Abstract
Abstract: :
Purpose: Our goal is to characterize the specific cell surface purinergic receptors that play a role in cell–cell communication following injury or activation. Previously we demonstrated that nucleotides and not EGF mediates the immediate response to injury and activation of ERK (Yang et al 2003, Klepeis et al 2001). Methods: Identification of P2Y receptors was performed on human corneal epithelial cells (HCE–T) and primary corneal epithelial cells using live cell imaging, western blot analysis, immunohistochemical localization and RT–PCR. Results: Live cell imaging using the Ca2+ indicator dye, Fluo–3AM, was used to characterize the response to purines and pyrimidines. In experiments involving single nucleotides we found that ATP elicited the strongest response, followed by UTP, ADP and UDP, and the responses were dose dependent. We have also shown that each nucleotide desensitized itself and that both ATP and UTP desensitized each other. Stimulation with 2MeSATP resulted in a small response compared to that of ATPγS, which resulted in a response analogous to ATP. Together these results and RT–PCR confirmed that P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors were present in both primary and epithelial cell lines. Localization of P2Y2 and P2Y4 was detected using antibodies and GFP constructs. Furthermore, we have shown that ATP and UTP caused rapid transient activation of paxillin on tyrosines 31 and 118. We demonstrated that nucleotides induced cell migration and that this was enhanced in the presence of EGF. Furthermore, western blots showed transactivation of the EGF receptors ErbB1 and 2 following ATP stimulation. In contrast, there was no response to α,ß–MeATP or ß,γ–MeATP in Ca2+ imaging or phosphorylation studies indicating that P2X receptors are not involved in these events. Conclusions:These results indicate that the initial response to injury and activation is mediated by specific P2Y receptors and that the regulation of later events may be mediated by cross–talk between EGF and P2Y receptors.
Keywords: cell–cell communication • receptors • cornea: epithelium