Extracellular nucleotides are released at high concentrations from damaged cells and function through P2 receptor activation. Intestinal epithelial restitution, which is defined as cell migration independent of cell proliferation, is an important initial step in the process of wound healing. In this study, we investigated the role of extracellular nucleotides in intestinal epithelial migratory responses.

Wound‐healing and trans‐well migration assays were performed with a rat intestinal epithelial cell line (IEC‐6). The concentrations of extracellular nucleotides released from injured IEC‐6 cells were measured by HPLC. TGF‐β expression was assessed by RT‐PCR and elisa.

Scratching the monolayer of IEC‐6 cells induced cell migration. Pretreatment with apyrase or MRS2578, a selective P2Y₆ antagonist, inhibited the wound‐induced cell migration. Among the cellular nucleotides, only ATP and uridine 5'‐diphosphate (UDP) were detected in the culture medium after cell wounding. Exogenously applied UDP dose‐dependently enhanced the migration more effectively than ATP but did not induce proliferation. In addition, cell wounding and UDP increased the expression of TGF‐β, and both the wound‐induced and UDP‐enhanced migration were inhibited by MRS2578 or ALK5Inhibitor (ALK5i), a TGF‐β receptor blocker. Furthermore, cell wounding and UDP stimulation up‐regulated the expression of P2Y₆ receptor mRNA, and this effect was suppressed by MRS2578 or ALK5i.

Wound‐induced UDP evokes intestinal epithelial restitution by activation of P2Y₆ receptors, which mediates de novo synthesis of TGF‐β. In addition, the expression of P2Y₆ receptors is increased by cell wounding and UDP, which constitutes a positive‐feedback loop for mucosal repair.