title: 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, 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.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Centre, Box 582, SE-751 23 Uppsala, Sweden.
authors.forename: Amos M
authors.initials: AM
authors.lastname: Sakwe
authors.forename: Lars
authors.initials: L
authors.lastname: Rask
authors.forename: Erik
authors.initials: E
authors.lastname: Gylfe
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D002412:Cations; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D051744:Protein Kinase C-epsilon; D044169:Receptors, Calcium-Sensing / Q000096:biosynthesis* / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
chemical_list: C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D002227:Carbazoles; D002412:Cations; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D020033:Protein Isoforms; D044169:Receptors, Calcium-Sensing; C081021:Go 6976; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D009355:Neomycin; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium