Purinergic signalling regulates airway defence mechanisms, suggesting that extracellular purines could serve as airway inflammation biomarkers in cystic fibrosis (CF).The purines adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine were measured in sputum from 21 adults (spontaneously expectorated from seven CF patients, induced from 14 healthy controls) to assess normal values and CF-associated changes. Subsequently, purine levels were measured in bronchoalveolar lavage fluid (BALF) from 37 children (25 CF patients, 12 disease controls) and compared with neutrophil counts, presence of airway infection and lung function. To noninvasively assess airway purines, ATP levels were measured using luminometry in exhaled breath condensate (EBC) from 14 children with CF and 14 healthy controls, then 14 CF children during a pulmonary exacerbation.Both ATP and AMP were elevated in sputum and BALF from CF subjects compared with controls. In BALF, ATP and AMP levels were inversely related to lung function and strongly correlated with neutrophil counts. In EBC, ATP levels were increased in CF relative to controls and decreased after treatment of CF pulmonary exacerbation.The purines adenosine triphosphate and adenosine monophosphate are candidate biomarkers of neutrophilic airways inflammation. Measurement of purines in sputum or exhaled breath condensate may provide a relatively simple and noninvasive method to track this inflammation.

Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y 14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Δ was reduced by deletion of the YEA4 UDP-N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation.Purinergic signaling pathways are present in nearly all mammalian cells (1) and regulate numerous critical physiologic functions, including control of extracellular volume (2,3) and inflammatory responses (4-7). The natural agonists of purinergic receptors are extracellular nucleosides, nucleotides, and nucleotide sugars such as adenosine triphosphate (ATP) 1 and uridine diphosphate (UDP) glucose (UDP-glucose). The physiological actions mediated by purinergic pathways depend on cellular release of these agonists into the extracellular space. † This work was supported by National Institutes of Health Grants NIH/NHLBI 5 P01 34322-18, HL34322, HL60280, HL074158, SCOR, and MTCC (R.C.B. and E.R.L.), NIH Grant RR17667-02, the Parker B. Francis Families Foundation, and the Cystic Fibrosis Foundation (C.R.E.). * To whom correspondence should be addressed: Pediatric Pulmonology, Fifth Floor Bioinformatics, CB#7217, University of North Carolina, Chapel Hill, NC 27599. Telephone: (919) 966-1055. Fax: (919) However, while vesicular release of ATP has been demonstrated in neuro-endocrine cells (1), the mechanisms by which ATP, UDP-glucose, and other purinergic agonists exit the cytoplasm in other cell types remain unclear.Several mechanisms have been pro...

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