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, Go6983, or Go6976 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-alpha or -epsilon augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-epsilon strongly decreased the release, but dominant negative PKC-alpha had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-alpha but had little effect on the expression of PKC-epsilon. 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.
Highlights
The Ca2ϩ receptor (CaR)1 is a G-protein-coupled receptor that senses the extracellular Ca2ϩ concentration ([Ca2ϩ]o) in parathyroid and other cell types
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
By maximally stimulating the CaR with 200 M neomycin in Ca2ϩ-deficient medium we studied the effect of PKC modulators on the amounts of Ca2ϩ, which could be released from intracellular stores
Summary
CaR, Ca2ϩ receptor; DAG, diacylglycerol; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; 2-APB, 2-aminoethoxydiphenyl borate; HEK, human embryonic kidney; wt, wild type; DN, dominant negative; BAPTA-AM, 1,2-bis(2aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis(acetoxymethyl ester). Depending on the cell type and the receptor that is activated, PKC activation inhibits or facilitates this process (14 –16), but it affects non-store-operated Ca2ϩ entry (17) and the Ca2ϩ storage capacity (18). It has been shown that TPA-induced PKC activation affects Ca2ϩ handling independently of the receptor via activation of plasma membrane Ca2ϩ-ATPases (22, 23). In accordance with such an effect, the amounts of mobilized intracellular Ca2ϩ were reduced when TPA-treated parathyroid cells were exposed to the Ca2ϩ ionophore ionomycin (24). Based on overexpression of PKC-␣ and -⑀ in HEK-CaR cells, we found that they differentially affected the release of Ca2ϩ from intracellular stores. These results urge for caution when interpreting data obtained with phorbol esters and/or PKC inhibitors
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