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Effect of AQP1 Expression Level on CO₂ Permeability in Bovine Corneal Endothelium

¹ From the School of Optometry, Indiana University, Bloomington, Indiana; and the ² Department of Ophthalmology, University of Arizona, Tucson, Arizona.

PURPOSE. Corneal endothelial fluid transport is dependent on HCO₃^- and CO₂ fluxes. CO₂ permeability (PCO₂) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO₂. An AQP1 knockout mouse model, however, showed no evidence for CO₂ transport through AQP1 in erythrocytes or lung. Because HCO₃^- and CO₂ fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO₂.

METHODS. BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1.

RESULTS. Expression of AQP1 and osmotic water permeability (control P_f = 0.046 ± 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P_f. Adenoviral empty virus (EV) infection did not change AQP1 expression or P_f. PCO₂ was determined by measuring the rate of intracellular pH decrease after exposure to CO₂/HCO₃^--rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO₂ of BCEC (0.0036 ± 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P_f could also be reduced more than 50% by 3 to 5 minutes’ exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease.

CONCLUSIONS. AQP1 does not contribute to PCO₂ in corneal endothelial cells.

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