Direct evidence for phorbol ester-stimulated accumulation of diacylglycerol derived from phosphatidylcholine.

A fluorescent analog of phosphatidylcholine, 1-acyl-2-[N-(4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl]phosphatidyl choline (NBD-PC), was inserted into the plasma membrane of HeLa cells. Treatment of NBD-PC-labelled cells with the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in the production of fluorescent diacylglycerol, 1-acyl-2-[N-(4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl]diacylglycerol (NBD-DG), suggesting that phorbol ester activates a PC-specific phospholipase C in these cells. Furthermore pretreatment of cells with phorbol-12,13-dibutyrate for 24 h, a process which depletes cellular protein kinase C activity, inhibited the ability of TPA to induce this effect. These results suggest that TPA stimulation of a phospholipase C specific for PC in HeLa cells is mediated via protein kinase C.