Cultured cokneal fibroblasts and myofibroblasts have functional gap junctions

Purpose: Gap junctional communication allows the diffusion of <lkD molecules between cells. In corneal fibroblasts cultured at high density, the gap junction protein, connexin43 (Cx43), is localized immunocytochemically to dense maculae at cell-cell contacts. In contrast, as myofibroblasts differentiate in low density culture, their cell-cell contacts have few Cx43 maculae; myofibroblast Cx43 is primarily intracellular (IOVS,I996; 37:1740-1748). Here we have investigated intercellular communication, the functional consequence of gap junctions, in phenotypically defined cultures of corneal myofibroblasts and fibroblasts. Methods: Gap junctionbased communication was evaluated by comparing the diffusion of two fluorescent dyes: gap junction-permeant Lucifer- yellow (LY), and non-permeant, 10 kD rhodamine dextran. Diffusion was evaluated both by scrape loading and microinjection. Results: In confluert cultures of fibroblasts or myofibroblasts, scrape loaded LY diffused into adjacent cells whereas dextran was restricted to the wounded cells. This paralleled the increased number of Cx43 maculae found with increasing numbers of adjacent cells in myofibroblast cultures. Furthermore, in nonconfluent cultures in which few Cx43 maculae are found between myofibroblasts, microinjected LY rapidly diffused to adjacent myofibroblasts or fibroblasts; dextran was limited to the injected cell. Treatrient with ct-glycyrrhetinic acid, an uncoupling agent, prevented the spread of LY. Conclusions: Gap junctional communication has previously been demonstrated for keratocytes in vivo . The current finding of functional gap junctions in cultured mysfibroblasts and fibroblasts suggests that after wounding, fibroblasts and myofibroblasts derived from keratocytes may use gap junctions for intercellular communication.