Human keratinocytes release ATP and utilize three mechanisms for nucleotide interconversion at the cell surface.
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, F(1)F(0) 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, F(1)F(0) 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, F(1)F(0) 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.
Burrell
Helen E
HE
Department of Human Anatomy & Cell Biology, School of Biomedical Sciences, University of Liverpool, The Sherrington Buildings, UK. H.E.Burrell@liv.ac.uk
Wlodarski
Brenda
B
Foster
Brian J
BJ
Buckley
Katherine A
KA
Sharpe
Graham R
GR
Quayle
John M
JM
Simpson
Alec W M
AW
Gallagher
James A
JA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000262:Adenylyl Cyclases / Q000378:metabolism; D002452:Cell Count; D002478:Cells, Cultured; D003470:Culture Media; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D012867:Skin / Q000201:enzymology
D003470:Culture Media; D009711:Nucleotides; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases; D000262:Adenylyl Cyclases