Na,K-ATPase α2 Inhibition Alters Calcium Responses in Optic Nerve Astrocytes

Experiments were conducted to test the effect of 1 μM 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 μM ouabain is sufficient to inhibit the α2 Na,K-ATPase, but not the α1. Immortalized astrocytes derived from Na,K-ATPase α2 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 μM ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 μM ouabain, a significantly greater calcium response to ATP was observed. 1 μM ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase α2 and α1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase α1 (rat kidney). In intact astrocytes, 1 μM ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na, K-ATPase α2 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 μM ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase α2 isoform has the potential to change calcium signaling and CCE.