Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.