Extracellular nucleotides and nucleosides mediate diverse signaling effects in virtually all organs and tissues. Most models of purinergic signaling depend on functional interactions between distinct processes, including (i) the release of endogenous ATP and other nucleotides, (ii) triggering of signaling events via a series of nucleotide-selective ligand-gated P2X and metabotropic P2Y receptors as well as adenosine receptors and (iii) ectoenzymatic interconversion of purinergic agonists. The duration and magnitude of purinergic signaling is governed by a network of ectoenzymes, including the enzymes of the nucleoside triphosphate diphosphohydrolase (NTPDase) family, the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, ecto-5'-nucleotidase/CD73, tissue-nonspecific alkaline phosphatase (TNAP), prostatic acid phosphatase (PAP) and other alkaline and acid phosphatases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). Along with "classical" inactivating ectoenzymes, recent data provide evidence for the co-existence of a counteracting ATP-regenerating pathway comprising the enzymes of the adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK/NME/NM23) families and ATP synthase. This review describes recent advances in this field, with special emphasis on purine-converting ectoenzymes as a complex and integrated network regulating purinergic signaling in such (patho)physiological states as immunomodulation, inflammation, tumorigenesis, arterial calcification and other diseases. The second part of this review provides a comprehensive overview and basic principles of major approaches employed for studying purinergic activities, including spectrophotometric Pi-liberating assays, high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analyses of purine substrates and metabolites, capillary electrophoresis, bioluminescent, fluorometric and electrochemical enzyme-coupled assays, histochemical staining, and further emphasizes their advantages, drawbacks and suitability for assaying a particular catalytic reaction.

Enzymes involved in metabolism of extracellular nucleotides and nucleosides: functional implications and measurement of activities.

Biologically active concentrations of potently vasoactive and platelet-active adenine nucleotides are generated in plasma by a variety of pathophysiological mechanisms. Although there is evidence that ATP and ADP are inactivated by endothelial ectonucleotidases, there has been little attempt to study the metabolic routes of their catabolism in blood or to assess the contribution of this process to their clearance in vivo. Therefore, we have studied the rates and patterns of catabolism of ATP, ADP, and AMP in whole blood, plasma, and isolated blood cells. Rates of degradation of each nucleotide in cell-free plasma ranged from 0.07-0.32 nmol/min/ml with 1 microM substrates to 1.1-3.6 nmol/min/ml with 100 microM substrates. The pattern of catabolism indicated that sequential dephosphorylation from ATP----ADP----AMP----adenosine occurs. In whole blood, the pattern was similar although ATP and ADP (but not AMP) breakdown was more rapid. This was due to leukocyte ectonucleotidase activity. The use of selective inhibitors demonstrated that catabolism was not due to nonspecific phosphatase activity and that plasma 5'-nucleotidase is distinct from ATPase or ADPase. In leukocytes, ATPase and ADPase activities were distinguishable, and each contributed substantially to the rates of catabolism in whole blood. Leukocyte 5'-nucleotidase did not measurably contribute to AMP dephosphorylation in blood. By comparison, ecto-ATPase and ecto-ADPase activities on cultured human umbilical vein endothelial cells were similar to those on leukocytes while endothelial 5'-nucleotidase per 10(6) cells was equivalent to the soluble activity in 1 ml of blood or plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of adenine nucleotides in human blood.

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5′-nucleotidases).




The activity of soluble ATPase was progressively increased from 1.62±0.27 to 12.7±1.0 pmoles ml−1 h−1 after 60 min of stimulation by shear stress.




The rate of [3H]-ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate-6-azophenyl-2′4′-disulphonic acid, and remained insensitive to the classic inhibitors of ion-pumping and intracellular ATPases.




Shear stress also increased the activity of 5′-nucleotidase in the medium from 2.0±0.5 to 27.2±2.8 pmoles ml−1 h−1. When shear stress was applied after removal of ecto-5′-nucleotidase by phosphatidylinositol-specific phospholipase C, the release of 5′-nucleotidase was drastically reduced.




These results show that soluble ATPase and 5′-nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.






British Journal of Pharmacology (2000) 129, 921–926; doi:10.1038/sj.bjp.0703136

/pdf/effect-of-shear-stress-on-the-release-of-soluble-ecto-1pvfshfwex.pdf

Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5′-nucleotidase along with endogenous ATP from vascular endothelial cells

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface.

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.

/pdf/the-evidence-for-two-opposite-atp-generating-and-atp-44g7r4roai.pdf

D.A. Hector

Papers

Quantitative thin-layer chromatography of ATP and the products of its degradation in meat tissue.