June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Effects of pH, HCO3 and carbonic anhydrase inhibitors on the corneal endothelial short-circuit current
Author Affiliations & Notes
  • Sangita P Patel
    Ophthalmology and SUNY Eye Institute, University at Buffalo, Buffalo, NY
    Research Service, VA Western New York Healthcare System, Buffalo, NY
  • Sarada P Vissapragada
    Ophthalmology and SUNY Eye Institute, University at Buffalo, Buffalo, NY
    Research Service, VA Western New York Healthcare System, Buffalo, NY
  • Sangwon Min
    Ophthalmology and SUNY Eye Institute, University at Buffalo, Buffalo, NY
    Research Service, VA Western New York Healthcare System, Buffalo, NY
  • Footnotes
    Commercial Relationships Sangita Patel, None; Sarada Vissapragada, None; Sangwon Min, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2469. doi:
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      Sangita P Patel, Sarada P Vissapragada, Sangwon Min; Effects of pH, HCO3 and carbonic anhydrase inhibitors on the corneal endothelial short-circuit current. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2469.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The corneal endothelial short-circuit current (Isc) represents active ion transport across the corneal endothelium and underlies mechanisms of fluid movement regulating corneal hydration. We have previously shown that Isc 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 Isc.

Methods: We measured corneal endothelial Isc 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 Isc. Percent effect of an intervention was calculated as the Isc value post-intervention divided by the difference between maximum Isc for the recording and post-ouabain baseline. Significance was calculated by Student’s t-test (p ≤ 0.05).

Results: Maximum Isc 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 Isc (% of Isc,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 Isc,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 Isc shifted towards pH 8.0 (3/5 recordings). While dorzolamide did not inhibit Isc 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.

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