Distribution of the plasmalemmal Ca2+-pump and caveolin in the corneal epithelium during the wound healing process

Purpose. Caveolae are small plasmalemmal invaginations which are assumed to play various physiological functions. In the present study, distribution of two caveolae-specific proteins, the plasmalemmal Ca2+-pump and caveolin, was examined in the corneal epithelium in the normal state and after artificial wounding. Methods. A central epithelial ablation was made in the mouse cornea by a razor blade. After various intervals, the corneas were excised, fixed, and rapidly frozen. The specimens were subjected to immunofluorescence microscopy and immunoelectron microscopy, using antibodies against the plasmalemmal Ca2+-pump or caveolin. Results. In the normal corneal epithelium, both plasmalemmal Ca2+-pump and caveolin were observed along the cell surface by immunofluorescence microscopy, and were localized to caveolae by immunogold electron microscopy. In the regenerating epithelium, 12-18 h after injury, plasmalemmal Ca2+-pump was seen as many dots in the cytoplasm by immunofluorescence microscopy; in contrast, caveolin persisted along the cell surface. Immunoelectron microscopy revealed that the labeling for the plasmalemmal Ca2+-pump was located around membranous structures in the cytoplasm and was scarce along the plasma membrane, while caveolin remained in caveolae. The Ca2+-pump regained normal distribution when the wound was closed. By quantitation in electron micrographs, the number of caveolae per unit plasma membrane length was found to be decreased in the wounded corneal epithelium. Conclusions. The present results indicate that caveolae undergo compositional modification during the wound healing process of the corneal epithelium. Considering putative caveolar functions, the phenomenon may be related to possible fluctuations of the intracellular Ca2+ concentration in the regenerating epithelium.