title: Protein kinase C isoform delta is involved in the stimulation of the Na(+)-H+ exchanger in C6 glioma cells.
abstract: Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, is a family consisting of at least 12 distinct isoforms. In C6 glioma cells, short term (10 min) treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) results in a dose-dependent translocation of PKC alpha, PKC delta and PKC theta. Long term (24 hr) treatment with appropriate doses of TPA results in the complete down-regulation of PKC delta but not of PKC alpha PKC theta. This property was used to determine which isoform might be involved in the activation of the glial cell Na(+)-H+ exchanger. It was found that (a) TPA has dose-dependent effects on PKC alpha, PKC delta, and PKC theta translocation and on Na(+)-dependent, EIPA-sensitive Na(+)-H+ exchanger stimulation; (b) the antiporter is blocked both by staurosporine and in cells in which PKC alpha, PKC delta, and PKC theta have been completely down-regulated; (c) the inactive form (alpha-TPA) of TPA, which does not include translocation of the three isoforms (alpha, delta, and theta) has no effect on the antiporter; (d) Western blot analysis demonstrated dose-dependent TPA (10, 30, 100, and 1000 nM)-induced translocation of PKC alpha, PKC delta, and PKC theta. TPA (10, 30, 100, and 1000 nM)-induced Na(+)-H+ exchanger activation was also found to be dose dependent. There was no difference in TPA-induced Na(+)-H+ exchanger activation between 30 and 100 nM; it correlated with the extent of TPA-induced PKC delta translocation over the same concentration range, suggesting that isoform responsible for the exchanger activation is PKC delta; (e) When 1 microM TPA is added after prior treatment of the cells with 10 nM TPA, an additive effect on Na(+)-H+ exchanger is seen that is not observed when the initial stimulus in 30 nM TPA, as would be expected if the PKC delta isoform were responsible for exchanger activation; (f) Finally, when PKC delta, but not PKC alpha and PKC theta, is completely down-regulated by 24-hr pretreatment with 10, 30, or 100 nM TPA, the Na(+)-H+ exchanger can no longer be stimulated by 1 microM TPA; when 1 nM TPA pretreatment is used, no down-regulation occurs and the exchanger still responds to 1 microM TPA. We have shown that the Na(+)-H+ exchanger in C6 glioma cells can be stimulated by TPA-induced PKC activation and, for the first time, that PKC delta is involved in the activation of this antiporter. Our results also suggest that different members of the PKC family within a single cell elicit specific physiological responses.
authors.affiliation: Department of Pharmacology, College of Medicine, National Taiwan University, Taipei.
authors.forename: C C
authors.initials: CC
authors.lastname: Chen
authors.forename: M L
authors.initials: ML
authors.lastname: Wu
mesh_terms: D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D005910:Glioma / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000502:physiology*; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014178:Translocation, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D007527:Isoenzymes; D017923:Sodium-Hydrogen Exchangers; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate