DIRECT DEMONSTRATION OF MECHANICALLY INDUCED RELEASE OF CELLULAR UTP AND ITS IMPLICATION FOR URIDINE NUCLEOTIDE RECEPTOR ACTIVATION

ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large fam ilies of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotide s, direct demonstration of the release of cellular UTP has not, been r eported, Pharmacological studies of the P2Y, receptor expressed in 132 1N1 human astrocytoma cells indicated that this receptor is activated by UTP but mot by ATP. Mechanical stimulation of 1321N1 cells also res ulted in release of a molecule that markedly activated the expressed P 2Y(4) receptor. This nucleotide was shown to be UTP by two means, Firs t, high performance liquid chromatography analysis of the medium from [P-33]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulatio n 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 sp ecifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mas s at low nanomolar concentrations was devised Based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechan ical 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 a min, Thi s increase was paralleled by a similar augmentation of extracellular A TP levels, Pn calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels cou ld be accounted for by cell lysis, Taken together, these results direc tly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y(4) recept ors.