title: Effect of acetazolamide on intracellular pH and bicarbonate transport in bovine corneal endothelium.
abstract: Carbonic anhydrase inhibitors are known to inhibit fluid transport by the corneal endothelium. This phenomenon could be due to a combination of effects involving disruption of intracellular pH regulation, reduced gradients for diffusion of CO2, substrate limitation to HCO3- transport systems or direct inhibition of membrane HCO3- transport. We examined the effects of the carbonic anhydrase inhibitor, Acetazolamide (ACTZ), on intracellular pH (pHi) and HCO3- transport in cultured bovine corneal endothelium. The pHi was measured utilizing the pH sensitive fluorescent dye, BCECF. Na+:HCO3- cotransport and Cl-/HCO3- exchange activities were studied by measuring the HCO3(-)-dependent flux of Na+ and Cl-, respectively. Na+ and Cl- fluxes were measured using the ion-sensitive dyes SBFI and SPQ, respectively. Application of 100 or 500 microM ACTZ to cells perfused under HCO3(-)-rich conditions, significantly reduced steady-state pHi by 0.06 +/- 0.01 (n = 14, P < 0.05). ACTZ also eliminated rapid pHi transients due to CO2 diffusion, significantly slowed the initial rate of pHi changes (50 +/- 10% of control, n = 7, P < 0.05) secondary to Na+:HCO3- cotransport or Cl-/HCO3- exchange (37 +/- 1% of control, P < 0.05, n = 7). However, the flux of the cotransported ions, Na+ and Cl-, and the steady-state levels of these ions were not affected by ACTZ. We conclude that the drop in steady-state pHi, the elimination of CO2 induced pHi transients and the slowed pHi changes secondary to HCO3- transport were due to inhibition of cytosolic carbonic anhydrase by ACTZ, i.e. slowing the equilibrium among CO2, HCO3- and H+ and not due to limitation of substrate availability or direct inhibition of the membrane transporters.
authors.affiliation: Morton D. Sarver Center for Cornea and Contact Lens Research, School of Optometry, University of California, Berkeley 94720, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Bonanno
authors.forename: S P
authors.initials: SP
authors.lastname: Srinivas
authors.forename: M
authors.initials: M
authors.lastname: Brown
mesh_terms: D000086:Acetazolamide / Q000494:pharmacology*; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D002417:Cattle; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D012964:Sodium / Q000378:metabolism
chemical_list: D001639:Bicarbonates; D002712:Chlorides; D012964:Sodium; D000086:Acetazolamide