Quantitation of extracellular UTP using a sensitive enzymatic assay

1 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 extracell ular accumulation has been addressed only recently due to the lack of a sen sitive assay for UTP mass. In the present study, we describe a method that is based on the uridinylation of [C-14]-glucose-1P by the enzyme UDP-glucos e pyrophosphorylase which allows quantification of UTP in the sub-nanomolar concentration range. 2 The UTP-dependent conversion of [C-14]-glucose-1P to [C-14]-UDP-glucose w as made irreversible by including the pyrophosphate scavenger inorganic pyr ophosphatase in the reaction medium and [C-14]-glucose-1P and [C-14]-UDP-gl ucose were separated and quantified by HPLC. Formation of [C-14]-UDP-glucos e was linearly observed between 1 and 300 nM UTP. The reaction was highly s pecific for UTP and was unaffected by a 1000 fold molar excess of ATP over UTP. 3 Release of UTP was measured with a variety of cells including platelets a nd leukocytes, primary airway epithelial cells, rat astrocytes and several cell lines. In most resting attached cultures, extracellular UTP concentrat ions were found in the low nanomolar range (1-10 nM in 0.5 mi medium bathin g 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 w ere 10-30% of the ATP levels in both resting and mechanically-stimulated cu ltured cells. In unstirred platelets, a 1:100 ratio UTP/ATP was observed. E xtracellular UTP and ATP increased 10 fold in thrombin-stimulated platelets . 4 Detection of UTP in nanomolar concentrations in the medium bathing restin g cultures suggests that constitutive release of UTP may provide a mechanis m of regulation of the basal activity of uridine nucleotide sensitive recep tors.