siRNA Knockdown of the Na+:HCO3– Cotransporter Reduces Transendothelial HCO3– Transport in Bovine Corneal Endothelium Cells

Abstract

Abstract: : 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.

Keywords: cornea: endothelium • gene modifiers • ion transporters