Effects of pH, HCO₃⁻ and carbonic anhydrase inhibitors on the corneal endothelial short-circuit current

Abstract

Purpose: The corneal endothelial short-circuit current (I_sc) represents active ion transport across the corneal endothelium and underlies mechanisms of fluid movement regulating corneal hydration. We have previously shown that I_sc is facilitated by carbonic anhydrase in bovine corneal endothelium but not human. The purpose of this study is to define the roles of pH and HCO₃⁻, substrates of carbonic anhydrase, in corneal endothelial I_sc.

Methods: We measured corneal endothelial I_sc with the Ussing chamber short-circuit current technique in fresh bovine corneas with epithelium removed. Recording solutions contained (in mM): 111.6 NaCl, 4.8 KCl, 1.0 CaCl₂, 0.8 MgCl₂, 0.9 NaH₂PO₄, 20 HEPES, 5 glucose, bubbled with air, 35°C. For pH experiments, we adjusted pH to 8.5 with 1M NaOH and added 3M HCl during the recording to reach desired pH endpoints. NaHCO₃ was added to achieve the desired concentrations of HCO₃⁻. Ouabain (10 µM) was added at the conclusion of recordings to determine baseline I_sc. Percent effect of an intervention was calculated as the I_sc value post-intervention divided by the difference between maximum I_sc for the recording and post-ouabain baseline. Significance was calculated by Student’s t-test (p ≤ 0.05).

Results: Maximum I_sc with 12 mM HCO₃⁻ was observed at pH 7.5 (8/10 recordings) during a stepwise decrease of pH from 8.5 to 6.5. At pH 7.5, increases in [HCO₃⁻] from 0 to 12 mM resulted in increases in I_sc (% of I_sc,max at 12 mM HCO₃⁻: 0 mM, 35.3 ± 3.3; 3 mM, 59.7 ± 5.0; n = 3; p = 0.002); however, at pH 8.5, that increase was absent (% of I_sc,max at 12 mM HCO₃⁻, pH 7.5: 0 mM, 26.5 ± 6.9, n = 8; 12 mM, 33.2 ± 8.1, n = 7; p = 0.108). In the presence of 100 µM dorzolamide (a carbonic anhydrase inhibitor) and 12 mM HCO₃⁻, maximum I_sc shifted towards pH 8.0 (3/5 recordings). While dorzolamide did not inhibit I_sc responses to changes in [HCO₃⁻] or pH, the responses to interventions were qualitatively blunted.

Conclusions: Both HCO₃⁻ and an optimal pH are necessary for maximizing transendothelial active ion transport in the cornea. These responses are facilitated by but not dependent upon carbonic anhydrase. Our data support the central role of [HCO₃⁻] and [H⁺] in both current models (HCO₃⁻ secretion and lactate transport) of fluid movement across the corneal endothelium. In future studies, we will establish the roles of HCO₃⁻ and pH in human corneal endothelium.