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 22.214.171.124) and ecto-5′-nucleotidase/CD73 (EC 126.96.36.199); (2) ecto-nucleotide kinases, adenylate kinase (EC 188.8.131.52) and nucleoside diphosphate kinase (EC 184.108.40.206); (3) ecto-adenosine deaminase (EC 220.127.116.11). Evidence for this was obtained by using enzyme assays with 3H-labelled nucleotides and adenosine as substrates, direct evaluation of γ-phosphate transfer from [γ-32P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20kDa surface protein was observed following incubation of Namalwa B cells with [γ-32P]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.
- ecto-adenosine deaminase
- ecto-nucleotide kinase
- purine metabolism
Abbreviations used: EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride; HUVEC, human umbilical vein endothelial cells; mAb, monoclonal antibody; NBTI, nitrobenzylthioinosine; NTPDase, NTP diphosphohydrolase; PBL, peripheral blood lymphocytes; PI-PLC, phosphatidylinositol-specific phospholipase C.
- The Biochemical Society, London ©2002