ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.The mucociliary clearance process that removes foreign particles and pathogens from the airways is the primary innate defense mechanism in the lung (1). Nucleotides and nucleosides within the airway surface liquid (ASL) 3 regulate key components of mucociliary clearance via activation of epithelial cell surface purinergic receptors (2, 3). ATP activates the G q -coupled P2Y 2 receptor that promotes mucin secretion and ciliary beat frequency and regulates electrolyte transport and ASL volume production by inhibiting sodium absorption (4 -10) and promoting cystic fibrosis transmembrane conductance regulator and Ca 2ϩ -activated Cl Ϫ channel activity (11-16). Adenosine, generated from the hydrolysis of ATP, activates the G scoupled A 2b receptor that promotes cyclic AMP-regulated cystic fibrosis transmembrane conductance regulator Cl Ϫ channel activity (17) and increases cilia beat frequency (5). Although ATP and adenosine are naturally occurring signaling molecules in ASL (18 -22), the mechanisms of airway epithelial ATP release into the ASL are poorly understood.The lung epithelia exhibit a complex cellular composition, and thus, several mechanisms and pathways are likely involved in the release of nucleotides onto airway surfaces. Our recent studies with goblet-like cell models indicate that ATP and other nucleotides are released concomitantly with MUC5AC, a secretory mucin...

The nucleotide-sugar-activated P2Y 14 receptor (P2Y 14 -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. ) that exhibited antagonist activity at the P2Y 14 -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 P2Y 14 -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 K B of 434 pM observed. In contrast, 1 mM PPTN exhibited no agonist or antagonist effect at the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , or P2Y 13 receptors. UDP-glucose-promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the K B determined with recombinant P2Y 14 -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 P2Y 14 -R that is useful for interrogating the action of this receptor in physiologic systems.

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In non-neuronal cells, inactivation of protein kinase D (PKD) blocks fission of trans-Golgi network (TGN) transport carriers, inducing the appearance of long tubules filled with cargo. We now report on the function of PKD1 in neuronal protein trafficking. In cultured hippocampal pyramidal cells, the transferrin receptor (TfR) and the low-density receptor-related protein (LRP) are predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive PKD1 or its depletion by RNA interference treatment dramatically and selectively alter the intracellular trafficking and membrane delivery of TfR-and LRP-containing vesicles, without inhibiting exit from the TGN or inducing Golgi tubulation. After PKD1 suppression, dendritic membrane proteins are mispackaged into carriers that transport VAMP2; these vesicles are distributed to both axons and dendrites, but are rapidly endocytosed from dendrites and preferentially delivered to the axonal membrane. A kinase-defective mutant of PKD1 lacking the ability to bind diacylglycerol and hence its Golgi localization does not cause missorting of TfR or LRP. These results suggest that in neurons PKD1 regulates TGN-derived sorting of dendritic proteins and hence has a role in neuronal polarity.

The G(i)-coupled P2Y(14) receptor (P2Y(14)-R) is potently activated by UDP-sugars and UDP. Although P2Y(14)-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration. We also demonstrate that UDP-glucose promotes rapid, robust, and concentration-dependent activation of RhoA in these cells. Ecto-nucleotidases expressed on neutrophils rapidly hydrolyzed extracellular ATP, but incubation with UDP-glucose for up to 1 h resulted in negligible metabolism of the nucleotide-sugar. HL60 human promyelocytic leukemia cells do not express the P2Y(14)-R, but neutrophil differentiation of HL60 cells with DMSO resulted in markedly enhanced P2Y(14)-R expression. Accordingly, UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine promoted Rho activation in differentiated but not in undifferentiated HL60 cells. Stable expression of recombinant human P2Y(14)-R conferred UDP-sugar-promoted responses to undifferentiated HL60 cells. UDP-glucose-promoted RhoA activation also was accompanied by enhanced cell migration in differentiated HL60 cells, and these responses were blocked by Rho kinase inhibitors. These results support the notion that UDP-glucose is a stable and potent proinflammatory mediator that promotes P2Y(14)-R-mediated neutrophil motility via Rho/Rho kinase activation.

Conflict of interest:SB is a cofounder of Kantum Pharma (previously "Kantum Diagnostics Inc."), a company developing a diagnostic and therapeutic combination to prevent and treat acute kidney injury. SB and her spouse own equity in the privately held company. SB and DB are inventors on a patent (US Patent 10,088,489) covering technology that has been licensed to the company through Massachusetts General Hospital (MGH). SB's and DB's interests were reviewed and are managed by MGH and Partners HealthCare in accordance with their conflict-of-interest policies.

Extracellular UDP-sugars promote cellular responses by interacting with widely distributed P2Y 14 receptors, but the mechanisms by which these molecules are released from cells are poorly understood. Given the active role of UDP-sugars in glycosylation reactions within the secretory pathway, we hypothesized that UDP-sugar release includes an exocytotic component. This hypothesis was tested by assessing the contribution of endoplasmic reticulum (ER)/Golgi-resident UDPGlcNAc transporters to the cellular release of their cognate substrates. A sensitive and highly selective assay for UDP-GlcNAc mass was developed using purified AGX2, an isoenzyme of human UDP-GlcNAc pyrophosphorylase. Robust constitutive release of UDP-GlcNAc was observed in yeast as well as in well differentiated human airway epithelial cells. The human UDPGlcNAc transporter HFRC1 was overexpressed in human bronchial epithelial cells and was shown to localize in the Golgi and to enhance the surface expression of N-acetylglucosamine-rich glycans. HFRC1-overexpressing cells also displayed increased constitutive and hypotonic stress-stimulated release of UDPGlcNAc. Yeast mutants lacking Yea4 (the ER UDP-GlcNAc transporter endogenously expressed in Saccharomyces cerevisiae) showed reduced UDP-GlcNAc release. Yea4-deficient cells complemented with Yea4 showed UDP-GlcNAc release rates at levels similar to or higher than wild type cells. Our results illustrate that ER/Golgi lumen constitutes a significant source of extracellular UDP-sugars and therefore plays a critical role in nucleotide sugar-promoted cell signaling.Extracellular nucleotides perform important signaling functions via activation of broadly distributed P2X and P2Y purinergic receptors (1, 2). P2X receptors comprise seven species (P2X 1 -P2X 7 ) of ATP-gated ion channels. P2Y receptors belong to the superfamily of G protein-coupled receptors. At least eight human P2Y receptor species have been identified, seven of which (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13 ) are activated by adenine and/or uridine nucleoside di-and triphos-

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 UDPsugar 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 coinstilled 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.