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A. Mandal, M. Shahidullah, N. A. Delamere; Ouabain Stimulation of Sodium-Hydrogen Exchange in Rat Optic Nerve Astrocytes Involves Calcium Entry. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3171.
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It was shown earlier that 1 µM ouabain, a concentration sufficient to inhibit the Na,K-ATPase alpha 2 isoform, stimulates capacitative calcium entry in rat optic nerve astrocytes (Hartford et al., 2004 Glia 45:229). Here we report altered intracellular pH (pHi) responses in cells exposed to ouabain.
Astrocytes were isolated from rat optic nerve and established in primary culture. Cells were exposed to ammonium chloride (20 mM) for 5 min to acid load the cells. BCECF and FURA-2 were used to measure intracellular pH (pHi) and calcium. Expression of sodium hydrogen exchanger (NHE) isoforms was studied by western blot.
On removal of ammonium chloride pHi fell abruptly then gradually recovered toward baseline. 1 µM ouabain increased the rate of pHi recovery both in the presence and in the absence of bicarbonate in the bathing medium. Western blot showed presence of NHE1 but not NHE2, NHE3 or NHE4. The NHE inhibitor DMA reduced the rate of pHi recovery by >50%. DMA also suppressed the effect of ouabain on the rate of pHi recovery. 1 µM ouabain did not detectably alter cytoplasmic calcium concentration but the calcium chelator BAPTA-AM as well as SKF96365 and 2-APB, inhibitors of capacitative calcium entry (CCE), reduced the magnitude of the ouabain effect on pHi recovery.
Ouabain-induced stimulation of pHi recovery was due, at least in part, to stimulation of NHE1. The findings suggest capacitative calcium entry is necessary for the NHE1 response to occur.
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