Reduction of water permeability by anisotonic solutions in frog corneal epithelium

Abstract

Purpose: To study the effects of bathing solution osmolarity and Cl- secretagogues on the diffusional water permeability (Pdw) of the isolated frog corneal epithelium.

Methods: Isolated frog corneas, with the endothelium scraped off, were mounted as a partition between Ussing-type hemichambers. Unidirectional diffusional water fluxes (Jdw) were measured by adding 3H2O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. Jdw was determined in control isosmotic conditions and after either changes in osmolarity of the bathing solutions or the additions of amphotericin B, epinephrine, Ca2+ ionophore, and other agents.

Results: Apical addition of 0.5 mM HgCl2 elicited an 11-fold increase in paracellular conductance and inhibited Jdw by 36%, suggesting that Jdw was predominantly transcellular and that there was a negligible contribution of the paracellular pathway. Pretreatment of corneas with 2-mercaptoethanol prevented the effects of Hg2+ on the paracellular conductance and Jdw. A hypotonic medium on the basolateral side reversibly reduced Jdw proportionately to the reduction in osmolarity, with 40 mOsm exerting a 29% decrease. Results from an Arrhenius plot suggest that water channels closed under this condition. Apical hypertonicity (350 mOsm) reduced Jdw by approximately 12%. Basolateral hypertonicity (450 mOsm), after permeabilization of the apical membrane with amphotericin B, reduced Jdw by 15%. Epinephrine was the only Cl- secretagogue that reduced Jdw, on average by 12%. This effect, which was also observed with amphotericin B-treated corneas, was not mediated by classical beta-receptors based on the results obtained with isoproterenol and propranolol.

Conclusions: Changes in basolateral osmolarity or the presence of an apical hypertonic solution decreased the diffusional water permeability (Pdw) of the corneal epithelium. Epinephrine also decreased Pdw, and this effect was localized to the basolateral membrane. The similarities, of a sequence motif found in potassium channels and beta-adrenergic receptor kinases that are regulated by the beta gamma subunit of G proteins with that found in aquaporins 2 and 5, could explain the link with epinephrine. Regardless of the mechanism, these results indicate that corneal epithelial water permeability can be regulated, presumably to protect cell volume from changes in solution osmolarity.