Impairment by activation of TRPA1 of gastric epithelial restitution in a wound model using RGM1 cell monolayer

We examined the influence of TRPA1 on the epithelial restitution using a rat gastric epithelial cell line RGM1 monolayer. RGM1 cells were inoculated in 24-well plates cultured for 24 hr, and then starved for 24 hr in a culture medium at 37 degrees C under 5 % CO(2) in air. After obtaining a confluent RGM1 cell monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. The repair process was monitored by quantitatively measuring the area of the epithelial wound (cell-free area). Immediately after the wound induction, cells at the edge of wound started to form lamellipodia, migrating toward the center of wound, and by so doing the cell-free area was decreased over time. The addition of icilin, the TRPA1 agonist, suppressed the recovery of the epithelial wound in a concentration-dependent manner. Likewise, another TRPA1 agonist, ally isothiocyanate, also significantly inhibited the wound repair. In addition, the recovery of the epithelial wound was potently inhibited when the ambient temperature was lowered to 17 degrees C, the threshold temperature where TRPA1 is known to be activated. By contrast, the wound healing was not affected by either menthol, the TRPM8 agonist, or capsaicin, the TRPV1 agonist. These results showed for the first time that the activation of TRPA1 inhibited the repair of the epithelial wound in the stomach, probably by the suppression of cell migration, and suggested the involvement of TRPA1 in the mechanism of gastric epithelial restitution.