Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions.
Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.
Yegutkin
G G
GG
MediCity Research Laboratory, University of Turku and National Public Health Institute, FIN-20520, Turku, Finland. gennady.yegutkin@utu.fi
Henttinen
T
T
Jalkanen
S
S
D015720:5'-Nucleotidase / Q000378:metabolism; D000225:Adenine / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism; D001081:Apyrase / Q000378:metabolism; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D013379:Substrate Specificity
D008565:Membrane Proteins; D010710:Phosphates; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); D000263:Adenylate Kinase; D009701:Nucleoside-Diphosphate Kinase; D015720:5'-Nucleotidase; D001081:Apyrase; D000225:Adenine; D000241:Adenosine