In-vitro Rat Retinal Pigment Epithelium (RPE) Electrophysiology

Abstract

Purpose:: To develop an in vitro rat preparation of RPE-choroid (RPE-Ch) and retina-RPE-choroid (R-RPE-Ch) which could be used to analyze light/dark induced changes in RPE physiology. In this study, we determined some of the membrane mechanisms that help determine trans-RPE and intracellular apical and basolateral membrane potentials (V_A, V_B) and transepithelial potential and resistance (TEP, R_t).

Methods:: RPE-Ch or R-RPE-Ch layer were isolated from adult Long Evans rat eyes and mounted in a modified Ussing chamber that allowed penetration of the basolateral membrane with intracellular microelectrodes and continuous and independent perfusion of the bathing solutions on either side of the tissue. This allowed continuous monitoring of V_A, V_B, TEP, R_t, ratio of apical to basolateral membrane resistance (R_A/R_B), and total trans-tissue voltage and resistance (TTP, TTR).

Results:: The diameter of the RPE-Ch preparation is only 2 mm; we confirmed the viability of the intact monolayer qualitatively using a live/dead assay kit. After mechanically mounting the tissue in a modified Ussing chamber, the TEP and R_t of the RPE-Ch was 1.9±0.8 mV and 100±14Ω •cm² (n =7), respectively. Apical or basal Ba²⁺ (1mM) altered TEP and Rt (n = 3). The R-RPE-Ch had a TTP of 3.2±0.9 mV and TTR of 167±53 Ω •cm² (n =14). V_B and V_A were -45±2.5mV and -48.2±2.8mV (n=5), respectively, indicating an apical positive TEP. R_A/R_B was 0.7±0.3 (n=5), indicating that R_B is considerably larger than R_A. Increasing basal [K]o from 5 to 25 mM depolarized the basolateral membrane by ~8mV (n=2). These changes were blocked by basal Ba²⁺. Decreasing [K]o in the apical bath from 5 to 2mM hyperpolarized the apical membrane by ~2 mV (n =1). Step increases in apical [K]o, from 5 to 25mM, depolarized the apical membrane by ~6mV (n=3). Apical and basal [K]o-induced voltage changes were 90% blocked (n =2) by the prior addition of Ba²⁺ to the apical or basal baths. Elevation of [Ca²⁺] in the basal bath from 1.8mM to 10 mM increased TTP by 0.6mV (p < 0.001; n= 5). This increase was inhibited by 50% (p< 0.01; n=3) in less than 1 minute after the addition of DIDS (1mM) to the basal bath.

Conclusions:: The rat R-RPE-Ch preparation shows evidence of Ba²⁺-sensitive K channels at the apical and basolateral membranes. The relative depolarization of these membranes suggests the presence of other conductive or electrogenic mechanisms whose equilibrium potential is more positive than E_K. DIDS and extracellular [Ca²⁺] -sensitive changes in V_B suggests the presence of Ca²⁺-activated Cl^- channels but more definitive experiments remain to be done to verify that possibility.

Keywords: retinal pigment epithelium • electrophysiology: non-clinical • ion channels