Increased diacylglycerol levels inhibit [20-3H]phorbol 12,13-dibutyrate binding and the glucocorticoid-mediated increase in glycerol phosphate phosphate dehydrogenase levels in C6 rat glioma cells.

I examined whether the phorbol ester-mediated inhibition of glycerol 3-phosphate dehydrogenase (GPDH) induction could be mimicked by raising the cellular diacylglycerol levels. Phorbol ester tumor promoters and diacylglycerols activate protein kinase C. An increase in radiolabeled diacylglycerol levels in C6 rat glioma cells was observed when cells were prelabeled overnight with [3H]arachidonic acid and treated with either phospholipase C (Clostridium perfringens) or 2-bromooctanoate. The increase was dose dependent. The diacylglycerols competed with [20-3H]phorbol 12,13-dibutyrate in binding to the phorbol ester receptor. A Scatchard analysis of the binding of cells treated with 0.1 unit/ml of phospholipase C demonstrated that the inhibition was mainly due to a decrease in binding affinity and not in the total number of binding sites. 2-Bromooctanoate and phospholipase C, but not the synthetic diacylglycerol 1-oleoyl 2-acetyl glycerol, inhibited the glucocorticoid induction of GPDH levels. Boiled phospholipase C, phospholipase A2, or phospholipase D was ineffective in inhibiting induction, a result suggesting that the inhibition was not due to nonspecific membrane perturbation. Thus, inhibition of the glucocorticoid-mediated increase in GPDH induction is most likely mediated by protein kinase C, and not by an alternate phorbol ester receptor.