Abstract
Purpose::
We have previously shown that ENaC-dependent plasma membrane potential depolarization (PMPD) and intracellular sodium increase of the border cells participate in the process of wound healing in monolayers of bovine corneal endothelial (BCE) cells in culture (Chifflet et al, Am. J. Physiol., 288:C1420, 2005). We have also recently reported (ICER, 2006) that a late slow calcium wave develops concomitantly to the PMPD and is dependent upon the increase in ENaC expression. The purpose of this work is to provide with novel evidence about the mechanisms involved in the generation of the late calcium wave.
Methods::
Wounds were produced on BCE cells cultured to confluence by scraping the monolayers with a thin needle. Fluorescent probes were employed to detect PMPD (oxonol V) and modifications in intracellular calcium and sodium concentrations (Fluo 4 and Sodium Green). Anti a and bENaC antibodies were used for IIF and Western Blot. Thapsigargin and EGTA were employed to assess the participation of the rapid calcium wave and intracellular calcium reservoirs in PMPD and late calcium wave development.
Results::
The fast calcium wave produced immediately after wounding was only completely inhibited by simultaneous application of thapsigargin and EGTA. Thapsigargin alone, incorporated 30 minutes prior to wounding, or EGTA alone, applied at the time of wounding, only partially inhibited the fast wave. Approximately three hours after wounding, both in thapsigargin-EGTA- and in thapsigargin-treated cells a significant decrease in the late Ca++ and Na+ waves could be observed, accompanied by an abolishment of the increase in ENaC expression. In EGTA-treated monolayers no modifications in the Ca++ and Na+ waves and in ENaC expression could be detected.
Conclusions::
The increase in ENaC expression and late calcium wave could be triggered by the second stage of the fast calcium wave and could be mediated by calcium release from intracellular calcium reservoirs.
Keywords: cornea: endothelium • wound healing • calcium