Sodium and Chloride Transport at the Mouse Ocular Surface Measured by Open–Circuit Potential Differences, and Analyzed Using an Electrokinetic Model of Ocular Surface Ion Transport

Abstract

Abstract: : Purpose: Corneal and conjunctival epithelia are capable of transcellular Na⁺ absorption and Cl^– secretion, which drives water movement across these tissues. Multiple Na⁺ and Cl^– transporters are involved in these processes. We recently demonstrated using a new open–circuit potential difference (PD) technique that Cl^– moves across the ocular surface in living mice through both Ca⁺⁺– and cAMP–sensitive Cl^– channels, the latter pathway being the cystic fibrosis transmembrane conductance regulator (CFTR). The purpose of this study was to identify the transporting mechanisms involved in Na⁺ absorption, and to apply a mathematical model of ocular surface ion transport to describe rigorously the electrochemical coupling among transporting processes. Methods: PDs across the fluid–bathed ocular surface were measured in anesthetized wild–type and cystic fibrosis (CF) mice in response to Na⁺ and Cl^– ion substitution with choline and gluconate, respectively, as well as transporter agonists, inhibitors, and substrates. An electrokinetic model of ocular surface epithelium was developed to simulate PD measurements, which involved computation of membrane potentials and cellular [Na⁺], [K⁺] and [Cl^–]. Results: Na⁺ replacement produced a 6 mV depolarization that was blocked in a dose–dependent manner by amiloride (Ki 1.0 µM) or benzamil (Ki 0.3 µM). In cystic fibrosis mice lacking functional CFTR, the Na⁺–dependent amiloride effect was significantly greater (19 +/– 5 vs. 6 +/– 2; P<0.01), in accordance with model predictions of Na⁺ hyperabsorption in CFTR deficiency. In wild–type mice, D–, but not L–glucose addition produced a maximal, phloridzin–sensitive 4 +/– 2 mV hyperpolarization in the presence of Na⁺ and amiloride, with a Kd of 3 mM. The amino acids glycine and L–arginine also produced small hyperpolarizations of 2.1 and 1.5 mV, respectively, that were reversed by Na⁺ replacement or amino acid removal. The epithelial transport model permitted quantitative analysis of the relative roles of multiple Na⁺ and Cl^– pathways, and investigation of proposed CFTR–ENaC interactions. Conclusions: Amiloride–sensitive, glucose–coupled, and amino acid–coupled Na⁺ transporters facilitate ocular surface Na⁺ absorption in mice, Amiloride–sensitive Na⁺ transport, probably involving ENaC Na⁺ channels, appears to be most relevant under physiologic conditions.

Keywords: cornea: epithelium • pump/barrier function • ion channels