Inhibition of the Na,K–Atpase Alpha 2 Isoform Alters Calcium Store Size and Bcl–2 Expression in Optic Nerve Astrocytes

Abstract

Abstract: : Purpose: Previously we showed that selective inhibition of Na,K–ATPase α2 alters capacitative calcium entry (CCE), the mechanism by which ER calcium stores refill following emptying (Hartford et al., 2000, Glia 24: 229–37). Studies were conducted to determine whether calcium store size is altered in ouabain–treated cells. Bcl–2 expression was examined because there have been reports that in other cell types, altered Bcl–2 expression elicits changes in CCE (Williams et al., 2000, Cancer Res., 60: 4358–61). Methods: Rat optic nerve astrocytes were established in primary culture. Na,K–ATPase α2 was selectively inhibited using a low concentration of ouabain. The ouabain concentration was not sufficient to inhibit the rat Na,K–ATPase alpha 1 isoform which is ouabain–resistant. Cytosolic calcium was detected using Fura–2. Bcl–2, Bax and PCNA were detected by Western analysis. Results: Endoplasmic reticulum (ER) calcium store size, measured as ionomycin–releasable calcium, was significantly increased in cells exposed to 1 µM ouabain. Cell sodium content and baseline cytoplasmic calcium concentration was not changed. In cells exposed to ouabain for 24 hours, Western analysis revealed a reduced band density for Bcl–2. PCNA (proliferating cell nuclear antigen) band density also was decreased but an increase in Bax was observed. Conclusions: The results suggest that inhibition of the Na,K–ATPase α2 isoform has the potential to change ER store size. The observed changes of Bcl–2, Bax and PCNA abundance point to a possible link between Na,K–ATPase inhibition and cell survival or proliferation.

Keywords: calcium • astrocytes: optic nerve head • apoptosis/cell death