Bradykinin B-2 receptors activate Na+/H+ exchange in mIMCD-3 cells via Janus kinase 2 aHld Ca2+/calmodulin

We used a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) to examine the regulation of the ubiquitous sodium-proton exchanger, Na+/H+ exchanger isoform 1 (NHE-1), by a prototypical G protein- coupled receptor, the bradykinin B, receptor. Bradykinin rapidly activates NHE-1 in a concentration-dependent manner as assessed by proton microphysio metry of quiescent cells and by 2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluo rescein fluorescence measuring the accelerated rate of pH, recovery from an imposed acid load. The activation of NHE-1 is blocked by inhibitors of the bradykinin B, receptor, phospholipase C, Ca2+/calmodulin (CaM), and Janus kinase 2 (Jak2), but not by pertussis toxin or by inhibitors of protein kin ase C and phosphatidylinositol 3'-kinase, Immunoprecipitation studies showe d that bradykinin stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1, CaM appears to be a direct substrate fo r phosphorylation by Jak2 as measured by an in vitro kinase assay. We propo se that Jak2 is a new indirect regulator of NHE-1 activity, which modulates the activity of NHE-1 by increasing the tyrosine phosphorylation of Ca2+ a nd most likely by increasing the binding of CaM to NHE-1.