Regulation of phospholipase D in L6 skeletal muscle myoblasts. Role of protein kinase c and relationship to protein synthesis.

The addition of vasopressin or 12-O-tetradecanoylphorbol-13-acetate (TPA) to prelabeled L6 myoblasts elicited increases in [14C]ethanolamine release, suggesting the activation of phospholipase D activity or activities. While the effects of both agonists on intracellular release were rapid and transient, when extracellular release of [14C]ethanolamine was measured, the effect of vasopressin was again rapid and transient, whereas that of TPA was delayed but sustained. Effects of both agonists on intra- and extracellular release were inhibited by the protein kinase C (PKC) inhibitor, Ro-31-8220, and PKC down-regulation by preincubation with TPA. The formation of phosphatidylbutanol elicited by vasopressin and TPA mirrored their effects on extracellular [14C]ethanolamine release in that the former was transient, whereas the latter was sustained. Responses to both agonists were abolished by PKC down-regulation. When protein synthesis was examined, the stimulation of translation by TPA and transcription by vasopressin were inhibited by Ro-31-8220. In contrast, down-regulation of PKC inhibited the synthesis response to TPA but not vasopressin. Furthermore, following down-regulation, the effect of vasopressin was still blocked by the PKC inhibitors, Ro-31-8220 and bisindolylmaleimide. Analysis of PKC isoforms in L6 cells showed the presence of alpha, epsilon, delta, mu, iota, and zeta. Down-regulation removed both cytosolic (alpha) and membrane-bound (epsilon and delta) isoforms. Thus, the elevation of phospholipase D activity or activities induced by both TPA and vasopressin and the stimulation of translation by TPA involves PKC-alpha, -epsilon, and/or -delta. In contrast, the increase in transcription elicited by vasopressin involves mu, iota, and/or zeta. Hence, although phospholipase D may be linked to increases in translation elicited by TPA, it is not involved in the stimulation of transcription by vasopressin.