REGULATION OF NA,K-ADENOSINE TRIPHOSPHATASE GENE-EXPRESSION BY SODIUM-IONS IN CULTURED NEONATAL RAT CARDIOCYTES

Na,K-ATPase (Na,K-pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na,K-ATPase alpha and beta subunits in cultured neonatal rat cardiocytes. We measured intracellular Na+ levels ([Na+]i) in cardiocytes using a Na+-sensitive fluorescence dye (SBFI). 1 mM ouabain caused a significant increase in [Na+]i in cardiocytes; from 12.8+/-0.3 to 28.8+/-1.8 mM. Exposure of cardiocytes to 1 mM ouabain resulted in a three- to fourfold increase in alpha1, alpha2, and alpha3 mRNA accumulation, and an approximate two-fold increase in beta1 mRNA accumulation. A maximum elevation was reached at 60 min in both cases. The ouabain-induced alpha1 mRNA accumulation was still observed in the Ca2+-free culture medium. Exposure of cardiocytes to 10 muM monensin in the absence of extracellular Ca2+ also resulted in a threefold increase in alpha1 mRNA accumulation. The increased alpha1 mRNA expression by 1 mM ouabain was associated with a fourfold increase in alpha1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha1, alpha2, and alpha3 isoform genes and a luciferase reporter gene revealed that 1 mM ouabain caused a twofold increase in luciferase activity in each alpha system. These results suggest that Na + directly regulates Na,K-ATPase gene expression in cardiocytes. The transfection study further supports the premise that Na+-responsive elements are located within the 5'-flanking sequences of each alpha isoform gene.