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 (T PA) to prelabeled L6 myoblasts elicited increases in [C-14]ethanolamin e release, suggesting the activation of phospholipase D activity or ac tivities, While the effects of both agonists on intracellular release were rapid and transient, when extracellular release of [C-14]ethanola mine was measured, the effect of vasopressin was again rapid and trans ient, whereas that of TPA was delayed but sustained, Effects of both a gonists on intra- and extracellular release were inhibited by the prot ein kinase C (PKC) inhibitor, Re-31-8220, and PKC down-regulation by p reincubation with TPA, The formation of phosphatidylbutanol elicited b y vasopressin and TPA mirrored their effects on extracellular [C-14]et hanolamine release in that the former was transient, whereas the latte r was sustained, Responses to both agonists were abolished by PKC down -regulation. When protein synthesis was examined, the stimulation of t ranslation by TPA and transcription by vasopressin were inhibited by R e-31-8220, In contrast, down-regulation of PKC inhibited the synthesis response to TPA but not vasopressin, Furthermore, following down-regu lation, the effect of vasopressin was still blocked by the PKC inhibit ors, Re-31-8220 and bisindolylmaleimide. Analysis of PKC isoforms in L 6 cells showed the presence of alpha, epsilon, delta, mu, iota, and ze ta. Down-regulation removed both cytosolic (alpha) and membrane-bound (epsilon and delta) isoforms, Thus, the elevation of phospholipase D a ctivity or activities induced by both TPA and vasopressin and the stim ulation of translation by TPA involves PKC-alpha, -epsilon, and/or -de lta, In contrast, the increase in transcription elicited by vasopressi n involves mu, iota, and/or zeta. Hence, although phospholipase D may be linked to increases in translation elicited by TPA, it is not invol ved in the stimulation of transcription by vasopressin.