Experiments were conducted to test the effect of 1 microM ouabain, an Na,K-ATPase inhibitor, on capacitative calcium entry (CCE) and calcium responses elicited by ATP in rat optic nerve astrocytes. In the rat, 1 microM ouabain is sufficient to inhibit the alpha2 Na,K-ATPase, but not the alpha1. Immortalized astrocytes derived from Na,K-ATPase alpha2 homozygous knockout (KO) mice and wild-type (WT) littermates were also used. Cytosolic calcium and sodium concentrations were measured using Fura-2 and SBFI, respectively. The magnitude of the increase in cytosolic calcium concentration during CCE was significantly greater in rat astrocytes exposed to 1 microM ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 microM ouabain, a significantly greater calcium response to ATP was observed. 1 microM ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase alpha2 and alpha1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase alpha1 (rat kidney). In intact astrocytes, 1 microM ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na,K-ATPase alpha2 KO astrocytes, the calcium increase during CCE was significantly higher than in WT cells, as was the magnitude of the calcium response to ATP. In KO astrocytes, but not WT, the cytosolic calcium increase during CCE was insensitive to 1 microM ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase alpha2 isoform has the potential to change calcium signaling and CCE.
Copyright 2003 Wiley-Liss, Inc.