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Role of carbonic anhydrase IV in corneal endothelial HCO3- transport.
- Source :
-
Investigative ophthalmology & visual science [Invest Ophthalmol Vis Sci] 2008 Mar; Vol. 49 (3), pp. 1048-55. - Publication Year :
- 2008
-
Abstract
- Purpose: Carbonic anhydrase activity has a central role in corneal endothelial function. The authors examined the role of carbonic anhydrase IV (CAIV) in facilitating CO(2) flux, HCO(3)(-) permeability, and HCO(3)(-) flux across the apical membrane.<br />Methods: Primary cultures of bovine corneal endothelial cells were established on membrane-permeable filters. Apical CAIV was inhibited by benzolamide or siRNA knockdown of CAIV. Apical CO(2) fluxes and HCO(3)(-) permeability were determined by measuring pH(i) changes in response to altering the CO(2) or HCO(3)(-) gradient across the apical membrane. Basolateral to apical (B-to-A) HCO(3)(-) flux was determined by measuring the pH of a weakly buffered apical bath in the presence of basolateral bicarbonate-rich Ringer solution. In addition, the effects of benzolamide and CAIV knockdown on steady state DeltapH (apical-basolateral compartment pH) after 4-hour incubation in DMEM were measured.<br />Results: CAIV expression was confirmed, and CAIV was localized exclusively to the apical membrane by confocal microscopy. Both 10 microM benzolamide and CAIV siRNA reduced apparent apical CO(2) flux by approximately 20%; however, they had no effect on HCO(3)(-) permeability or HCO(3)(-) flux. The steady state apical-basolateral pH gradient at 4 hours was reduced by 0.12 and 0.09 pH units in benzolamide- and siRNA-treated cells, respectively, inconsistent with a net cell-to-apical compartment CO(2) flux.<br />Conclusions: CAIV does not facilitate steady state cell-to-apical CO(2) flux, apical HCO(3)(-) permeability, or B-to-A HCO(3)(-) flux. Steady state pH changes, however, suggest that CAIV may have a role in buffering the apical surface.
- Subjects :
- Animals
Basement Membrane metabolism
Benzolamide pharmacology
Biological Transport
Carbon Dioxide metabolism
Carbonic Anhydrase IV antagonists & inhibitors
Carbonic Anhydrase Inhibitors pharmacology
Cattle
Cell Membrane Permeability
Cells, Cultured
Endothelium, Corneal cytology
Fluorescent Antibody Technique, Indirect
Hydrogen-Ion Concentration
Immunoblotting
Intracellular Membranes
Microscopy, Confocal
RNA, Small Interfering genetics
Reverse Transcriptase Polymerase Chain Reaction
Sodium-Bicarbonate Symporters metabolism
Transfection
Bicarbonates metabolism
Carbonic Anhydrase IV physiology
Endothelium, Corneal enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0146-0404
- Volume :
- 49
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Investigative ophthalmology & visual science
- Publication Type :
- Academic Journal
- Accession number :
- 18326729
- Full Text :
- https://doi.org/10.1167/iovs.07-1188