Dye spread through gap junctions in the corneal epithelium of the rabbit

Purpose. Microelectrode dye injection of 5,6-carboxyfluorescein was used to investigate gap junctional communication in the corneal epithelium. Methods. Dye injection started in the superficial layer and proceeded stepwise into the underlying epithelial layers until spread was observed. Intracellular [Ca²⁺] was manipulated by exposing the cornea to the calcium ionophore A23187 (global increase) or by increasing the [Ca²⁺] in the injection electrode (source cell increase). Intracellular pH was manipulated by exposing the cornea to nigericin in a low-pH KC1 Ringer's (global decrease) or by lowering the pH in the injection electrode (source cell decrease). Heptanol was tested for its ability to uncouple gap junctions. Gap junctional communication was based on the layer at which spread was first observed and on the apparent dye travel distance from the point of injection. Results. Control dye spread occurred, on average, in the third layer from the surface. Increased [Ca²⁺] in the source cell resulted in an initial spread occurring in the second layer. Globally increasing [Ca²⁺] with A23187 resulted in no change in the average initial spread layer. Lowering intracellular pH of the source cell did not affect the initial dye spread layer. Globally lowering intracellular pH resulted in significant gap junctional inhibition in a time-dependent manner. Dye spread distance was not significantly affected by [Ca²⁺] or pH manipulations. Heptanol (2.5 mM) completely inhibited dye coupling. Conclusion. All cell layers of the corneal epithelium contain functional gap junctions, although it appears that intercellular communication in the superficial layers does not occur under our control conditions. Intercellular communication through junctions can be altered by various manipulations of [Ca²⁺] and pH.