Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Abstract

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.

Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin

“…Further study is needed to determine the extent to which the two isoforms contribute to CMEC Na/H exchange activity under control, as well as ischemic, conditions. Our observation that the Na/H exchange-mediated J H ϩin CMEC is 0.23 Ϯ 0.01 mM/min, which is equivalent to 0.003 pH unit/s in our studies, is comparable to Na/H exchange-mediated J H ϩreported for other cells, including rat optic nerve astrocytes (0.003 pH unit/s) (38) and cortical astrocytes (0.005 pH unit/s) (32).…”

Genetic variation of the alpha subunit of the epithelial Na+ channel influences exhaled Na+ in healthy humans

“…Na + concentrations were measured using an atomic absorption spectrophotometer (Analyst 100; Perkin-Elmer, Norwalk, CT) with a detection limit of 0.01 μmol/L. emission wavelength of 566.5 nm Na + as previously described (Mandal et al, 2008; Wheatley et al, 2010). Samples were diluted as needed in order for absorbance to be measured within the prepared standard solution curve with known concentrations of sodium diluted with Milli-Q water (Millipore, Burlington, MA) to concentrations of 0.01, 0.05, 0.10, 0.15, 0.20 mmol/L.…”

Variability in Measures of Exhaled Breath Na+, Influence of pulmonary Blood Flow and salivary Na+

“…Sodium concentrations were measured using an atomic absorption spectrophotometer (Analyst 100; Perkin-Elmer, Norwalk, CT) with a detection limit of 0.01 μmol/L. emission wavelength of 566.5 nm Na + as previously described 22. Briefly, the Na + cation lamp provided absorbance measurements of samples.…”