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Quantitation of extracellular UTP using a sensitive enzymatic assay
Author(s) -
Lazarowski Eduardo R,
Harden T Kendall
Publication year - 1999
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0702654
Subject(s) - extracellular , p2y receptor , uridine triphosphate , biochemistry , nucleotide , enzyme , chemistry , intracellular , receptor , pyrophosphate , biology , purinergic receptor , gene
The wide distribution of the uridine nucleotide‐activated P2Y 2 , P2Y 4 and P2Y 6 receptors suggests a role for UTP as an important extracellular signalling molecule. However, direct evidence for UTP release and extracellular accumulation has been addressed only recently due to the lack of a sensitive assay for UTP mass. In the present study, we describe a method that is based on the uridinylation of [ 14 C]‐glucose‐1P by the enzyme UDP‐glucose pyrophosphorylase which allows quantification of UTP in the sub‐nanomolar concentration range. The UTP‐dependent conversion of [ 14 C]‐glucose‐1P to [ 14 C]‐UDP‐glucose was made irreversible by including the pyrophosphate scavenger inorganic pyrophosphatase in the reaction medium and [ 14 C]‐glucose‐1P and [ 14 C]‐UDP‐glucose were separated and quantified by HPLC. Formation of [ 14 C]‐UDP‐glucose was linearly observed between 1 and 300 n M UTP. The reaction was highly specific for UTP and was unaffected by a 1000 fold molar excess of ATP over UTP. Release of UTP was measured with a variety of cells including platelets and leukocytes, primary airway epithelial cells, rat astrocytes and several cell lines. In most resting attached cultures, extracellular UTP concentrations were found in the low nanomolar range (1–10 n M in 0.5 ml medium bathing 2.5 cm 2 dish). Up to a 20 fold increase in extracellular UTP levels was observed in cells subjected to a medium change. Extracellular UTP levels were 10–30% of the ATP levels in both resting and mechanically‐stimulated cultured cells. In unstirred platelets, a 1 : 100 ratio UTP/ATP was observed. Extracellular UTP and ATP increased 10 fold in thrombin‐stimulated platelets. Detection of UTP in nanomolar concentrations in the medium bathing resting cultures suggests that constitutive release of UTP may provide a mechanism of regulation of the basal activity of uridine nucleotide sensitive receptors.British Journal of Pharmacology (1999) 127 , 1272–1278; doi: 10.1038/sj.bjp.0702654