Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C.

Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with 3Hinositol. GTP gamma S stimulated the formation of 3Hinositol mono-, bis-, and trisphosphate (3HInsP, 3HInsP2, and 3HInsP3) with a half-maximal effect on 3HInsP formation at 5 microM. Carbachol increased the accumulation of 3Hinositol phosphates only in the presence of added guanine nucleotide. Calcium increased 3HInsP3 accumulation over a range of concentrations (10 nM-3 mM free calcium). When 1321N1 cells were treated with phorbol ester (100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)) prior to preparation of the membranes, the maximal 3HInsP formation induced by GTP gamma S or GTP gamma S plus carbachol was decreased by 50-75%. In contrast, the response to a maximal calcium concentration presumed to activate phospholipase C directly was minimally inhibited (approximately 15%). PMA treatment did not affect muscarinic receptor affinity for carbachol or the effect of GTP on agonist binding. PMA treatment was also without effect on the breakdown of exogenous 3HInsP3 in homogenates, permeabilized cells, and membranes, indicating that the InsP3-phosphatase was not the site of phorbol ester action. PMA treatment inhibited 3H InsP3 formation only in membranes and not in cytosol prepared from the same cells, suggesting a membrane site of PMA action. Membranes were also required to demonstrate GTP gamma S-stimulated 3HInsP3 formation although calcium-stimulated 3HInsP3 formation was demonstrable in both membranes and cytosol. The addition of purified protein kinase C to the membranes mimicked the effect of PMA treatment to decrease GTP gamma S-stimulated 3HInsP3 production. These data indicate that the effect of PMA on phosphoinositide metabolism is demonstrable in a cell-free system and that it can be mimicked by protein kinase C. We suggest that the ability of PMA to block GTP gamma S-stimulated formation of 3HInsP3 results from inhibition of the G protein interaction with phospholipase C.