Use of a fluorescent cyanine dye for electrophysiological studies on the frog cornea

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Abstract
A dye, diS-C3-(5), used for potential difference (PD) studies on cells and vesicles, was used in an intact bullfrog [Rana catesbeiana] cornea. The cornea was mounted in a chamber, the tear side outward, and placed in a spectrofluorometer. The aqueous side was perfused with regular or modified Ringer solution. Two pairs of electrodes were used: 1 pair for transcorneal PD measurement and 1 pair for transcorneal current sending. For elevated K+ Ringer solution, K+ replaced Na+. After mounting, Ringer solution containing 4 .mu.M of dye was perfused through the aqueous side for about 10 min and then perfusion was switched to a regular Ringer solution. A decrease in fluorescence (F) and an increase in PD (aqueous normally positive) resulted when current was sent from the aqueous side to the tear side. Current in the opposide direction produced an increase in F and a decrease in PD. No effect on F occurred with currents of the same magnitude in either direction after removal of the epithelium. A current would produce the same .DELTA.PD across the stroma and endothelium, whether the epithelium was present or not; the change in F in intact corneas is apparently due to the epithelium. A concurrent decrease in PD and an increase in F results when K+ was increased on the aqueous side (2.5-79 mM). After removal of the epithelium increasing K+ has quantitatively the same effect on F, but a negligible effect on the PD. The K+ response of fluorescence is due to the endothelium. A fluorescent dye can be used to study the properties of the endothelium of the cornea in contrast to conventional techniques.