Quantitation of extracellular UTP using a sensitive enzymatic assay

Abstract

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 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 [ ]-UDP-glucose was linearly observed between 1 and 300 nM UTP. The reaction was highly speci®c for UTP and was unaected by a 1000 fold molar excess of ATP over UTP. 3 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 nM 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. 4 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.

Endotoxin Activation of Macrophages Does Not Induce ATP Release and Autocrine Stimulation of P2 Nucleotide Receptors

“…Serotype 0111:B4 (List Biologicals) was found to contain very little ATP and was, therefore, suitable for these studies. Because medium agitation or removal can cause lytic or mechanically induced nucleotide release (23)(24)(25)(26), cells were allowed to recondition their media for at least 45 min after all washes. Each on-line analysis of adherent macrophages included direct calibration with known amounts of ATP, permeabilization to liberate total intracellular ATP, and treatment with apyrase to determine background luminescence; these steps permitted rigorous quantitative evaluation of any changes in extracellular ATP accumulation or catabolism.…”

“…This may be due in part to the fact that routine experimental procedures, such as solution exchange and cell washing, have been shown to elicit nucleotide release from a variety of cell types (23)(24)(25)(26). During prolonged endotoxin activation, macrophages produce and secrete a variety of cytotoxic mediators, including reactive oxygen and nitrogen species (1,3).…”

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

“…Basal constitutive release of nucleotides occurs from most, if not all, cell types [17][18][19][20], and this release is counterbalanced by ectonucleotidase-catalyzed degradation [19,21]. Baseline apoptosis in the presence of TNFα depends on extracellular calcium, and ATP is released from most eukaryotic cells into the extracellular environment [22].…”

Endoplasmic Reticulum/Golgi Nucleotide Sugar Transporters Contribute to the Cellular Release of UDP-sugar Signaling Molecules

“…Given that the pyrophosphorolysis reaction displays an equilibrium constant of 5 Ϯ 1.6 (42), our assay conditions ensured that the direction of the reaction would be highly favorable toward [ 32 P]UTP synthesis. Indeed, the reaction product UTP, at concentrations in the range observed in extracellular solutions (43), had no significant effect on the UDPGlcNAc-dependent conversion of [ 32 P]PP i to [ 32 P]UTP (data not shown). Moreover, the reaction was not affected by the presence of 100 nM UDP-GalNAc, UDP-Glc, UDP-Gal, UDPGlcA, ATP, ADP, or UDP (data not shown).…”