The Mechanisms of Cell Membrane Resealing in Rabbit Corneal Epithelial Cells

Abstract

Purpose: To examine membrane repair mechanisms in rabbit corneal epithelial (RCE) cells. Methods: Microneedle puncture and fluorescent dye loss were used to wound membranes and assay resealing, respectively. Different repair mechanisms were detected pharmacologically and with antisense oligonucleotides. Results: The RCE cells rapidly reseal plasma membranes by calcium-dependent exocytotic mechanisms that exhibit both facilitated and potentiated responses to multiple wounding. The facilitated response was inhibited by specific inhibitors of protein kinase C (PKC) and brefeldin A, and the potentiated response was blocked by inhibitors of cAMP-dependent protein kinase (PKA). Reduction of myosin IIA inhibited the facilitated response, and reduction of IIB inhibited the initial resealing. Conclusions: RCE cells rapidly repair plasma membrane disruptions. At a second wound at the same site, PKC stimulated vesicle formation from the Golgi apparatus, resulting in more rapid membrane resealing for a facilitated response. The RCE cell also contains a PKA-dependent global potentiation of membrane resealing.

Key Words:

• exocytosis
• membrane resealing
• myosin IIA and IIB
• protein kinase A
• protein kinase C