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 (N CX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization (60-80% inhibiti on) induced by cr-thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant with t he calmodulin (CaM)-binding domain deleted (amino acids 637-656), sugg esting that the effect of NCX1 transfection involves Ca2+-CaM binding. Expression of NCX1 only slightly inhibited platelet-derived growth fa ctor BB-induced alkalinization and did not affect hyperosmolarity-or p horbol 12-myristate 13-acetate-induced alkalinization. Downregulation of protein kinase C (PKC) inhibited thrombin-induced alkalinization pa rtially in control cells and abolished it completely in NCX1-transfect ed cells, suggesting that the thrombin effect is mediated exclusively via Ca2+ and PKC. On the other hand, deletion mutant study revealed th at PKC-dependent regulation occurs through a small cytoplasmic segment (amino aids 566-595). These data suggest that a mechanism involving d irect Ca2+-CaM binding lasts for a relatively long period after agonis t stimulation, despite apparent short-lived Ca2+ mobilization, and fur ther support our previous conclusion that Ca2+- and PKC-dependent mech anisms are mediated through distinct segments of the NHE1 cytoplasmic domain.