Role of NBC1 in apical and basolateral HCO3− permeabilities and transendothelial HCO3− fluxes in bovine corneal endothelium

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 5–15 nM siRNA decreased NBC1 expression by 80–95%, 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·min−1·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.