LEUPEPTIN INHIBITS PHOSPHOLIPASE-D AND PHOSPHOLIPASE-C ACTIVATION IN RAT HEPATOCYTES

The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact he patocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation , a specific marker of phospholipase D activity. Leupeptin (10-1500 mu M) inhibited PETH formation induced by vasopressin, but was ineffecti ve in response to phorbol ester or calcium. Leupeptin also inhibited t he formation of inositol phosphates in intact cells in response to vas opressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membra ne-associated phospholipase D did not require calcium and was insensit ive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the in hibitory effects of leupeptin on phospholipase D occurred at doses tha t far exceed those required to maximally inhibit proteolysis. These da ta highlight a central role for phospholipase C in the activation of p hospholipase D, and a minor role for a direct G-protein activation. Th e findings also demonstrate a novel use of leupeptin as an inhibitor o f phospholipases D and C, perhaps at the level of a G protein.