Presence of Net Fluorescein Flux Across Cultured Human Corneal Endothelial Layers

Abstract

Abstract: : Purpose: We have presented evidence suggesting that endothelial fluid transport results from electro-osmosis across intercellular junctions. If so, fluid movement might drag solute along the paracellular route. In this study, we used fluorescein as a paracellular marker and determined its unidirectional fluxes across cultured human corneal endothelial (HCE) cells. Methods: SV-40-transformed HCE layers (J. Bednarz's line) were grown to confluence on 24 mm permeable membrane inserts (Transwell_® Costar_® #3450). We used DMEM with high glucose (4.5g/l) plus 6% FBS, penicillin (100U/ml) and streptomycin (100 ng/ml). For the experiments, DMEM had no phenol red. Na₂-fluorescein was dissolved in the medium (0.15 mg/ml). Fluorescein labeled medium was added either on the basolateral or apical sides of the insert, while the other side carried unlabeled medium. The inserts were held in a CO₂ incubator for one hour (37 C), after which the entire volume of the sides originally unlabeled was collected. The amount of fluorescein in an aliquot (50 µL) of each sample (mixed with 2.95 mL saline and placed in a standard fluorometer vial) was determined with a Photon Technology International fluorometer (excitation 380 nm; emission 550 nm; 2 nm bwth) using Felix^TM software. Samples were read for 60 s. Results: The cells utilized are known to transport fluid in vitro (Aboalchamat et al., Exp. Eye Res. 69:547-53, 1999). We used 4 groups of inserts (total: 20 inserts). We found a net flux of fluorescein from the basolateral to the apical side; the ratio of the two fluxes was 1.104 0.034. Conclusion: The larger fluorescein flow occurs in the same direction as that of fluid transport, which is consistent with solvent drag along a paracellular route for fluid transport. CR: None Support: EY 06178 (JF) and RPB, Inc 

Keywords: 446 ion transporters • 370 cornea: basic science • 371 cornea: endothelium