The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells.
Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5'-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with (3)H-labelled nucleotides and adenosine as substrates, direct evaluation of gamma-phosphate transfer from [gamma-(32)P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20 kDa surface protein was observed following incubation of Namalwa B cells with [gamma-(32)P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature.
Yegutkin
Gennady G
GG
MediCity Research Laboratory, Turku University and National Public Health Institute, Tykistökatu 6A, FIN-20520 Turku, Finland. gennady.yegutkin@utu.fi
Henttinen
Tiina
T
Samburski
Sergei S
SS
Spychala
Jozef
J
Jalkanen
Sirpa
S
D015720:5'-Nucleotidase / Q000096:biosynthesis; D000241:Adenosine / Q000378:metabolism; D000243:Adenosine Deaminase / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011687:Purines / Q000378:metabolism; D011869:Radioligand Assay; D013997:Time Factors; D014407:Tumor Cells, Cultured
D011687:Purines; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D000243:Adenosine Deaminase; D000241:Adenosine