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J. Biol. Chem., Vol. 280, Issue 33, 29667-29676, August 19, 2005

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Human Keratinocytes Release ATP and Utilize 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

From the Department of Human Anatomy & Cell Biology, School of Biomedical Sciences, University of Liverpool, The Sherrington Buildings, Ashton Street, Liverpool L69 3GE and the ^¶Dermatology Unit, Department of Medicine, UCD Building, University of Liverpool, Liverpool, L69 3GA, United Kingdom

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 utilizing AMP. In normal human skin, F₁F₀ ATP synthase and NDPK were differentially localized, 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.

Received for publication, May 17, 2005

^* Funding for the confocal microscope was obtained from the Biotechnology and Biological Sciences and Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 44-0-151-794-5468; Fax: 44-0-151-794-5517; E-mail: H.E.Burrell{at}liv.ac.uk.

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