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A. Crooke, A. Mediero, A. Guzman-Aranguez, A. Peral, J. Pintor; Role of P2Y2 Nucleotide Receptor in Ap4A-Induced Migration of Rabbit Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3395.
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© ARVO (1962-2015); The Authors (2016-present)
In previous work we have demonstrated the Ap4A-accelerating effect on rabbit corneal wound healing and provided pharmacological evidence for P2Y2 nucleotide receptor mediation. The aim of this study is to know the molecular identity and functional role of rabbit P2Y2 receptor in Ap4A-reepithelization process.
The full-length of rabbit P2Y2 receptor cDNA was cloned using a combination of degenerate reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) method. Function of the rabbit P2Y2 receptor was assessed using P2Y2 receptor-specific siRNA. Transfection of siRNA was examined by incubating establish rabbit corneal epithelial cell line (SIRC) with Cy3-labeled non-silencing siRNA and fluorescence microscopy. SIRC monolayers were transfected with P2Y2 receptor siRNA and non-silencing siRNA. At different times after transfection, cells were extracted for quantitative real-time RT-PCR (qrtPCR) assay and wounded with a pipette tip in the presence or absence of Ap4A 100µM. Migration studies were performed at 0, 2, 4, 6, 8 and 10 hours after wound injury.
Blast database searches revealed that the deduced amino acid sequence of rabbit P2Y2 had high homology (93%) with human P2Y2 receptor. Cellular uptake of Cy3-labeled siRNA was confirmed. Prominent silencing of rabbit P2Y2 gene was detected in SIRC transfected with P2Y2 siRNA by quantitative mRNA expression analysis. In contrast, no change in P2Y2 mRNA level was observed in SIRC transfected with non-silencing siRNA. Cell migration experiments showed that Ap4A accelerated the rate of healing in cells transfected with non-silencing siRNA when compared with the rate of Ap4A in P2Y2 siRNA-transfected cells.
P2Y2 receptor-specific siRNA counteract the effect of Ap4A on rabbit corneal wound healing. These results confirm the involvement of P2Y2 receptor in Ap4A-reepithelization process.
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