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.

Extracellular ATP protects endothelial cells against DNA damage

“…Other explanation could be difference in purine-inactivating cell surface enzymes between ECs and cancer cells. While CD39 is the main ATPhydrolyzing enzyme in ECs, cancer cells have additional phosphatases, which could explain the differential response to CD39 inhibition with POM-1 [35]. Our results with cancer cells, where γH2AX expression in POM-1-pretreated cells was increased after DOX-induced DNA damage, fit well to these previous findings.…”

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

“…Apart from transiently induced ATP release (e.g., by agonists and hormones), cells have basal release of ATP and other nucleotides, which can involve a number of releasing mechanisms and ecto-enzymatic modifications, and serve auto/paracrine modulation of cellular functions [34,35]. Our experiments indicate that there were no significant differences in basal extracellular ATP between cells with/without dexamethasone, which would suggest that this was independent of VNUT expression.…”

Adenosine Inhibits Tumor Cell Invasion via Receptor-Independent Mechanisms

“…7A), possess high ecto-5 0 -nucleotidase/CD73 activity (Fig. 7B), and, in addition, are capable of further deaminating adenosine (29), with subsequent uptake of generated nucleosides and their interconversion into intracellular [ 3 H]ADP/ATP (data not shown).…”