Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger.
To assess the role of Ca2+ in regulation of the Na+/H+ exchanger (NHE1),we used CCL-39 fibroblasts overexpressing the Na+/Ca2+ exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization (60-80% inhibition) induced by alpha-thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant with the calmodulin (CaM)-binding domain deleted (amino acids 637-656), suggesting that the effect of NCX1 transfection involves Ca2+-CaM binding. Expression of NCX1 only slightly inhibited platelet-derived growth factor BB-induced alkalinization and did not affect hyperosmolarity- or phorbol 12-myristate 13-acetate-induced alkalinization. Downregulation of protein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely in NCX1-transfected cells, suggesting that the thrombin effect is mediated exclusively via Ca2+ and PKC. On the other hand, deletion mutant study revealed that PKC-dependent regulation occurs through a small cytoplasmic segment (amino aids 566-595). These data suggest that a mechanism involving direct Ca2+-CaM binding lasts for a relatively long period after agonist stimulation, despite apparent short-lived Ca2+ mobilization, and further support our previous conclusion that Ca2+- and PKC-dependent mechanisms are mediated through distinct segments of the NHE1 cytoplasmic domain.
Department of Molecular Physiology, National Cardiovascular Research Institute, Osaka, Japan.
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Ikeda
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Iwamoto
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Wakabayashi
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Shigekawa
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D004285:Dogs; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D016257:Fura-2; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D010982:Platelet-Derived Growth Factor; D019831:Sodium-Calcium Exchanger; D017923:Sodium-Hydrogen Exchangers; D013395:Sucrose; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2