Simultaneous administration of cycloheximide (CHX) with ethanol (EtOH) prevents ethanol-induced increases in StAR protein, as well as plasma and brain steroid levels, but does not affect ethanol-induced ACTH. Cycloheximide (20 mg/mL, i.p.) was administered with ethanol (2 g/kg, i.p.) and tissue was collected 60 min later. (a) Adrenal StAR protein expression was measured by western blot analysis as shown in a representative blot. StAR was normalized to b-actin and presented as % control values. (b) Plasma ACTH and (c) plasma progesterone levels. *p < 0.001 compared to control and #p < 0.001 compared to ethanol (ANOVA followed by Newman–Keuls test), n = 6 in duplicate.

Fig. 6

Acute ethanol administration increases phosphorylation of adrenal StAR protein. Rats were administered ethanol (2 g/kg, i.p.) and tissues were collected 60 min later. Adrenal fractions were immunoprecipitated with phospho-PKA substrate antibody, run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with StAR. Results are reported as fold increase of phospho-StAR (EtOH vs. saline) compared to fold increase of total StAR (EtOH vs. saline) *p < 0.01 (Student's t-test), n =4.