Isoform-specific up-regulation by ouabain of Na+,K+-ATPase in cultured rat astrocytes

Abstract

There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na+,K+- ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5-1.0 mM) in- creased the levels of α1 and β1 mRNAs, whereas it de- creased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6-48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K+ and monensin (20 μM) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 μM), the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (30 μM), and the cal- cineurin inhibitor FK506 (1 nM). These findings indicate thai chronic inhibition of Na+,K+-ATPase up- regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes suggesting a functional coupling of α1/β1 complex. They also suggest that intracellular Na+, Ca2+, and calcineurin may be involved in ouabain- induced up-regulation of the enzyme in astrocytes.