Functional gap junctions in corneal fibroblasts and myofibroblasts.
OBJECTIVE
Within the corneal stroma, keratocytes communicate through gap junctions. These plasma membrane channels, which connect the cytoplasm of adjacent cells, are composed of connexins. In a cell culture model, an investigation was conducted to determine whether connexin-based gap junction intercellular communication is present in fibroblasts and myofibroblasts, both of which replace keratocytes after wounding.


METHODS
Fibroblasts and myofibroblasts were grown according to preestablished methods. Phenotype was determined by immunocytochemistry. A gap junction-permeant dye, Lucifer yellow or Cascade blue, and nonpermeant 10-kDa Texas red-dextran were used. Tracer fluorescent dyes were introduced by scrape-loading or by microinjection, and their diffusion into adjacent cells was recorded photographically. Inhibition of gap junction dye transfer was elicited by treatment with 18-alpha-glycyrrhetinic acid (AGA).


RESULTS
In confluent fibroblast or myofibroblast cultures, the scrape-loaded dextran probe remained within wounded cells, whereas the Lucifer yellow or Cascade blue dye diffused into adjacent intact cells. Similarly, in nonconfluent fibroblast and myofibroblast cultures, microinjected Lucifer yellow rapidly diffused from the microinjected cell to adjacent cells. Treatment with 2 microM AGA, an uncoupling agent, blocked the spread of Lucifer yellow in fibroblast and myofibroblast cultures.


CONCLUSIONS
Cultured fibroblasts and myofibroblasts have functional gap junctions as has previously been demonstrated for keratocytes in vivo. Thus, fibroblasts and myofibroblasts have the ability to establish and maintain intercellular communication with themselves and with nonactivated keratocytes. This property may be critical in the wound-healing process, especially in the avascular corneal environment.
Spanakis
S G
SG
Department of Ophthalmology, Mount Sinai School of Medicine of the City University of New York, NY 10029-6574, USA.
Petridou
S
S
Masur
S K
SK
D000199:Actins / Q000378:metabolism; D000818:Animals; D002450:Cell Communication / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology*; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D007546:Isoquinolines / Q000378:metabolism; D008845:Microinjections; D009942:Organometallic Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011817:Rabbits; D014966:Xanthenes / Q000378:metabolism
D000199:Actins; D005456:Fluorescent Dyes; D007546:Isoquinolines; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D014966:Xanthenes; C066850:Cascade Blue; C034657:Texas red; C017475:lucifer yellow; D006034:Glycyrrhetinic Acid