Demonstration of the sodium-hydrogen exchanger NHE-1 in corneal endothelial cells

Abstract

Purpose: Maintenance of corneal hydration and transparency requires the presence of many membrane transporter proteins in the corneal endothelium, including, it is suggested, a sodium-hydrogen (Na+/H+) exchanger. This study investigates the presence of the Na+/H+ exchanger (NHE) in bovine and sheep corneal endothelium. Methods: A polyclonal antibody, specific for type 1 Na+/H+ exchanger (NHE-1), was used to screen bovine and sheep corneal endothelial cells for the presence of NHE activity by the Western blotting technique. Using immunoassay quantification techniques (ELISA), the quantity of NHE-1 present was measured. Results: Western blot analysis, using the NHE-1 antibody, recognized proteins with a mass of 70 kDa and 56 kDa in bovine and sheep corneal endothelial cells respectively. From the measured activity of NHE-1 detected using ELISA and the previously measured cell density, an estimate of the number of NHE-1 molecules present in each corneal endothelial cell was calculated to be 40 000 per cell. Conclusion: Bovine and sheep corneal endothelium contain type I Na+/H+ exchanger (NHE-1). Future immunochemical studies will confirm its precise membrane location. This data provides direct evidence of its existence and the quantitative estimate of its concentration in the membrane provides further opportunity to resolve its role in transendothelial bicarbonate ion transport. Acknowledgement: The polyclonal antibody was a generous gift from Prof. L. Fliegel, Department of Biochemistry and Pediatrics, University of Alberta.