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J.A. Hernandez, S. Chifflet, S. Grasso; ENaC Participation in Plasma Membrane Depolarization During Wound Healing in Cultured Bovine Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4713.
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Purpose: In previous work we showed that narrow linear wounds produced on cultured bovine corneal endothelial (BCE) monolayers heal by a mechanism that simultaneously combines actin cable formation and cell crawling. Also that, concomitantly to this, plasma membrane potential (PMP) depolarization occurs at the leading edge of the wounds and gradually extends inwardly towards the neighboring cells. With the purpose of studying the underlying mechanism of depolarization, we obtained evidence that suggests that the PMP depolarization mainly occurs by an increase in epithelial sodium channel (ENaC)–mediated sodium permeability. Methods: Narrow linear wounds were produced on BCE cells cultured to confluence by carefully scraping the monolayers with a thin needle. Fluorescent probes were employed to detect modifications in PMP (oxonol V), intracellular sodium concentration (Sodium Green), F–actin distribution (rhodamin–phalloidin) and ENaC localization (IIF). ENaC participation in PMP depolarization was assessed by substitution of extracellular sodium by choline or lithium and by inhibition with phenamil. Results: The replacement of extracellular sodium by choline provokes a marked decrease in the depolarization areas and in actin cable formation. Phenamil (an ENaC inhibitor) determines analogous effects to the ones produced by the replacement of the ambient sodium by choline and a marked decrease in the lamellar activity of the wound edges and in the velocity of healing. The replacement of extracellular sodium by lithium (an ENaC–permeant cation) produces similar depolarization and cytoskeletal responses to the ones determined in normal sodium ambient. Finally, there is an increase in the ENaC IIF signal which is spatially correlated with the PMP depolarization areas. Conclusions: The results suggest that PMP depolarization participates in the cytoskeletal modifications that take place during the healing of narrow wounds in BCE monolayers and that this depolarization is mediated by an ENaC–dependent increase in sodium permeability.
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