Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium |
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Authors: | Li Jinhua Sun Xing Cai Bonanno Joseph A |
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Affiliation: | Indiana University School of Optometry, Bloomington, IN 47405, USA. jbonanno@indiana.edu |
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Abstract: | Corneal transparency and hydration control are dependent on HCO3 transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na+-3HCO3 cotransporter (NBC1) in addition to a basolateral 1Na+-2HCO3 cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO3 permeability and whether the cotransporter participates in transendothelial net HCO3 flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 515 nM siRNA decreased NBC1 expression by 8095%, 4 days posttransfection. Apical and basolateral HCO3 permeabilities were determined by measuring the rate of pHi change when HCO3 was removed from the bath under constant pH or constant CO2 conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO3 permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO3 permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO3 flux was 0.707 ± 0.009 mM·min1·cm2 in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO3 flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO3 flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO3 permeability, basolateral-to-apical fluxes, and net HCO3 flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO3 flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport |
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