Fluid transport by cultured corneal epithelial cell layers

Abstract

Background/aims - Fluid transport across the in vitro corneal epithelium is short lived, hence difficult to detect and characterise. Since stable rates of fluid transport across several cultured epithelial cell layers have been demonstrated, the behaviour of confluent SV40 transformed rabbit corneal epithelial cells (tRCEC) grown on permeable supports was examined. Methods - Fluid transport was determined with a nanoinjector volume clamp; the specific electrical resistance of the layers was 184 (SEM 9) Ω cm2, tRCEC layers transported fluid (from basal to apical) against a pressure head of 3 cm H2O for 2-3 hours. Results - In the first hour, the rate of fluid transport was 5.2 (0.5) μl/h/cm-2 (n = 23), which is comparable with that found in other epithelia. Fluid transport was completely inhibited in 15-30 minutes by either 100 μM ouabain (n = 6), 50 μM bumetanide (n = 6), or 1 μM endothelin-1 (ET-1; n = 6). Preincubation with 10 μM BQ123 (an ET(A) receptor antagonist) obviated inhibition by ET-1 (n = 6). ET-1 also caused a 22% decrease in specific resistance. Conclusions - Fluid transport appears to depend on transepithelial Cl- transport since (1) their directions are the same (stroma→tear), and (2) both bumetanide and ouabain inhibit it with similar time course. tRCEC appear useful to investigate aspects of the physiology and pharmacology of fluid transport across this layer, including receptor mediated control of this process.