The role of purinoceptors in wound healing

Abstract

Purinoceptors are membrane‐bound receptors for adenosine, purines and pyrimidines that are expressed in nearly all cell types throughout the organism. Previous studies demonstrated that they are involved in the regulation of proliferation of most target cells. Since it is well known that several purinoceptors are expressed in skin keratinocytes, we were interested in examining their involvement in wound healing. Primary skin keratinocytes from four different mouse strains were isolated and cultured for in vitro studies. The expression patterns of metabotropic purinoceptors of the primary cells as well as a murine cell line were determined by RT‐PCR analysis. The specificity of the amplification products was verified by sequence analysis. Proliferation assays with various purinoceptor agonists and antagonists were performed using the murine cell line MSC‐P5. Cell proliferation was determined by incorporation of 5‐bromo‐2‐deoxyuridine (BrdU). Female NMRI mice were used for in vivo trials. To inhibit the wound healing process, animals were treated once daily with dexamethasone. After a week of treatment, full thickness wounds were set with biopsy punches in depilated back skin. The wound healing process was measured by determination of the wound area and the incorporation of BrdU in vivo. The purinoceptor agonists and antagonists were administered topically on the wound area. Among the examined metabotropic purinoceptors, the murine cell line MSC‐P5 expressed the receptors A2b, P2Y1, P2Y2 and P2Y6. Primary cultured murine keratinocytes possessed the same expression profile and they additionally expressed the P2Y4 receptor. Furthermore, three of the four primary cultures showed a splicing variant of the P2Y2 receptor. The purinoceptor agonists ATP, UTP and 5’‐(N‐ethyl)‐carboxamidoadenosine (NECA) enhanced the cell growth of the murine cell line MSC‐P5. The mitogenic effect of ATP and UTP was inhibited by the P2Y1, P2Y2 and P2Y6 receptor antagonist suramin, while the effect of NECA was inhibited by the selective A2b receptor antagonist enprofylline. Taking into account that other tested purinoceptor agonists, 2‐Me‐S‐ATP and UDP, exhibited no proliferative activity on MSC‐P5 cells, it can be concluded that ATP and UTP act on proliferation via the P2Y2 receptor, and NECA via the A2b receptor. In vivo trials in an animal model of impaired wound healing confirm that pharmacological actions via purinoceptors offer an intriguing possibility in the treatment of wounds. Nevertheless, further investigations are needed to fully elucidate the role of purinergic mechanisms involved in wound healing. Funding: Self‐funded.