Role of protein kinase C in 1,25(OH)2‐vitamin D3 modulation of intracellular calcium during development of skeletal muscle cells in culture

Regulation of muscle cell Ca2+ metabolism by 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] is mediated by the classic nuclear mechanism and a fast, nongenomic mode of action that activates signal transduction pathways. The role of individual protein kinase C (PKC) isoforms in the regulation of intracellular Ca2+ levels ([Ca2+]i) by the hormone was investigated in cultured proliferating (myoblasts) and differentiated (myotubes) chick skeletal muscle cells. 1,25(OH)2D3 (10−9 M) induced a rapid (30- to 60-s) and sustained (>5-min) increase in [Ca2+]i which was markedly higher in myotubes than in myoblasts. The effect was suppressed by the PKC inhibitor calphostin C. In differentiated cells, PKC activity increased in the particulate fraction and decreased in cytosol to a greater extent than in proliferating cells after 5-min treatment with 1,25(OH)2D3. By Western blot analysis, these changes were correlated to translocation of the PKC α isoform from cytosol to the particulate fraction, which was more pronounced in myotubes than in myoblasts. Specific inhibition of PKC α activity using antibodies against this isoform decreased the 1,25(OH)2D3-induced [Ca2+]i sustained response associated with Ca2+ influx through voltage-dependent calcium channels. Neomycin, a phospholipase C (PLC) inhibitor, blocked its effects on [Ca2+]i, PKC activity, and translocation of PKC α. Exposure of myotubes to 1,2-dioleyl-rac-glycerol (1,2-diolein), also increased [Ca2+]i, PKC activity, and the amount of PKC α associated with the particulate fraction. Changes in [Ca2+]i induced by diolein were inhibited by calphostin C and nifedipine. The results indicate that PKC α activation via PLC-catalyzed phosphoinositide hydrolysis is part of the mechanism by which 1,25(OH)2D3 regulates muscle intracellular Ca2+ through modulation of the Ca2+ influx pathway of the Ca2+ response to the sterol. J. Cell. Biochem. 77:200–212, 2000. © 2000 Wiley-Liss, Inc.