Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells overexpressing the calcium sensing receptor

Abstract

This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Gö6983, or Gö6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-α or -ε augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-ε strongly decreased the release, but dominant negative PKC-α had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-α but had little effect on the expression of PKC-ε. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.