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A. Mediero, A. Peral, J. Pintor; Role of Intracellular Pathways (mapk and Cytoskeleton) in Rabbit Corneal Epithelial Wound Healing Mediated by Ap4a and Ap3a. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3489. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previously studies in primary cultures of rabbit corneal epithelial cells, have demonstrated that dinucleotides produce two actions in wound healing: Ap4A accelerates the rate of healing with the activation of a P2Y2 receptor, and Ap3A and Ap5A delays this process with the activation of a P2Y6 receptor. The aim of this study is to understand the role of the main intracellular pathways in the re-epithelization process involving Ap4A and Ap3A.
In wounded confluent primary corneal epithelial cells monolayers and in the presence or absence of Ap4A or Ap3A 100 µM, we assayed a battery of antagonist of MAPK and cytoskeleton pathways (U0126 100 µM, Y27632 100 µM, PAO (phenylarsine oxide) 5 µM, (-)-Blebblistatin 10 µM and ML7 25 µM), and we also assayed a general inhibitor of Tyrosin kinases (AG1478 100 µM) (n = 8 each). A rabbit corneal epithelial establish cell line (SIRC) has been used to check the activation of ERK1/2 and ROCKI in western blot assay.
In the presence of Ap4A 100 µM, U0126, Y27632, AG1478 and (-)- Blebblistatin, produced a delay in the migration rate when compare with Ap4A 100 µM alone, and caused an increase in the healing time, while PAO and ML7 produced a lethal effect, causing cell death within the next 2 and 6 hours after incubation. In the presence of Ap3A 100 µM, U0126 and Y27632 accelerated the migration rate when compare with the rate of Ap3A 100 µM alone, while AG1478 and (-)-Blebblistatin produced a high delay in this migration rate. PAO and ML7 produced the same lethal effect as previously described. In the absence of agonists, all of the tested antagonist delay the migration rate when compared with a control. The Western Blot assays have demonstrated that both dinucleotides activate ERK1/2 pathway but only Ap4A activates ROCKI pathway.
The activation of Ap4A /P2Y2 receptor, produce its accelerating effect with the activation of both MAPK and cytoskeleton pathways, while activation of Ap3A/P2Y6 receptor activates only the MAPK pathway.
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