1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells

It has recently been proposed that degradation products of sphingolipids may serve as physiologic inhibitors of protein kinase C. The present study was performed to determine the effect of 1,2-diacylglycerols and phorbol esters, known activators of protein kinase C, on sphingomyelin metabolism. 1,2-Dioctanoylglycerol (diC8) caused time- and concentration-dependent reduction in the level of sphingomyelin labeled to equilibrium with 3H]choline. diC8 (200 micrograms/ml) reduced 3H]sphingomyelin to 81 +/- 3% of control (p less than 0.005) by 15 min, and the level was 58 +/- 5% of control after 1 h; an EC50 for this event was 56 micrograms/ml. To evaluate the mechanisms of stimulated hydrolysis, the sphingoid base backbone of sphingomyelin was labeled with 14C] serine, and the effects of diC8 were quantitated. diC8 (100 micrograms/ml) reduced the level of sphingomyelin to 66 +/- 7% of control by 1 h from 375 +/- 12 pmol/10(6) cells to 245 +/- 26 pmol/10(6) cells. There was a concomitant increase in ceramide from 89 +/- 4 pmol/10(6) cells to 252 +/- 27 pmol/10(6) cells consistent with activation of the enzyme, sphingomyelinase (EC 3.1.4.12). In support of this contention, 1,2-diacylglycerols appeared to enhance the activity of an acid, but not a neutral, sphingomyelinase in homogenates of GH3 cells. The 1,2-diacylglycerol, 1-oleyl-2-acetylglycerol, produced similar effects. In contrast, the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate, failed to stimulate sphingomyelin hydrolysis. Further, these effects of the 1,2-diacylglycerols occurred in cells down-modulated for protein kinase C. These studies demonstrate that 1,2-diacylglycerols stimulate sphingomyelin hydrolysis by a mechanism independent of the protein kinase C which mediates phorbol ester action. This is the first report of stimulated sphingomyelin hydrolysis by a physiologic effector molecule.