title: Enzyme-coupled assays for simultaneous detection of nanomolar ATP, ADP, AMP, adenosine, inosine and pyrophosphate concentrations in extracellular fluids.
abstract: Purinergic signaling cascade includes the release of endogenous ATP and other agonists by chemical and mechanical stimuli, modulation of diverse cellular functions and subsequent ectoenzymatic inactivation. Basal release of extracellular purines and its physiological relevance remain controversial. Here we employed a combination of enzyme-coupled approaches for simultaneous bioluminescent (ATP, ADP, PP(i)) and fluorometric (AMP), adenosine, inosine, hypoxanthine) measurements of ATP and its metabolites without additional manipulations or derivatization of sampled biological fluids. By using these sensing techniques, extracellular purines were determined in various cells and tissues both at resting and pro-inflammatory conditions. The results obtained revealed the ability of endothelial, lymphoid and tumor cells to maintain extracellular ATP, ADP and adenosine at certain characteristic nanomolar levels. By quantifying the amounts of endogenously released and/or exogenously applied purines and their metabolites, these sensing techniques may be applied for evaluating purine-converting pathways on the cell surfaces and also for ex vivo analysis of purine homeostasis in the intact tissues. Furthermore, we provide novel insight into the mechanisms underlying tumorigenic effects of ATP by demonstrating the ability of metastatic prostate carcinoma PC3 and breast cancer MDA-MB-231 cells to maintain PP(i), which derives from extracellular ATP in the course of nucleotide pyrophosphatase/phosphodiesterase reaction. Collectively, the results imply a complex pattern of nucleotide turnover where extracellular ATP, ADP and adenosine are maintained at steady-state levels via conunterbalanced release and inactivation of ATP and other purines, and further suggest the importance of basal agonist release for continuous activation and/or desensitization of purinergic receptors.
authors.affiliation: Medicity Research Laboratory, University of Turku, Finland.
authors.forename: Mikko
authors.initials: M
authors.lastname: Helenius
authors.forename: Sirpa
authors.initials: S
authors.lastname: Jalkanen
authors.forename: Gennady
authors.initials: G
authors.lastname: Yegutkin
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D045744:Cell Line, Tumor; D002855:Chromatography, Thin Layer; D011756:Diphosphates / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D045604:Extracellular Fluid / Q000378:metabolism*; D005470:Fluorometry; D006706:Homeostasis; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D007288:Inosine / Q000378:metabolism; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000378:metabolism; D009362:Neoplasm Metastasis; D009705:Nucleosides / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism
chemical_list: D011756:Diphosphates; D009705:Nucleosides; D009711:Nucleotides; D000249:Adenosine Monophosphate; D007288:Inosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase; D000241:Adenosine