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J.A. Bonanno, J. Li; siRNA Knockdown of the Na+:HCO3– Cotransporter Reduces Transendothelial HCO3– Transport in Bovine Corneal Endothelium Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1089.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the role of the Na+:HCO3– cotransporter (NBC–1) in transendothelial HCO3– transport in bovine corneal endothelial cells (BCEC). Methods: BCEC were transfected with NBC–1 targeted siRNA and grown to confluence on permeable Anodiscs for 4 days. Total protein was subjected to western blot analysis using anti–NBC polyclonal antibody. Apical and basolateral HCO3– permeabilities were determined by removing HCO3– from either the apical or basolateral sides using a constant–pH or a constant–CO2 protocol, while measuring pHi. To test the effect of treatment on transendothelial HCO3– fluxes, basolateral to apical (B to A) and apical to basolateral (A to B) fluxes were determined by measuring the initial ΔpH in each compartment while a HCO3– gradient was imposed. Results: Western blot analysis showed that NBC expression was reduced ∼80% after 4 days transfection. Under the constant–CO2 protocol, there was a sharp acidification upon removing HCO3– on the basolateral side of control (0.005±0.0000017 pHi/sec) and oligofectamine–only treated cells (0.0041±2.36E–07 pHi/sec, p>0.1), but a significantly slower acidification in cells treated with 5 nM siRNA (0.00087±3.44E–08 pHi/sec, p<0.001). However, there was no difference in the rate of pHi decrease on the apical side among the controls and siRNA treated cells, consistent with NBC having a basolateral location only. Similar results were obtained with the constant–pH protocol (0.0038±0.0000014 pHi/sec, 0.00387±1.27E–06 pHi/sec, and 0.00083±5.31E–08 pHi/sec for control, oligofectamine–only, and 5 nM siRNA treated cells, respectively, p<0.001). Net initial HCO3– flux was 0.707±0.042 mM/min×cm2, basolateral to apical, and it was increased to 1.74±0.15 when cells were stimulated with 2 µM forskolin, p<0.001. In siRNA treated cells, B to A fluxes were reduced 67%, while A to B fluxes were unaffected, giving a net initial HCO3– flux reduced to 0.236±0.0024, p<0.001. Conclusions: The basolateral Na+/2HCO3– cotransporter has a major role in net HCO3– fluxes across the bovine corneal endothelium.
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