title: Regulation of phospholipase D in L6 skeletal muscle myoblasts. Role of protein kinase c and relationship to protein synthesis.
abstract: 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.
authors.affiliation: Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom.
authors.forename: M G
authors.initials: MG
authors.lastname: Thompson
authors.forename: S C
authors.initials: SC
authors.lastname: Mackie
authors.forename: A
authors.initials: A
authors.lastname: Thom
authors.forename: R M
authors.initials: RM
authors.lastname: Palmer
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D010739:Phospholipase D / Q000096:biosynthesis*; D014176:Protein Biosynthesis / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014667:Vasopressins / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D004983:Ethanolamines; D007211:Indoles; D014667:Vasopressins; D019856:Ethanolamine; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; C064758:Ro 31-8220