Mechanisms of corticotropin action in rat adrenal cells. I. The effects of inhibitors of protein synthesis and of microfilament formation on corticosterone synthesis.

The contributions of protein synthesis and formation of microtubules and microfilaments to corticotropin-stimulated steroidogenesis in rat adrenal cell suspensions has been assessed by use of a series of inhibitors to each function. Five inhibitors of protein synthesis (cycloheximide, puromycin, blastocidin S, anisomycin, and trichodermin) each exhibited time-dependent inhibition of corticotropin-stimulated steroidogenesis. For the first 30 min, steroidogenesis was more extensively inhibited than protein synthesis, after which the effectiveness of the inhibitors diminished on steroidogenesis but not on protein synthesis. The reversal effect was not observed at high levels of inhibitors. One inhibitor of microfilament formation (cytochalasin B) and four inhibitors of microtubule formation (colchicine, podophyllotoxin, vinblastine sulfate and griseofulvin) inhibited steroidogenesis without inhibiting protein synthesis and without any reversal effect with prolonged incubation. The actions of all ten inhibitors were shown to be fully reversible. Cell superfusion of adrenal cells showed that the decay of steroidogenesis upon addition of all the protein synthesis inhibitors was similar to decay upon removal of corticotropin from the medium (t1/2 = 4--6 min). Recoveries from inhibition upon removal of the inhibitors were similar to each other and comparable to initial corticotropin stimulation of the cells (lag of 3--5 min, t1/2=7--9 min). Similar kinetics of inhibition and recovery were observed for vinblastine sulfate while a direct inhibition of cytochrome P-450scc by aminoglutethimide was complete within 1 min and was rapidly reversed. Injection of each inhibitor (all classes) into hypophysectomized rats inhibited the elevation of plasma corticosterone by corticotropin. The extent of cholesterol combination with cytochrome P-450scc in adrenal mitochondria isolated from these rats was also decreased by all of the inhibitors. Decreases in plasma corticosterone correlated directly with decreases in cholesterol combination with cytochrome P-450scc (r=0.94). It is concluded that protein synthesis and steroidogenesis must be intimately coupled probably due to the requirement of a labile protein for cholesterol transport to cytochrome P-450scc. An involvement of microtubules and microfilaments in this process is clearly indicated.