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J. Biol. Chem., Vol. 280, Issue 6, 4436-4441, February 11, 2005

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Protein Kinase C Modulates Agonist-sensitive Release of Ca²⁺ from Internal Stores in HEK293 Cells Overexpressing the Calcium Sensing Receptor^*

Amos M. Sakwe, Lars Rask, and Erik Gylfe¶

From the Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Centre, Box 582, SE-751 23 Uppsala, Sweden and the ^¶Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23 Uppsala, Sweden

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- or - augmented the neomycin-induced release of Ca²⁺ from internal stores, whereas dominant negative PKC- strongly decreased the release, but dominant negative PKC- had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC- but had little effect on the expression of PKC-. 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.

Received for publication, October 14, 2004

^* This work was supported by Grants 15029 and 6240 from the Swedish Research Council (Medicine). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medical Biochemistry and Microbiology Biomedical Centre, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden. Tel.: 4618-4714335; Fax: 4618-4714975; E-mail: Lars.Rask{at}imbim.uu.se.

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