Protein kinase C (PKC) has been shown to exert broad actions in modulating Ca2+ in cardiac myocytes, however, the effect of PKC in skeletal muscle cells is largely unknown. In this Study, we examined the effect of the PKC inhibitor calphostin C (CC) and the PKC agonist phorbol 12-myristate 13-acetate (PMA) on intracellular Ca2+ handling in C2C12 skeletal myotubes and skinned skeletal muscle fibers of the rat. CC (250 nM) significantly prolonged (P = 0.01, n = 6), and the PKC agonist PMA (500 nM; P = 0.03, n = 6) significantly shortened the decay phase of electrically induced Ca2+ transients in C2C12 myotubes without affecting the amplitude or the time to peak of the transients. Skinned fiber studies showed that CC significantly inhibits SR Ca2+, uptake in skeletal muscle Cells. PMA had no effect. CC also increased the peak of ATP-induced Ca2+ transients release by 94.2% (P < 0.0001) in the presence of extracellular Ca2+ and 54.5% (P = 0.04) without external Ca2+ via IP3-Ca2+ release pathway in C2C12 myotubes, while PMA had no effect, Suggesting that CC may modulate IP3-induced Ca2+ release via a PKC-independent mechanism. CC at a concentration of 1 mu M was able to induce a large sustained elevation in basal [Ca2+](i) that was blocked by Ca2+ store depletion and the IP3 receptor blocker 2-APB. These results indicate that PKC plays a role in modulation of SR function in skeletal muscle cells, and the PKC inhibitor CC may alter Ca2+ handling via both PKC-dependent and PKC-independent pathways. (c) 2005 Elsevier Inc. All rights reserved.