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In the steroidogenic pathway, 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) catalyzes the formation of hormonally active delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. In the present study the regulation of 3 beta HSD by ACTH action on bovine adrenocortical (BAC) cells in primary culture was evaluated. Western blot analysis was accomplished using an antibody against human placental 3 beta HSD. The relative molecular mass of 3 beta HSD in these cells was 45K, which was similar to that in human placenta. A significant effect of ACTH was not detected until day 6 of culture due to the high basal levels of the enzyme in BAC cells. Treatment of cells with ACTH on day 8 of culture resulted in a marked increase in the amount of 3 beta HSD protein, and this effect was correlated directly with enzymatic activity. The effects of ACTH were time and dose dependent, with an increase detectable only after 48 h of treatment; the maximal response was obtained with 10(-9) M ACTH. As demonstrated by Northern analysis, ACTH action was manifested by increasing the steady state level of 3 beta HSD mRNA. A human 3 beta HSD cDNA probe, which was used in this study, hybridized to a 1.7-kilobase species of BAC RNA. The effects of ACTH on 3 beta HSD activity and increases in 3 beta HSD protein and mRNA in BAC cells were mimicked by treatment with (Bu)2cAMP. The findings of this study suggest that ACTH controls 3 beta HSD gene expression in BAC cells by a cAMP-dependent mechanism similar to that involved in the expression of steroid hydroxylase genes. However, because the different stabilities of 3 beta HSD and hydroxylase proteins and/or mRNAs may play a critical role in determining the zone-specific steroids secreted from the adrenal cortex, other cAMP-dependent or independent regulatory mechanisms may also be important in regulating the expression of adrenal 3 beta HSD.

The regulation of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) was studied in primary cultures of rat adrenocortical cells. In the absence of ACTH, this enzymic activity was found to decay with a half-life of 3.1 days, which was similar to the half-life of the enzyme activity induced by ACTH in vitro (3.5 days). The increase in 3 beta-HSD activity was highly specific for ACTH and dibutyryl cAMP; the activity was not increased by other hormones known to affect adrenocortical growth or function. The induction of 3 beta-HSD activity by ACTH or dibutyryl cAMP required a lag period of approximately 4 h and was dependent on RNA and protein syntheses. The increase in 3 beta-HSD activity observed after ACTH treatment was not a result of ACTH-induced inhibition of degradation of the enzyme, nor was it due to the synthesis of a soluble intermediate which could directly activate the enzyme. ACTH stimulated the incorporation of [35S]methionine into a protein associated with 3 beta-HSD activity detected on polyacrylamide gels after electrophoresis of Triton X-100 extracts of adrenocortical cells. The induction of this protein by ACTH was inhibited by actinomycin D. A protein band of a partially purified preparation of rat adrenal 3 beta-HSD was found to comigrate with the ACTH-induced protein on sodium dodecyl sulfate-polyacrylamide gel. These results suggest that ACTH caused the de novo synthesis of 3 beta-HSD by a mechanism dependent on RNA synthesis.