Effects of adrenocorticotropin on 17 alpha-hydroxylase activity and cytochrome P-450(17 alpha) synthesis in bovine adrenocortical cells.
The effects of adrenocorticotropin (ACTH) on 17 alpha-hydroxylase activity and cytochrome P-450(17 alpha) synthesis have been studied utilizing bovine adrenocortical cells in primary monolayer culture. A 20-fold stimulation of the conversion of pregnenolone to 17 alpha-hydroxypregnenolone was observed in postmitochondrial supernatant fractions from cells treated with ACTH as compared to controls. This increase in 17 alpha-hydroxylase activity was found to be due to a change in the Vmax and not a change in the Km(app). By immunoisolation of newly synthesized protein from an RNA-directed cell-free translation system we found that the level of P-450(17 alpha) was many-fold greater when RNA from ACTH-stimulated cells was used, as compared to RNA from control cells. A similar pattern was obtained when the rate of P-450(17 alpha) synthesis was analyzed by immunoisolation from radiolabeled cellular protein from ACTH-stimulated cells. When the total amount of P-450(17 alpha) was measured by immunoblotting we found that the levels of enzyme present correlated with the 17 alpha-hydroxylase activity. In addition, we found that these ACTH-mediated effects could be mimicked by treatment of cells with analogs of cyclic AMP. These results indicate that the activity of P-450(17 alpha) is regulated primarily by cyclic AMP-mediated changes in synthesis, probably at the transcriptional level, which in turn has a profound effect on the pattern of steroid secretion. Thus, we believe cytochrome P-450(17 alpha) to be a key regulatory enzyme in the steroidogenic pathway.
Zuber
M X
MX
Simpson
E R
ER
Hall
P F
PF
Waterman
M R
MR
D006907:17-alpha-Hydroxypregnenolone / Q000378:metabolism; D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D007158:Immunologic Techniques; D007700:Kinetics; D011284:Pregnenolone / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*
D006907:17-alpha-Hydroxypregnenolone; D003994:Bucladesine; D011284:Pregnenolone; D000324:Adrenocorticotropic Hormone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase