Corticotropin regulation of the synthesis of a specific rat adrenal cytosolic protein. Effects of hypophysectomy and actinomycin D

Abstract

The mechanism of corticotropin stimulation of the synthesis of a specific rat adrenal cytosolic protein was investigated. This protein (protein E) has a mol.wt. of approx. 30000. It is detected by polyacrylamide-gel electrophoresis of cytosol prepared from adrenal slices from rats treated with corticotropin in vivo and control rats, the slices being incubated with [3H]-and ['4C]-leucine respectively. In rats 1-15 days after hypophysectomy, corticotropin, like dibutyryl cyclic AMP, induces an increase in protein E similar to that induced in control rats, even though both compounds no longer stimulate total protein synthesis. Corticotropin stimulation of protein E synthesis is mediated by cyclic AMP but not by corticosterone, since aminoglutethimide, a steroidogenic inhibitor, does not affect corticotropin stimulation, and dexamethasone alone has no effect. Actinomycin D, when injected in vivo 1 h before or after corticotropin injection, prevents the effect of corticotropin on protein E synthesis, which is interpreted as evidence that mRNA synthesis is necessary for the stimulation of protein E synthesis. When injected more than 2h after corticotropin, actinomycin D does not prevent corticotropin stimulation of protein E synthesis, but completely blocks corticotropin stimulation of total protein synthesis. This is interpreted as meaning that, after stimulation of mRNA coding for protein E, corticotropin has no effect on the synthesis of protein E. On the other hand, corticotropin stimulation of protein E synthesis persists after hypophysectomy even though it no longer stimulates total protein synthesis. These data suggest that the factor(s) involved in the synthesis of protein E are more stable than those involved in total protein synthesis.Hormonal induction of specific proteins has been studied in great detail for model systems that are under steroid-hormone control (Katzenellenbogen & Gorski, 1972;Schimke et al., 1975; Feigelson etal., 1975). Less work has been done with polypeptide hormones acting via cyclic AMP. Janszen et al. (1977) have shown that, in rat interstitial cell, lutropin induces an increased synthesis of a protein with an apparent mol.wt. of 21000 (lutropin-induced protein). It was further shown that the stimulatory effect of lutropin on this protein was mediated by cyclic AMP through an increase in mRNA (Janszen et al., 1978), but there is no evidence that this protein is involved in lutropin stimulation of testosterone biosynthesis. As far as corticotropin is concerned, the involvement of a rapidly turning-over protein mediating the steroidogenic action of the hormone was postulated several years ago (Garren et al., 1971;Schulster et al., 1974), but the identity of such a protein is still unknown. We have shown (Dazord et al., 1978) that, besides a general tropic effect on Vol. 182 most adrenal proteins, corticotropin produces a marked stimulation of a specific adrenal cytosolic protein (protein E) with mol.wt. approx. 30000 and p15.5. From our kinetic results (lack of effect of corticotropi...

ACTH Regulation of Adrenal mRNA Coding for Total and Specific Adrenal Proteins

“…This study confirms and extends previous work from our laboratory [2,3] indicating preferential synthesis of protein E in response to ACTH. In the present study, by using in vitro translation assays, we demonstrate that the increased synthesis of protein E is due to an increased level of its mRNA.…”

“…Radioactive amino acid incorporation into adre nal slices from control and ACTH-treated rats was performed as previously described [2,3] except that both ['H]-leucine 200pCi/ml and [,5S]-methionine 200 pCi/ml were used in the incubation. Adrenal cyto solic preparations from control and ACTH-treated rats prepared as detailed elsewhere [2,3] were submit ted to slab polyacrylamide gel electrophoresis.…”

“…Adrenal cyto solic preparations from control and ACTH-treated rats prepared as detailed elsewhere [2,3] were submit ted to slab polyacrylamide gel electrophoresis. The slabs were dried and exposed to Kodak X-Omat XR-I films for at least 2 days at -7 0 "C. Protein E was cut out from the appropriate region of the gel and eluted, in water, overnight at 4 °C.…”

Mechanism of Corticotropin Action on Adrenal Protein Synthesis

“…In the present experiments in order to increase the duration of the action of Synacthene Retard it was dissolved in saline at pH 10. Under these conditions using the same amount of Synacthene we were able to stimulate total protein synthesis in hypophysectomized rats to about the same extent as in control rats, contrary to what was found when Synacthene Retard was dissolved in saline at pH 7 [10] where no stimulation of total protein synthesis was found in hypophysectomized rats. Most of the experiments were repeated at least three times.…”

“…In vitro, corticotropin stimulates the synthesis of a small peptide in a cultured mouse adrenocortical cell line Y( [8], In vivo cortico tropin produces a marked stimulation of the synthesis of a specific adrenal cytosolic protein (protein E) [9], Previous work from this laboratory [10] has shown that the synthesis of this protein is cyclic AMP-dependent and requires mRNA synthesis.…”