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

• Published in: American Journal of Physiology: Cell Physiology Vol. 288; no. 3; pp. C739 - C746
• Main Authors: Li, Jinhua, Sun, Xing Cai, Bonanno, Joseph A
• Format: Journal Article
• Language: English
• Published: United States 01.03.2005
• Subjects: Animals; Bicarbonates - metabolism; Cattle; Cell Polarity; Cells, Cultured; Colforsin - pharmacology; Cornea - anatomy & histology; Cornea - metabolism; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Enzyme Inhibitors - pharmacology; Humans; Ouabain - pharmacology; Permeability; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Sodium-Bicarbonate Symporters - antagonists & inhibitors; Sodium-Bicarbonate Symporters - genetics; Sodium-Bicarbonate Symporters - metabolism
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00405.2004

Indiana University School of Optometry, Bloomington, Indiana Submitted 18 August 2004 ; accepted in final form 9 November 2004 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 µM 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 µM 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. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport Address for reprint requests and other correspondence: J. A. Bonanno, Indiana Univ. School of Optometry, Bloomington, IN 47405 (E-mail: jbonanno{at}indiana.edu )