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

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