Voltage-dependent Anion Channel-1 (VDAC-1) Contributes to ATP Release and Cell Volume Regulation in Murine Cells

Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b−) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (−/−)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (−/−) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (−/−) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (−/−) and WT mice after luminal hypotonic challenge, but VDAC-1 (−/−) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (−/−) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function

UDP-glucose promotes neutrophil recruitment in the lung

In addition to their role in glycosylation reactions, UDP-sugars are released from cells and activate widely distributed cell surface P2Y 14 receptors (P2Y 14 R). However, the physiological/pathophysiological consequences of UDP-sugar release are incompletely defined. Here, we report that UDP-glucose levels are abnormally elevated in lung secretions from patients with cystic fibrosis (CF) as well as in a mouse model of CF-like disease, the βENaC transgenic (Tg) mouse. Instillation of UDP-glucose into wild-type mouse tracheas resulted in enhanced neutrophil lung recruitment, and this effect was nearly abolished when UDP-glucose was co-instilled with the P2Y 14 R antagonist PPTN [4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl-2-naphthoic acid]. Importantly, administration of PPTN to βENaC-Tg mice reduced neutrophil lung inflammation. These results suggest that UDP-glucose released into the airways acts as a local mediator of neutrophil inflammation

Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin (~7 μm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1–10 nM. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ~250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ~1 μM independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intra-cellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation

Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery

BACKGROUND: Uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5'-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems. METHODS: The perforated-patch clamp technique was used to record the Ca(2+)-dependent, Cl(- )current (I(Cl,Ca)) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student's t test or one way ANOVA. RESULTS: ATP, UTP and UDP (10(-4)M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min(-1)) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10(-4)M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10(-4)M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA. CONCLUSION: At least two P2Y subtypes couple to I(Cl,Ca )in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y(11 )receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction

Nucleotide Release Provides a Mechanism for Airway Surface Liquid Homeostasis

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca2+- and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A2b adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N6-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolayers that endogenously express a luminal A2b adenosine receptor, we found that basal as well as forskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A2b receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis

UDP Is a Competitive Antagonist at the Human P2Y14 Receptor

G protein-coupled P2Y receptors (P2Y-R) are activated by adenine and uracil nucleotides. The P2Y14 receptor (P2Y14-R) is activated by at least four naturally occurring UDP sugars, with UDP-glucose (UDP-Glc) being the most potent agonist. With the goal of identifying a competitive antagonist for the P2Y14-R, UDP was examined for antagonist activity in COS-7 cells transiently expressing the human P2Y14-R and a chimeric Gα protein that couples Gi-coupled receptors to stimulation of phosphoinositide hydrolysis. UDP antagonized the agonist action of UDP-Glc, and Schild analysis confirmed that the antagonism was competitive (pKB = 7.28). Uridine 5′-O-thiodiphosphate also antagonized the human P2Y14-R (hP2Y14-R) with an apparent affinity similar to that of UDP. In contrast, no antagonist activity was observed with ADP, CDP, or GDP, and other uracil analogs also failed to exhibit antagonist activity. The antagonist activity of UDP was not observed at other hP2Y-R. In contrast to its antagonist action at the hP2Y14-R, UDP was a potent agonist (EC50 = 0.35 μM) at the rat P2Y14-R. These results identify the first competitive antagonist of the P2Y14-R and demonstrate pharmacological differences between receptor orthologs

A selective high-Affinity antagonist of the P2Y14 receptor inhibits udp-glucose-stimulated chemotaxis of human neutrophilss

The nucleotide-sugar–activated P2Y14 receptor (P2Y14-R) is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. A series of molecules recently were identified by Gauthier et al. (Gauthier et al., 2011) that exhibited antagonist activity at the P2Y14-R. We synthesized one of these molecules, a 4,7-disubstituted 2-naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase in C6 glioma cells stably expressing the P2Y14-R was shifted to the right in a concentration-dependent manner by PPTN. Schild analyses revealed that PPTN-mediated inhibition followed competitive kinetics, with a KB of 434 pM observed. In contrast, 1 μM PPTN exhibited no agonist or antagonist effect at the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, or P2Y13 receptors. UDP-glucose–promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the KB determined with recombinant P2Y14-R. In contrast, the chemotactic response evoked by the chemoattractant peptide fMetLeuPhe was unaffected by PPTN. UDP-glucose–promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. In summary, this work establishes PPTN as a highly selective high-affinity antagonist of the P2Y14-R that is useful for interrogating the action of this receptor in physiologic systems

Identification of an ATP/P2X7/Mast Cell Pathway Mediating Ozone-Induced Bronchial Hyperresponsiveness

Ozone is a highly reactive environmental pollutant with well-recognized adverse effects on lung health. Bronchial hyperresponsiveness (BHR) is one consequence of ozone exposure, particularly for individuals with underlying lung disease. Our data demonstrated that ozone induced substantial ATP release from human airway epithelia in vitro and into the airways of mice in vivo and that ATP served as a potent inducer of mast cell degranulation and BHR, acting through P2X7 receptors on mast cells. Both mast cell-deficient and P2X7 receptor-deficient (P2X7-/-) mice demonstrated markedly attenuated BHR to ozone. Reconstitution of mast cell-deficient mice with WT mast cells and P2X7-/- mast cells restored ozone-induced BHR. Despite equal numbers of mast cells in reconstituted mouse lungs, mice reconstituted with P2X7-/- mast cells demonstrated significantly less robust BHR than mice reconstituted with WT mast cells. These results support a model where P2X7 on mast cells and other cell types contribute to ozone-induced BHR

Positive Inotropic Effects by Uridine Triphosphate (UTP) and Uridine Diphosphate (UDP) via P2Y2 and P2Y6 Receptors on Cardiomyocytes and Release of UTP in Man During Myocardial Infarction

The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y2/4 agonist UTPγS increased contraction by 52%, similar to β1-adrenergic stimulation with isoproterenol (65%). The P2Y6-agonist UDPγS also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y6-blocker MRS2578. The phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 inhibited both the UDPγS and the UTPγS-induced inotropic effect, indicating an IP3-mediated effect via P2Y6 receptors. The P2Y14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y2 receptors in man. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y6 receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines (UTP and UDP) could be important inotropic factors involved in the development of cardiac disease