INVIVO EFFECTS OF ADRENOCORTICOTROPIN ON HAMSTER ADRENAL STEROIDOGENIC ENZYMES

The hamster, a rodent possessing adrenal 17alpha-hydroxylase activity, was used to study the effect of ACTH on the regulation of cortisol formation in vivo. The characterization of the mRNA and protein of hamster adrenal steroidogenic enzymes revealed close similarities between this animal and other mammalian species. The hamster adrenal RNA hybridized in a single band to cDNA probes for bovine adrenal P450scc, P450(17alpha), P450c21, to mouse adrenal P450(11beta), and to pig testis 3beta-hydroxysteroid dehydrogenase (3betaHSD) in the areas of 2.2, 2.0, 2.3, 2.0, and 2.1 kilobases, respectively. Immunoblotting analyses revealed the presence of single protein bands reacting with antibodies to bovine P450scc, P450c21, porcine P450(17alpha), or human placental 3betaHSD in the areas of 52, 55, 5 1, and 41 kilodaltons, respectively, whereas two protein bands were detected at 48 and 52 kilodaltons with the antibody to bovine P450(11beta). After stimulation with ACTH injected at 5-h intervals over 20 h, plasma cortisol levels, which were already increased 2.5 h after the first injection, remained elevated for the duration of treatment and returned to control values 15 h after the last injection. The ratios of plasma cortisol to corticosterone were 1.5, 3.9, and 7 at 0, 2.5, and 5 h after the first injection and continued to rise to a value of 11 at 15 h after multiple injections. This ratio returned to control values 15 h after cessation of either the short term (one injection) or long term (five injections) treatment, indicating a control mechanism favoring cortisol formation upon ACTH stimulation. Of the adrenal enzyme systems examined, only three were directly affected by ACTH treatment. The mRNA level of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase, the key precholesterol regulatory step, increased after ACTH administration within 2.5 h and remained elevated during the entire study period. ACTH provoked a rapid and sustained increase in P450scc mRNA levels, which decreased very slowly after cessation of treatment without reaching control values 30 h after the last injection. Meanwhile, ACTH treatment caused no changes in the amount of adrenal cytochrome P450scc protein during treatment and 30 h after its cessation. Therefore, we postulate that factors other than newly synthesized P450scc protein participate in the control of this rate-limiting step. The high P450scc, mRNA levels observed suggest stabilization of mRNA and posttranscriptional events affecting its catabolism. In contrast, both the mRNA and protein contents of the adrenal cytochrome P450(17alpha) increased after ACTH administration, thereby showing a direct control by ACTH of the synthesis of this protein. The P450(17alpha) mRNA level was already maximally increased 2.5 h after the first injection, whereas the protein level as well as enzyme activity increased slowly between 5 and 10 h to attain a plateau after 15 h. This indicates that newly transcribed P450(17alpha) mRNA was not translated into protein immediately and suggests a posttranscriptional control at this level. Both P450(17alpha) mRNA and protein levels had returned to control values 15 h after the last ACTH injection. Although the levels of P450c21 mRNA and protein remained unchanged, ACTH potentiated 21-hydroxylase activity. No changes occurred in either the mRNA levels or the content and activity of 3betaHSD and cytochrome P450(11beta) during the ACTH treatment, showing no immediate participation of these systems in the ACTH stimulation of cortisol secretion in vivo. Aldosterone synthase activity was decreased by ACTH injection, indicating different regulations of cortisol and aldosterone syntheses. In conclusion, our results indicate that in a situation closely akin to the physiological condition when circulating ACTH is increased during the diurnal rhythm, cytochrome P450(17alpha) plays a preponderant role over other steroidogenic enzymes in regulation of the synthesis of cortisol by ACTH in the hamster adrenal. We, therefore, suggest that the hamster should be used as an important intermediary in vivo model to validate results from in vitro studies before extending them to human physiology.