Regulation of Na,K-adenosine triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes

Abstract

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 α and β 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+]j 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 α1, α2, and α3 mRNA accumulation, and an approximate two-fold increase in β1 mRNA accumulation. A maximum elevation was reached at 60 min in both cases. The ouabain-induced α1 mRNA accumulation was still observed in the Ca2+-free culture medium. Exposure of cardiocytes to 10 μM monensin in the absence of extracellular Ca2+ also resulted in a threefold increase in al α1 MRNA accumulation. The increased al mRNA expression by 1 mM ouabain was associated with a fourfold increase in al subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5-flanking sequences of al, α2, and α3 isoform genes and a luciferase reporter gene revealed that 1 mM ouabain caused a twofold increase in luciferase activity in each α 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′-flankine sequences of each a isoform gene.