Abstract
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.
Highlights
Sis have been studied utilizing bovine adrenocortical Measurement of the conversion of progesterone to 17a-hycells in primary monolayer culture
The synthesis of several ment of cells with analogsof cyclic AMP. These results indicate that the activityof P-45017, is regulated primarily by cyclic AMP-mediated changes in synthesis, probably at the transcriptionalel vel, whichin turn has a profound effect on the pattern of steroid secretion
Webelievecytochrome P-45017, tobe a key components of the adrenocortical steroidogenic pathway is increased by treatment of bovine adrenocortical cells with ACTH (8), and such an action of ACTH could explain the above mentioned increase in cytochrome P-45Ol7, activity in response to stimulationby ACTH
Summary
4501,, was many-fold greater when RNA from ACTH- and progesterone to their respective 17a-hydroxylated derivstimulated cells was used, as compared to RNA from atives, cytochrome P-45OI7,,of both pig neonatal testis and control cells. In order to establish a suitable range of substrate concentrations for kinetic studies, the time course of 17a-hydroxypregnenolone formation at three different substrate concentrations was determined (Fig. 2 A ) Examination of these results shows that even at substrate concentrationsof 0.2 PM the reaction rate remained linear up to 40% conversion. Fig.2Bshows that the reaction was linear for the chosen periods of time when PMS fractions from gland cortex as well as PMS fractions from ACTH-stimulated or control bovine adrenocortical cells were utilized. There was not a significant difference in the values obtained for the apparent K , for pregnenolone in PMS fractions derived from either control or ACTH-stimulated cells or from fresh adrenal cortex (Table I).
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