Key result
Expression of NCX1 in fibroblasts markedly inhibited the transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization (60-80% inhibition) induced by alpha-thrombin.
The study demonstrates that Ca2+- and PKC-dependent mechanisms regulate the Na+/H+ exchanger through distinct segments of its cytoplasmic domain.
No immediate clinical application; leaves open Ca2+-CaM regulation of NHE1 in cardiovascular cells.
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.
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Ikeda et al. (1998) studied this question. Overexpression of Na+/Ca2+ exchanger (NCX1) vs. Control cells was evaluated on Transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization induced by alpha-thrombin (60-80% inhibition). Expression of NCX1 in fibroblasts markedly inhibited the transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization (60-80% inhibition) induced by alpha-thrombin.
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