Abstract
ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotides, direct demonstration of the release of cellular UTP has not been reported. Pharmacological studies of the P2Y4 receptor expressed in 1321N1 human astrocytoma cells indicated that this receptor is activated by UTP but not by ATP. Mechanical stimulation of 1321N1 cells also resulted in release of a molecule that markedly activated the expressed P2Y4 receptor. This nucleotide was shown to be UTP by two means. First, high performance liquid chromatography analysis of the medium from [33P]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulation resulted in a large increase in a radioactive species that co-eluted with authentic UTP. This species was degraded by incubation with the nonspecific pyrophosphohydrolase apyrase or with hexokinase and was specifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/10(6) cells within 2 min. This increase was paralleled by a similar augmentation of extracellular ATP levels. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis. Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors.
Highlights
ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors
These include the P2Y1 receptor, which is activated by adenine nucleotides [2, 3]; the P2Y2 receptor, which is activated equipotently by ATP and UTP [4]; the P2Y4 receptor, which is potently activated by UTP [5, 6]; and the P2Y6 receptor, which is selectively activated by UDP [7, 8]
A large body of evidence exists demonstrating that ATP is released in a regulated fashion from most cell types and that its availability for target receptor activation is tightly controlled by cell surface nucleotidases [9, 10]
Summary
ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. A sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/106 cells within 2 min. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors. We have shown that 1321N1 cells release a soluble mediator that activates the expressed P2Y4 receptor and have identified this extracellular mediator as UTP These results constitute the first direct demonstration of cellular release of UTP and provide biochemical approaches by which the cellular content and the release and extracellular presence of this molecule can be established in other tissues
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