Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium.
Corneal transparency and hydration control are dependent on HCO(3)(-) transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na(+)-3HCO(3)(-) cotransporter (NBC1) in addition to a basolateral 1Na(+)-2HCO(3)(-) cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO(3)(-) permeability and whether the cotransporter participates in transendothelial net HCO(3)(-) 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 HCO(3)(-) permeabilities were determined by measuring the rate of pH(i) change when HCO(3)(-) was removed from the bath under constant pH or constant CO(2) conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO(3)(-) permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO(3)(-) permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO(3)(-) flux was 0.707 +/- 0.009 mM.min(-1).cm(2) in the basolateral-to-apical direction and increased to 1.74 +/- 0.15 when cells were stimulated with 2 muM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO(3)(-) flux to 0.236 +/- 0.002. NBC1 siRNA treatment or 100 muM ouabain also eliminated steady-state HCO(3)(-) flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO(3)(-) permeability, basolateral-to-apical fluxes, and net HCO(3)(-) flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO(3)(-) flux and is functional only at the basolateral membrane.
Li
Jinhua
J
Indiana University School of Optometry, Bloomington, IN 47405, USA. jbonanno@indiana.edu
Sun
Xing Cai
XC
Bonanno
Joseph A
JA
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002417:Cattle; D016764:Cell Polarity; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003315:Cornea / Q000033:anatomy & histology* / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D010042:Ouabain / Q000494:pharmacology; D010539:Permeability; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D001639:Bicarbonates; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D027982:Sodium-Bicarbonate Symporters; D005576:Colforsin; D010042:Ouabain