Effects Of Inhibitors Of Chloride Channels And Carbonic Anhydrase (iv) On Fluid Transport By Corneal Endothelium

Abstract

Abstract: : Purpose: The mechanisms involved in corneal endothelial fluid transport remain unclear. We examined the role of chloride channels and membrane–bound carbonic anhydrase IV (CA–IV) in fluid transport. Methods: De–epithelialized rabbit corneas were mounted in a Dikstein–Maurice chamber (36.5 C) and viewed with a specular microscope. The stromal side was covered with mineral oil, and the endothelial side was superfused (1.41cc/h). We used a HEPES–HCO₃^– Ringer’s solution (pH: 7.4; osmolality: 290 mOsm). Reagents were: ouabain, 5–nitro–2–(3–phenylpropylamino) benzoic acid (NPPB) and parabromophenacyl bromide (pBPB) (Sigma, St. Louis, MO); dextran–bound acetazolamide (DBAZ, Synthelec AB, Lund, Sweden). Benzolamide and CFTR inhibitor were gifts (Drs. Swenson and Verkman, respectively). All reagents except ouabain were dissolved in in dimethyl sulfoxide (DMSO; solvent final concentration 0.1% or less). Results: Using a the HEPES–HCO₃^– Ringer’s superfusion solution plus inhibitor, with ouabain (1 mM) the stroma swelled at a rate of 32.3 um/h. In controls without inhibitor, spontaneous swelling was 3.2 um/h. With chloride inhibitors NPPB or pBPB (100 uM), the rate of swelling was 6.2 um/h. CFTR inhibitor had no significant effect compared to spontaneous swelling. With niflumic acid, a calcium–activated chloride channel (CACC) inhibitor, we observed a rate of swelling of 9.81 um/h. Inhibition of apical extracellular CA–IV with the relatively impermeable inhibitor benzolamide (10 uM) produced a rate of swelling of 11.3 um/h. Addition of CA enzyme (20 uM) arrested the inhibitory action of benzolamide. The impermeant DBAZ inhibitor (100 uM) acted in the same fashion. Conclusions: The effect on fluid transport by inhibition of chloride channels was 16% or less, and the effect by inhibition of CA–IV less than 30%. Hence, membrane–bound CA (IV) and chloride channels appear to have only relatively modest roles in unstimulated endothelial fluid transport.

Keywords: cornea: basic science • cornea: endothelium • ion channels