Vesicular and conductive mechanisms of nucleotide release

Extracellular nucleotides and nucleosides promote a vast range of physiological responses, via activation of cell surface purinergic receptors. Virtually all tissues and cell types exhibit regulated release of ATP, which, in many cases, is accompanied by the release of uridine nucleotides. Given the relevance of extracellular nucleotide/nucleoside-evoked responses, understanding how ATP and other nucleotides are released from cells is an important physiological question. By facilitating the entry of cytosolic nucleotides into the secretory pathway, recently identified vesicular nucleotide and nucleotide-sugar transporters contribute to the exocytotic release of ATP and UDP-sugars not only from endocrine/exocrine tissues, but also from cell types in which secretory granules have not been biochemically characterized. In addition, plasma membrane connexin hemichannels, pannexin channels, and less-well molecularly defined ATP conducting anion channels have been shown to contribute to the release of ATP (and UTP) under a variety of conditions

Quantification of extracellular UDP-galactose

The human P2Y14 receptor is potently activated by UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid. Recently, cellular release of UDP-Glc and UDP-GlcNAc has been reported, but whether additional UDP-sugars are endogenous agonists for the P2Y14 receptor remains poorly defined. In the present study, we describe an assay for the quantification of UDP-Gal with sub-nanomolar sensitivity. This assay is based on the enzymatic conversion of UDP-Gal to UDP, using 1–4-β-galactosyltransferase. UDP is subsequently phosphorylated by nucleoside diphosphokinase in the presence of [γ32P]ATP and the formation of [γ32P]UTP is monitored by high performance liquid chromatography. The overall conversion of UDP-Gal to [γ32P]UTP was linear between 0.5 and 30 nM UDP-Gal. Extracellular UDP-Gal was detected on resting cultures of various cell types, and increased release of UDP-Gal was observed in 1321N1 human astrocytoma cells stimulated with the protease-activated receptor agonist thrombin. Occurrence of regulated release of UDP-Gal suggests that, in addition to its role in glycosylation reactions, UDP-Gal is an important extracellular signaling molecule

Purinergic receptors in airway epithelia

Nucleotides and nucleosides within the airway surface liquid regulate mucociliary clearance (MCC) activities, the primary innate defense mechanism that removes foreign particles and pathogens from airway surfaces. Nucleotide and nucleoside actions in the airways are mediated mainly by two purinergic receptor subtypes, the Gq-coupled ATP/UTP-sensing P2Y2 receptor and the Gs-coupled A2b adenosine receptor. Activation of the A2b receptor results in cyclic AMP-dependent activation of the cystic fibrosis transmebrane regulator (CFTR) Cl- channel and stimulation of ciliary beat frequency. Agonist occupation of the P2Y2 receptor promotes inhibition of Na+ absorption as well as CFTR-dependent and CFTR-independent Cl-secretion, ciliary beating, and mucin secretion

Constitutive Release of ATP and Evidence for Major Contribution of Ecto-nucleotide Pyrophosphatase and Nucleoside Diphosphokinase to Extracellular Nucleotide Concentrations

Nucleotides are important extracellular signaling molecules. At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP. Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t(1/2);1 approximately;230-40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration. Although the base-line concentration of ATP remained stable for up to 12 h, [gamma-(32)P]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20-200 fmol x min(-)(1) x cell(-)(6)). High performance liquid chromatography analysis revealed that the gamma-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP. Conversely, addition of [gamma-(32)P]UTP to resting cells resulted in transient formation of [gamma-(32)P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final (32)P-products of [gamma-(32)P]ATP metabolism were [(32)P]orthophosphoric acid and a (32)P-labeled species that was further purified and identified as [(32)P]inorganic pyrophosphate. In C6 cells, the formation of [(32)P]pyrophosphate from [gamma-(32)P]ATP at steady state exceeded by 3-fold that of [(32)P]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides. In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase

Cystic Fibrosis Transmembrane Regulator-independent Release of ATP: ITS IMPLICATIONS FOR THE REGULATION OF P2Y2RECEPTORS IN AIRWAY EPITHELIA

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP

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 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

Identification of an Ecto-nucleoside Diphosphokinase and Its Contribution to Interconversion of P2 Receptor Agonists

The P2Y4 receptor is selectively activated by UTP. Although addition of neither ATP nor UDP alone increased intracellular Ca2+ in 1321N1 human astrocytoma cells stably expressing the P2Y4 receptor, combined addition of these nucleotides resulted in a slowly occurring elevation of Ca2+. The possibility that the stimulatory effect of the combined nucleotides reflected formation of UTP by an extracellular transphosphorylating activity was investigated. Incubation of cells with [3H]UDP or [3H]ADP under conditions in which cellular release of ATP occurred or in the presence of added ATP resulted in rapid formation of the corresponding triphosphates. Transfer of the gamma-phosphate from [gamma-33P]ATP to nucleoside diphosphates confirmed that the extracellular enzymatic activity was contributed by a nucleoside diphosphokinase. The majority of this activity was associated with the cell surface of 1321N1 cells, suggesting involvement of an ectoenzyme. Both ADP and UDP were effective substrates for transphosphorylation. Since ecto-nucleotidase(s) has been considered previously to be the primary enzyme(s) responsible for metabolism of extracellular nucleotides, the relative rates of hydrolysis of ATP, ADP, UTP, and UDP also were determined for 1321N1 cells. All four nucleotides were hydrolyzed with similar Km and Vmax values. Kinetic analyses of the ecto-nucleoside diphosphokinase and ecto-nucleotidase activities indicated that the rate of extracellular transphosphorylation exceeds that of nucleotide hydrolysis by up to 20-fold. Demonstration of the existence of a very active ecto-nucleoside diphosphokinase together with previous observations that stress-induced release of ATP occurs from most cell types indicates that transphosphorylation is physiologically important in the extracellular metabolism of adenine and uridine nucleotides. Since the P2Y receptor class of signaling proteins differs remarkably in their respective specificity for adenine and uridine nucleotides and di- and triphosphates, these results suggest that extracellular interconversion of adenine and uridine nucleotides plays a key role in defining activities in nucleotide-mediated signaling

Calcium-dependent release of adenosine and uridine nucleotides from A549 cells

Extracellular nucleotides play an important role in lung defense, but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined. In this study, to minimize cell surface hydrolysis, we used a low-volume, flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50% hypotonic shock. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (Ado) were quantified in high-performance liquid chromatography (HPLC) analysis of fluorescent etheno derivatives, and uridine triphosphate (UTP) and uridine diphosphate (UDP) were measured using HPLC-coupled radioenzymatic assays. After the onset of hypotonic shock, ATP, ADP, UTP, and UDP in the perfusates increased markedly and peaked at approximately 2.5 min, followed by a gradual decay in the next 15–20 min; peak changes in Ado and AMP were relatively minor. The peak concentrations and fold increment (in parentheses) were: 34 ± 13 nM ATP (5.6), 11 ± 5 nM ADP (3.7), 3.3 ± 1.2 nM AMP (1.4), 23 ± 7 nM Ado (2.1), 21 nM UTP (>7), and 11 nM UDP (27). Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Under isotonic conditions, elevation of intracellular calcium with the calcium ionophore ionomycin (5 μM, 3 min) also released nucleotides with kinetics and relative abundance as above, albeit less robust. ADP:ATP (1:3) and UDP:UTP (1:2) ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species, suggesting that a nucleotide diphosphate (NDP)-rich compartment, e.g., the secretory pathway, contributed to nucleotide release. Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment, consistent with enhanced vesicular exocytosis under these conditions. In summary, our results strongly suggest that calcium-dependent exocytosis is responsible, at least in most part, for adenosine and uridine nucleotide release from A549 cells

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

Background: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intraand extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). Methods: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin–luciferase based real-time luminometry. Results: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. Conclusions: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General significance: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.Fil: Leal Denis, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Incicco, Juan Jeremías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Espelt, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Verstraeten, Sandra Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Lazarowski, Eduardo R.. University of North Carolina at Chapel Hill. Cystic Fibrosis/Pulmonary Research and Treatment Center; Estados UnidosFil: Schwarzbaum, Pablo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentin

Neutrophilic inflammation is associated with altered airway hydration in stable asthmatics

Airway dehydration is a potential trigger of bronchoconstriction in exercise-induced asthma; however, its role in stable asthma has not been explored. Using sputum percent solids, as an indicator of airway hydration, we sought relationships between airway hydration and other known markers of neutrophilic (TH1) and allergic (TH2) inflammation in stable asthma.Thirty-seven atopic subjects with stable asthma and 15 healthy controls underwent sputum induction. Sputum was analyzed for percent solids, cell counts, cellular and biochemical markers of inflammation and purines.Sputum percent solids was significantly elevated in stable asthmatics vs. controls and positively correlated with markers of neutrophilic/TH1-type inflammation (neutrophils, IL-8 and AMP). Sputum percent solids were not correlated with markers of allergic/TH2-type inflammation. These data suggest a direct relationship between neutrophil inflammation and airway hydration in stable asthmatics