Protein kinase-mediated regulation of the Na+/H+ exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways

We examined regulation of the Na+/H+ exchanger isoform 1 by phosphorylation in the rat myocardium. We utilized cell extracts from adult rat hearts, ad ult rat extracts fractionated by fast performance liquid chromatography, an d extracts from cultured neonatal cardiac myocytes. The carboxyl-terminal 1 78 amino acids of the Na+/H+ exchanger were expressed in Escherichia coil f used with glutathione S-transferase. The purified protein was used as a sub strate for in vitro phosphorylation and in-gel kinase assays. Unfractionate d extracts from neonatal myocytes or adult hearts phosphorylated the COOH-t erminal domain of the antiporter. Western blot analysis revealed that mitog en-activated protein (MAP) kinase (44 and 42 kDa) and p90(rsk) (90 kDa) wer e present in specific fractions of cardiac extracts that phosphorylated the COOH-terminal protein, In-gel kinase assays confirmed that protein kinases of approximately 44 and 90 kDa could phosphorylate this domain. MAP kinase and p90(rsk)-dependent phosphorylation of the antiporter could be demonstr ated by immunoprecipitation of these kinases from extracts of neonatal card iac myocytes. PD98059, a mitogen-activated protein kinase kinase inhibitor, decreased MAP kinase and p90(rsk) phosphorylation of the antiporter and ab olished serum and endothelin 1-stimulated increases in steady-state pH(i). These results confirm the presence of MAP kinase-dependent phosphorylation in the regulation of the Na+/H+ exchanger in the rat myocardium and suggest an important role for p90(rsk) phosphorylation in regulation of the protei n by endothelin-mediated stimulation of the antiporter.