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V Trinkaus-Randall, V Klepeis; The Role Of Purinergic Receptors In Corneal Epithelial Cell Injury . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4214.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously we demonstrated that injury caused the propagation of a rapid intercellular Ca2+ wave. The response was enhanced by EGF but not by FGF-2, PDGF, VEGF or NGF. Our goal is to evaluate if propagation of the wave following injury is mediated by P2Y receptors and if there is an interaction between the P2Y and EGF receptor signaling pathways. Methods: Cells were cultured and live cell imaging of cells loaded with Fluo-3AM (2 uM) was performed. Heterologous desensitization experiments were performed using ATP, ATPgammaS, ADP, UTP, UDP or EGF and compared to single stimuli. In addition both ATP and EGF were added simultaneously to cells. All experiments were performed over 250 seconds and repeated a minimum of three times, and change in average fluorescence was plotted over time. Migration experiments were performed where cells were either pretreated with the above agents or stimulated at the time of migration. Results: Media from injured cells possessed a 3.5 fold increase in extracellular ATP over controls and addition of 30 U/ml apyrase diminished the injury-induced wave and inhibited the calcium response elicited by addition of conditioned media to cells. U73122, a phospholipase C inhibitor, prevented propagation of the calcium wave. Cells responded similarily to either 2.5 uM ATP or UTP. The response to 50 uM ATPgammaS, a nonhydrolyzable form of ATP, was similar to that for 5 uM ATP. Cells responded to 50 uM ADP and 50 uM UDP with an intensity comparable to that for 0.5 uM ATP. Pretreatment with ATP for 3 hrs decreased the Ca2+ response to ATP compared to nonpretreated cells. Pretreatment with EGF abolished the Ca2+ response to EGF in a time dependent manner. When added simultaneously, there was a rapid increase associated with ATP followed by an activation of the EGF receptor. Experiments with tyrphostin have further elucidated this relationship. In migration experiments preincubation with 10 uM ATPgammaS caused a 3-fold increase in cell migration to EGF while ATPgammaS or EGF alone did not. Conclusion: The results suggest that intercellular communication is required to coordinate cellular activities to produce changes responsible for wound repair such as migration. Both P2Y and EGF receptors mediate the injury response and there is strong evidence for cooperativity between the receptors.
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