ISOFORM-SPECIFIC UP-REGULATION BY OUABAIN OF NA-ATPASE IN CULTURED RAT ASTROCYTES(,K+)

There are two alpha-subunit isoforms (alpha 1 and alpha 2) and two bet a-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 af ter addition of the inhibitor. Immunochemical analyses showed that oua bain increased alpha 1 and beta 1, but not alpha 2 and beta 2, protein s, 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 cyclohexi mide (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 th at intracellular Na+, Ca2+, and calcineurin may be involved in ouabain -induced up-regulation of the enzyme in astrocytes.