DYE SPREAD THROUGH GAP-JUNCTIONS IN THE CORNEAL EPITHELIUM OF THE RABBIT

Purpose. Microelectrode dye injection of 5,6-carboxyfluorescein was us ed to investigate gap junctional communication in the corneal epitheli um. Methods. Dye injection started in the superficial layer and procee ded stepwise into the underlying epithelial layers until spread was ob served. 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 KC l Ringer's (global decrease) or by lowering the pH in the injection el ectrode (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 trav el distance from the point of injection. Results. Control dye spread o ccurred, on average, in the third layer from the surface. Increased [C a2+] in the source cell resulted in an initial spread occurring in the second layer. Globally increasing [Ca2+] with A23187 resulted in no c hange in the average initial spread layer. Lowering intracellular pH o f the source cell did not affect the initial dye spread layer. Globall y lowering intracellular pH resulted in significant gap junctional inh ibition in a time-dependent manner. Dye spread distance was not signif icantly affected by [Ca2+] or pH manipulations. Heptanol (2.5 mM) comp letely inhibited dye coupling. Conclusion. All cell layers of the corn eal epithelium contain functional gap junctions, although it appears t hat intercellular communication in the superficial layers does not occ ur under our control conditions. Intercellular communication through t hese junctions can be altered by various manipulations of [Ca2+] and p H.