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A. Mandal, M. Shahidullah, N. A. Delamere; Effect of Ouabain on pH Recovery in Cultured Rat Optic Nerve Astrocytes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2815.
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In rat optic nerve astrocytes two isoforms of Na,K-ATPase (alpha1 and alpha2) are present and alpha2 Na,K-ATPase is selectively inhibited by 1 µM ouabain (Hartford et al, 2004, Glia, 45:229-37). The sodium gradient across the cell membrane established by Na,K-ATPase is the driving force for many cellular mechanisms, including sodium hydrogen exchange (NHE) which contributes to cytoplasmic pH (pHi) regulation by exporting protons. Here we examined pHi regulation after selectively inhibiting Na,K-ATPase alpha2 activity with 1 µM ouabain.
Ammonium chloride (20 mM) was applied for 5 min to acid load the cells. BCECF and Fura-2 fluorescence imaging were used to measure cytoplasmic pH recovery and cytoplasmic calcium concentration. Expression of NHE isoforms was studied by western blot. Cell sodium was measured by atomic absorption spectrophotometry.
On removal of ammonium chloride pHi fell abruptly then gradually recovered toward baseline. In the presence of 1 µM ouabain the rate of pHi recovery increased by 68%. Baseline pHi was not altered by 1 µM ouabain. Higher ouabain concentrations caused a similar increase in pHi recovery rate. Dimethyl amiloride (DMA), a potent inhibitor of NHE, reduced the rate of pHi recovery. Western blot showed an NHE-1 immunoreactive band. NHE-2, NHE-3 and NHE-4 were not detected. Cell sodium content remained unaltered by 1 µM ouabain but increased significantly at higher concentrations of ouabain. The effect of 1 µM ouabain on pHi recovery rate was abolished by H-89, an inhibitor of protein kinase A. 8-Br-cAMP increased the recovery rate significantly in controls (no ouabain) but the recovery rate was not further increased when added with 1 µM ouabain. No significant difference in calcium concentration was observed between control and the ouabain-treated groups.
Ouabain at a concentration sufficient to inhibit Na,K-ATPase alpha2 but not alpha1 does not alter cell sodium but increases the pHi recovery rate after an acid load. Proton export via NHE-1 appears to be important for pHi recovery. The results suggest 1 µM ouabain activates NHE-1 by a mechanism linked to cAMP and protein kinase A.
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