Opposing effects of calcium entry and phorbol esters on fusion of chick muscle cells

Abstract

Studies utilizing cultured muscle cells have shown that myoblast fusion requires extracellular Ca 2+ and involves transient coordinated changes in cell membrane topography and cytoskeletal organization. However, neither the mechanisms by which Ca 2+ influences these changes nor its cellular sites of action are known. We have investigated the effects of Ca 2+ channel modulators and phorbol esters on fusion of embryonic chick myoblasts in culture. Myoblast fusion was inhibited by the Ca 2+ channel blockers D600 and nitrendipine and stimulated by the Ca 2+ channel activator Bay K 8644. We have obtained evidence that the tumor promoting phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA) inhibits fusion through activation of protein kinase C. Myoblasts prevented from fusing by Ca 2+ channel blockers or TPA display a distinctive elongated morphology that is characteristic of cells prevented from fusion by Ca 2+ deprivation. The inhibition of fusion by D600 and TPA is significantly diminished in the presence of the Ca 2+ ionophore A23187. TPA arrest of myoblast fusion was found to be accompanied by an increase in phosphorylation of the 20-kDa light chain of cytoplasmic myosin in a dose- and time-dependent manner. The effects of TPA on myoblast fusion and phosphorylation of myosin light chain were mimicked by the cell permeant diacylglycerol sn-1,2-dioctanoylglycerol, a potent activator of protein kinase C. The present results suggest that activators of protein kinase C block fusion by interfering with a Ca 2+ signal transduction pathway and that this interference may be associated with a protein kinase C catalyzed inhibitory phosphorylation of myosin light chain.