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Papers In Press, published online ahead of print June 15, 2005
J. Biol. Chem, 10.1074/jbc.M505381200
Submitted on May 17, 2005
Accepted on June 15, 2005

Human keratinocytes release ATP and utilise three mechanisms for nucleotide interconversion at the cell surface

Helen E. Burrell, Brenda Wlodarski, Brian J. Foster, Katherine A. Buckley, Graham R. Sharpe, John M. Quayle, Alec W. M. Simpson, and James A. Gallagher

Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3GE

Corresponding Author: H.E.Burrell{at}liv.ac.uk

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP-generation at the cell surface. Addition of ADP, GTP or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin and UDP, suggesting the involvement of cell surface adenylate kinase, F1F0 ATP synthase and nucleoside diphosphokinase (NDPK) respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F1F0 ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilising AMP. In normal human skin, F1F0 ATP synthase and NDPK were differentially localised, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.


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