Exogenous sphingosine l-phosphate and sphingosylphosphorylcholine do not stimulate phospholipase D in C6 glioma cells

In the present study we investigate the effect of exogenous sphingosine, sphingosine 1-phosphate and sphingosylphosphorylcholine on phsopholipase D (PLD) activity in glioma C6 cells. The cells were prelabeled with [1-C-14]palmitic acid and PLD-mediated synthesis of [C-14]phosphatidylethanol was measured. Sphingosine 1-phosphate and sphingosylphosphorylcholine did not stimulate [C-14]phosphatidylethanol formation either at low (0.1-10 mu M) or high (25-100 mu M) concentrations. On the other hand, sphingosine at concentrations of 100-250 mu M strongly stimulated PLD activity as compared to the effect of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), known as a PLD activator. The effect of TPA on PLD is linked to the activation of protein kinase C. The present study also shows that sphingosine additively enhances TPA-mediated PLD activity. This is in contrast to the postulated role of sphingosine as a protein kinase C inhibitor. These results demonstrate that in glioma C6 cells sphingosine not only affects PLD independently of its effect on protein kinase C, but also is unable to block TPA-mediated PLD activity.