Junctional Permeability for Neutral Solutes Across Cultured Rabbit Corneal Endothelial Cell Layers

Abstract

Abstract: : Purpose: We are developing an original model for fluid transport across epithelial layers based on electro–osmotic coupling at leaky tight junctions (TJs) with charge–selective pathways. We have now measured the permeation of several solutes (urea, mannitol, sucrose) analyzed in terms of steric hindrance using the Renkin equation to determine the width of rabbit corneal endothelial TJs, and the effects of perturbation by palmitoyl–DL–carnitine (PC) on the permeation of these solutes. Methods: Experiments were done with cells grown for two weeks on 24 mm Costar Transwell inserts. Determination of 3H mannitol flux from the basal to the apical side was done using 2.5 uCi/mL of 3H mannitol (20 Ci/mM) in bicarbonate Hepes buffer (BH). Samples from the basal (donor) side were 0.1 mL at 0 and 180 min. Samples from the apical (receiver) side were 0.1 mL taken every 30 min for 180 min. They were replaced with 0.1 mL BH each time. Each sample was added to 5 mL liquid scintillation solution and counted. Similar procedures were used to determine fluxes of 14C–urea and 14C–sucrose. In each perturbant study, cells were preteated with 0.2 mM PC on the apical side for 90 min, and the isotopes sample volume on the receiver side was replaced with 0.1 mL BH containing 0.2 mM PC. Results: The permeability (Pf) of three isotopes in the unperturbed RCE cell monolayer was: urea: 4.96 x 10–5 cm/s; mannitol: 3.33 x 10–5 cm/s; and sucrose: 2.54 x 10–5 cm/s. Pf values decreased with molecular radii (urea: 2.67 Å; mannitol: 4.1 Å and sucrose:5.12 Å ). PC decreased electrical resistance across the layers, and increased the Pf values for these solutes by 6.3X (urea), 3.7X (mannitol), and 3.9X (sucrose). Conclusions: The pore radius in the unperturbed monolayer was estimated with very good accuracy as 19 ± 0.2 Å, in agreement with a value of 19.5 Å previously reported for the junctional slit half width. The layers treated with PC appear to be heterogeneous in pore size.

Keywords: cornea: endothelium • cell adhesions/cell junctions • ion channels