Effects of Ca2+ on the activation of conventional and new PKC isozymes and on TPA and endothelin-1 induced translocations of these isozymes in intact cells

AbstractThe effects of Ca2+ on the translocation of conventional and new protein kinase C isozymes in intact cells were studied by using C6 glioma cells as a model system. Two conditions which monitor intracellular Ca2+ were performed: one is extracellular Ca2+-depletion by treating the cells with physiological saline solution (PSS) without Ca2+ but containing 0.5 mM EGTA, the other is treating the cells with 1 μM ionomycin to induce Ca2+-influx. In addition, the TPA and endothelin-1 induced translocations of conventional and new PKC isozymes under these two conditions were also comparatively studied. When the intact cells were treated with Ca2+-free, EGTA containing PSS, the membrane-bound conventional PKCα (cPKCα) was greatly reduced and cytosolic cPKCα was slightly increased. However, neither membrane bound nor cytosolic new PKCδ (nPKCδ) was affected by extracellular Ca2+-depletion. On the other hand, when the cells were treated with 1 μM ionomycin, the translocation of cPKCα itself was observed while nPKCδ was not affected. In extracellular Ca2+-depletion, the translocation of cPKCα induced by 100 nM TPA still occurred although the extent of translocation was smaller than that induced by TPA under normal Ca2+ conditions; however, that induced by 30 nM ET-1 was blocked. After the cells were treated with 1 μM ionomycin, the translocation of cPKCα induced by 30 nM TPA was further increased compared to 1 μM ionomycin or 30 nM TPA alone, while that induced by ET-1 was only slightly further increased. All these results suggested that in intact cells, the activation of cPKCα was operated by both the intracellular Ca2+ level and diacylglycerol and that of nPKCδ was operated by diacylglycerol alone as predicted by their properties from purified enzyme or cDNA. In addition, the translocation of cPKCα induced by the natural activator ET-1 seemed to be more dependent on Ca2+ than TPA in intact cells