Transepithelial electrical measurements on the isolated bovine Iris-Ciliary Body (I-CB) preparation

Purpose: The purpose of the study is to develop a stable in vitro bovine isolated iris-ciliary body (I-CB) preparation in an modified Ussing type chamber with continuous perfusion, and to study the effect of ouabain and furosemide on the transepithelial parameters of the preparation. Methods : Sections of fresh bovine I-CB tissues were isolated from the sclera. ICB preparations were mounted in Ussing typed chambers (0.35cm2) for measurements of potential difference (p.d) and short circuited current (SCC). Ouabain and furosemide of various concentrations were applied to the aqueous, blood or both sides. Their effects on the transepithelial electrical parameters were recorded. Results : The average values of p.d. and SCC were 0.51±0.05mV and 3.89±0.04μAcm-2 (mean±SEM, n=35) respectively, with the aqueous-side consistently negative with respect to the blood-side. When 1mM ouabain was applied to apical, basal or both sides, biphasic responses were observed 1mM ouabain hyperpolarized (SCC increased by 113%) the preparations which followed by depolarization (SCC decreased by 70%). At lower ouabain concentration (0.01mM), apical application of ouabain hyperpolarized (40%) the ICB, whereas basal application caused depolarization (69%). No biphasic response was observed and the preparations remained either hyperpolarized or depolarized for over 60 minutes. Hyperpolarization of the preparations was observed when ouabain (0.01mM) was added to the apical side of preparations which were previously depolarized with basal application of ouabain (0.01mM). Conversely, depolarization of the preparation was evident when ouabain (0.01mM) was applied to the basal side of I-CB which was previously hyperpolarized by apical administration of ouabain (0.01mM). 1mM and 0.1mM furosemide depolarized the preparation when applied to aqueous, blood or both sides. Conclusions : Viable bovine I-CB preparations were successfully developed with stable p.d and apparent SCC. The biphasic responses induced by 1mM ouabain suggest a dual Na/K-ATPase model of the ciliary epithelium bilayer, one at the pigmented epithelium and the other one at the nonpigmented epithelium. This biphasic response can be segregated with low ouabain concentration (0.01mM). A furosemide sensitive transmural anion pathway was found at the ciliary epithelium bilayer.