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

Abstract

The contribution of protein synthesis and formation of microtubules and microfilaments to corticotropin-stimulated steriodogenesis 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, steroidegenesis was more extensively inhibited than protein synthesis, after which the effectiveness of the inhibitors diminished on steroidegenesis but not on protein synthesis. The reversal effects was not observed at high levels of inhibitors. One inhibitor of microfilament fromation (cytochalasin B) and four inhinitors 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 inhinitors 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 ( t 1 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, f 1 2 = 7–9 min ). Similar kinetics of inhibition and recovery were observed for vinblastine sulfate while a direct inhbition of cytochrome P-450 sec by an aminoglutethimide was complete within 1 min and was rapidly reversed. Injection of each inhibitors (all classes) into hypophysectomized rats inhibited the elevation of plasma corticosterone by corticotropin. The extent of cholesterol combination with cytochrome P-450 sec in adrenal mitochondria isolated from these rats was also decreased by all inhbitors. Decreases in plasma corticosterone correlated directly with decreases in cholesterol combination with cytochrome P-450 sec ( r = 0.94). It is concluded that protein synthesis and steroidogenesis must be intimately coupled propbably due to the requirement of a labile protein for cholesterol transport to cytochrome P-450 sec. An involvement of microtubules and microfilaments in this process is clearly indicated.