REGULATION OF GUINEA-PIG ADRENAL P450C21 MESSENGER-RNA, PROTEIN AND ACTIVITY BY RU486

The role of ACTH, forskolin and 8Br-cAMP on the regulation of mRNA abu ndance, protein levels and enzymatic activity of cytochrome P450 21-hy droxylase (P450c21, CYP21) were investigated in guinea pig adrenal cel l cultures. In untreated cells, 21-hydroxylase activity was diminished throughout a 48 h period of incubation. Although incubation with fors kolin and 8Br-cAMP restored 21-hydroxylase activity to normal levels, the addition of ACTH did not prevent the decrease of 21-hydroxylase ac tivity. Treatment of cells with RU486 for 24 h inhibited 21-hydroxylas e activity by 93%; however, after removal of the drug a slight increas e of enzyme activity was observed; this rise was enhanced by the addit ion of ACTH. Forskolin and 8Br-cAMP increased the levels of 21-hydroxy lase activity to the same range as seen in untreated cells. In cells t hat were not pretreated with RU486, incubation with cycloheximide for 1 h had no effect on 21-hydroxylase activity and could not prevent the modest increase of 21-hydroxylase activity induced by forskolin or 8B r-cAMP after 48 h of incubation. In RU486-treated cells, cycloheximide blocks the stimulation of enzyme activity induced by ACTH, forskolin and 8Br-cAMP. Our findings indicate that 21-hydroxylase activity can b e stimulted by ACTH, forskolin or 8Br-cAMP solely in the presence of r educed enzymatic activity. Western immunoblot analysis of P450c21 prot ein levels in untreated or RU486-treated adrenal cells indicate that P 450c21 protein levels were in the same range and further incubation wi th ACTH caused a similar elevation of P450c21 protein levels in both t he untreated and RU486-treated cells. Northern blot analysis on RNA is olated from adrenal cells showed that RU486 did not alter the basal st eady state levels of P450c21 mRNA. As well, incubation with ACTH or 8B r-cAMP increased the levels of P450c21 transcript to the same extent i n both untreated and RU486-treated cells. These results taken together provide additional evidence for the presence of an adrenal specific p rotein factor(s); modulating 21-hydroxylase activity.