Rapuano M, Ross A F, Prives J
Cellular and Developmental Biology Program, State University of New York, Stony Brook 11794.
Dev Biol. 1989 Aug;134(2):271-8. doi: 10.1016/0012-1606(89)90099-7.
Studies utilizing cultured muscle cells have shown that myoblast fusion requires extracellular Ca2+ and involves transient coordinated changes in cell membrane topography and cytoskeletal organization. However, neither the mechanisms by which Ca2+ influences these changes nor its cellular sites of action are known. We have investigated the effects of Ca2+ channel modulators and phorbol esters on fusion of embryonic chick myoblasts in culture. Myoblast fusion was inhibited by the Ca2+ channel blockers D600 and nitrendipine and stimulated by the Ca2+ 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 Ca2+ channel blockers or TPA display a distinctive elongated morphology that is characteristic of cells prevented from fusion by Ca2+ deprivation. The inhibition of fusion by D600 and TPA is significantly diminished in the presence of the Ca2+ 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 Ca2+ signal transduction pathway and that this interference may be associated with a protein kinase C catalyzed inhibitory phosphorylation of myosin light chain.
利用培养的肌肉细胞进行的研究表明，成肌细胞融合需要细胞外钙离子（Ca2+），并且涉及细胞膜拓扑结构和细胞骨架组织的瞬时协调变化。然而，钙离子影响这些变化的机制及其细胞作用位点均尚不清楚。我们研究了钙离子通道调节剂和佛波酯对培养的胚胎鸡成肌细胞融合的影响。钙离子通道阻滞剂D600和尼群地平抑制成肌细胞融合，而钙离子通道激活剂Bay K 8644则刺激成肌细胞融合。我们已获得证据表明，促肿瘤佛波酯12-O-十四烷酰佛波醇-13-乙酸酯（TPA）通过激活蛋白激酶C来抑制融合。被钙离子通道阻滞剂或TPA阻止融合的成肌细胞呈现出一种独特的细长形态，这是被钙离子剥夺阻止融合的细胞所特有的。在钙离子载体A23187存在的情况下，D600和TPA对融合的抑制作用显著减弱。发现TPA阻止成肌细胞融合伴随着细胞质肌球蛋白20 kDa轻链磷酸化的剂量和时间依赖性增加。细胞可渗透的二酰基甘油sn-1,2-二辛酰甘油（一种蛋白激酶C的有效激活剂）模拟了TPA对成肌细胞融合和肌球蛋白轻链磷酸化的影响。目前的结果表明，蛋白激酶C激活剂通过干扰钙离子信号转导途径来阻止融合，并且这种干扰可能与蛋白激酶C催化的肌球蛋白轻链抑制性磷酸化有关。
