Nucleotide receptors mediate protection against neonatal sepsis and meningitis caused by alpha-hemolysin expressing Escherichia coli K1.

Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.

Impact of P2X7 Purinoceptors on Goblet Cell Function: Implications for Dry Eye.

By providing ~70% of the eye's refractive power, the preocular tear film is essential for optimal vision. However, its integrity is often jeopardized by environmental and pathologic conditions that accelerate evaporation and cause sight-impairing dry eye. A key adaptive response to evaporation-induced tear film hyperosmolarity is the reflex-triggered release of tear-stabilizing mucin from conjunctival goblet cells. Here, we review progress in elucidating the roles of ion channels in mediating this important exocytotic response. Much is now known about the modulatory impact of ATP-sensitive potassium channels, nonspecific cation channels and voltage-gated calcium channels. Recently, we discovered that during unremitting extracellular hyperosmolarity, P2X7 receptor/channels also become activated and markedly impair goblet cell viability. However, our understanding of possible adaptive benefits of this P2X7 activation remains limited. In the present study, we utilized high-temporal resolution membrane capacitance measurements to monitor the exocytotic activity of single goblet cells located in freshly excised rat conjunctiva. We now report that activation of P2X7 purinoceptors boosts neural-evoked exocytosis and accelerates replenishment of mucin-filled granules after exocytotic depletion. Thus, P2X7 activation exerts a yin-yang effect on conjunctival goblet cells: the high-gain benefit of enhancing the supply of tear-stabilizing mucin is implemented at the high-risk of endangering goblet cell survival.

Impact of Diverse Ion Channels on Regulatory T Cell Functions.

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

ATP signaling in the integrative neural center of Aplysia californica.

ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica-a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis.

The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechanisms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1-deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA-activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immunosuppressive P2RX1-negative neutrophils.

Cross-Talk between P2X and NMDA Receptors.

Purinergic P2X receptors (P2X) are ATP-gated ion channels widely expressed in the CNS. While the direct contribution of P2X to synaptic transmission is uncertain, P2X reportedly affect N-methyl-D-aspartate receptor (NMDAR) activity, which has given rise to competing theories on the role of P2X in the modulation of synapses. However, P2X have also been shown to participate in receptor cross-talk: an interaction where one receptor (e.g., P2X2) directly influences the activity of another (e.g., nicotinic, 5-HT3 or GABA receptors). In this study, we tested for interactions between P2X2 or P2X4 and NMDARs. Using two-electrode voltage-clamp electrophysiology experiments in Xenopus laevis oocytes, we demonstrate that both P2X2 and P2X4 interact with NMDARs in an inhibited manner. When investigating the molecular domains responsible for this phenomenon, we found that the P2X2 c-terminus (CT) could interfere with both P2X2 and P2X4 interactions with NMDARs. We also report that 11 distal CT residues on the P2X4 facilitate the P2X4-NMDAR interaction, and that a peptide consisting of these P2X4 CT residues (11C) can disrupt the interaction between NMDARs and P2X2 or P2X4. Collectively, these results provide new evidence for the modulatory nature of P2X2 and P2X4, suggesting they might play a more nuanced role in the CNS.

TMEM163 Regulates ATP-Gated P2X Receptor and Behavior.

Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs in vivo and a potential target for the discovery of drugs for treating pain.

Comparison of mRNA Expression of P2X Receptor Subtypes in Different Arterial Tissues of Rats.

This study aims to compare the expression of P2X receptor subtype mRNA in different arterial tissues of rats. After the rats were sacrificed, the internal carotid, pulmonary, thoracic aorta, mesenteric and caudal arteries were dissected out. Then, the P2X receptor mRNA expression in different blood vessels was detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction. The P2X1, P2X4 and P2X7 receptor mRNA amplification products revealed specific bands of the same size as the amplified target fragment in their respective lanes, while the P2X2, P2X3, P2X5 and P2X6 receptor mRNA amplification products did not reveal significant specific bands in their respective lanes by RT-PCR. Based on the P2X1 receptor mRNA expression of the mesenteric artery, there were no significant differences in the internal carotid, pulmonary and thoracic aorta (0.64 ± 0.07, 0.17 ± 0.11 and 1.49 ± 0.65, respectively). However, the P2X1 receptor mRNA expression level in the caudal artery significantly increased (11.06 ± 1.99, P < 0.01). Furthermore, there was no difference in P2X4 receptor mRNA expression among these five blood vessels (P > 0.05). The P2X7 receptor mRNA expression level was significantly different: pulmonary artery < tail artery = thoracic aorta < internal carotid artery < mesenteric artery. The relative P2X1 receptor mRNA expression in the caudal artery was observed to be elevated when compared to that of the internal carotid, pulmonary and thoracic aorta as well as the mesenteric arteries. The P2X7 receptor mRNA expression level is pulmonary artery < caudal artery = thoracic aorta < internal carotid artery < mesenteric artery. P2X4 receptor mRNA expression was not significantly different among these five blood vessels.

Heterologous Expression and Patch-Clamp Recording of P2X Receptors in HEK293 Cells.

P2X receptors (P2XRs) are ligand-gated ion channels gated by extracellular adenosine 5'-triphosphate (ATP) and play a critical role in mediating ATP-induced purinergic signaling in physiological and pathological processes. Heterologous expression of P2XR in human embryonic kidney 293 (HEK293) cells and measurement of P2XR-mediated currents using patch-clamp recording technique have been widely used to study the biophysical and pharmacological properties of these receptors. Combination of electrophysiology with site-directed mutagenesis and structural information has shed light on the molecular basis for receptor activation and mechanisms of actions by receptor antagonists and modulators. It is anticipated that such methodologies will continue helping us to provide more mechanistic understanding of P2XRs and to test novel receptor antagonists and allosteric modulators for therapeutical purposes. In this chapter, we describe protocols of transiently or stably expressing the P2XR in HEK293 cells and measuring P2XR-mediated currents by using whole-cell recording.

Purinergic receptor mediated calcium signalling in urothelial cells.

Non-neuronal ATP released from the urothelium in response to bladder stretch is a key modulator of bladder mechanosensation. Whilst non-neuronal ATP acts on the underlying bladder afferent nerves to facilitate sensation, there is also the potential for ATP to act in an autocrine manner, modulating urothelial cell function. The aim of this study was to systematically characterise the functional response of primary mouse urothelial cells (PMUCs) to ATP. PMUCs isolated from male mice (14-16 weeks) were used for live-cell fluorescent calcium imaging and qRT-PCR to determine the expression profile of P2X and P2Y receptors. The majority of PMUCs (74-92%) responded to ATP (1 µM-1 mM), as indicted by an increase in intracellular calcium (iCa2+). PMUCs exhibited dose-dependent responses to ATP (10 nM-1 mM) in both calcium containing (2 mM, EC50 = 3.49 ± 0.77 µM) or calcium free (0 mM, EC50 = 9.5 ± 1.5 µM) buffers. However, maximum iCa2+ responses to ATP were significantly attenuated upon repetitive applications in calcium containing but not in calcium free buffer. qRT-PCR revealed expression of P2X1-6, and P2Y1-2, P2Y4, P2Y6, P2Y11-14, but not P2X7 in PMUCs. These findings suggest the major component of ATP induced increases in iCa2+ are mediated via the liberation of calcium from intracellular stores, implicating functional P2Y receptors that are ubiquitously expressed on PMUCs.

Role of opioid receptors in modulation of P2X receptor-mediated cardiac sympathoexcitatory reflex response.

Myocardial ischemia evokes powerful reflex responses through activation of vagal and sympathetic afferents in the heart through the release of ischemic metabolites. We have demonstrated that extracellular ATP stimulates cardiac sympathetic afferents through P2 receptor-mediated mechanism, and that opioid peptides suppress these afferents' activity. However, the roles of both P2 receptor and endogenous opioids in cardiac sympathoexcitatory reflex (CSR) responses remain unclear. We therefore hypothesized that activation of cardiac P2 receptor evokes CSR responses by stimulating cardiac sympathetic afferents and these CSR responses are modulated by endogenous opioids. We observed that intrapericardial injection of α,ß-methylene ATP (α,ß-meATP, P2X receptor agonist), but not ADP (P2Y receptor agonist), caused a graded increase in mean arterial pressure in rats with sinoaortic denervation and vagotomy. This effect of α,ß-meATP was abolished by blockade of cardiac neural transmission with intrapericardial procaine treatment and eliminated by intrapericardial A-317491, a selective P2X2/3 and P2X3 receptor antagonist. Intrapericardial α,ß-meATP also evoked CSR response in vagus-intact rats. Furthermore, the P2X receptor-mediated CSR responses were enhanced by intrapericardial naloxone, a specific opioid receptor antagonist. These data suggest that stimulation of cardiac P2X2/3 and P2X3, but not P2Y receptors, powerfully evokes CSR responses through activation of cardiac spinal afferents, and that endogenous opioids suppress the P2X receptor-mediated CSR responses.

The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

Characterization of the antagonist actions of 5-BDBD at the rat P2X4 receptor.

P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC50 of 0.75 µM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 µM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 µM concentration but not 0.75 µM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.

Adherent-Invasive E. coli enhances colonic hypersensitivity and P2X receptors expression during post-infectious period.

Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) are related gastrointestinal disorders characterized by abdominal pain associated with colonic hypersensitivity (CHS). Studies in humans have reported an abnormal colonization of Adherent-Invasive E. coli (AIEC) in the ileum of Crohn's disease (CD) patients associated with overexpression of the bacterial colonizing receptor CEACAM6. The aim of the present study was to investigate whether AIEC reference strain LF82 could induce intestinal impairment during infectious and/or post-infectious periods and subsequently the development of CHS. Transgenic mice overexpressing human CEACAM6 protein (TG) and their wild-type littermates were gavaged by CD-associated AIEC bacteria (reference strain LF82) or PBS for 3 d. Colonic hypersensitivity was assessed by colorectal distension (CRD) test during infectious (D4) and post-infectious periods (D21). Several markers of intestinal inflammation were monitored and the colonic expression of purinergic P2X receptors was quantified. At D4, an increased visceromotor response (VMR) to the CRD test was observed in TG mice infected with CD-associated AIEC LF82 in comparison with non-infected TG mice and persisted in a subgroup of infected animals at D21 after bacteria clearance. Increased VMR was associated with low-grade intestinal inflammation, increased intestinal permeability and expression of P2X 3, 4 and 7. This study shows that certain susceptible hosts infected with CD-associated AIEC bacteria can develop persistent CHS associated with low-grade inflammation and increased P2X receptors expression. Thus, CD-associated AIEC infection in CEACAM6 transgenic mice could be used as a novel post-infectious mouse model mimicking quiescent IBD with IBS-like symptoms such as visceral pain.

Interactions of Pannexin1 channels with purinergic and NMDA receptor channels.

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

The therapeutic potential of purinergic signalling.

This review is focused on the pathophysiology and therapeutic potential of purinergic signalling. A wide range of diseases are considered, including those of the central nervous system, skin, kidney, musculoskeletal, liver gut, lower urinary tract, cardiovascular, airways and reproductive systems, the special senses, infection, diabetes and obesity. Several purinergic drugs are already on the market, including P2Y12 receptor antagonists for stroke and thrombosis, P2Y2 receptor agonists for dry eye, and A1 receptor agonists for supraventricular tachycardia. Clinical trials are underway for the use of P2X3 receptor antagonists for the treatment of chronic cough, visceral pain and hypertension, and many more compounds are being explored for the treatment of other diseases. Most experiments are 'proof of concept' studies on animal or cellular models, which hopefully will lead to further clinical trials. The review summarises the topic, mostly referring to recent review articles.

P2X receptor overexpression induced by soluble oligomers of amyloid beta peptide potentiates synaptic failure and neuronal dyshomeostasis in cellular models of Alzheimer's disease.

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aß). Because Aß increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aß and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aß oligomers (0.5 µM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aß. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aß. Taken together, our findings suggest that Aß disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.

P2X Receptor Activation.

Extracellular ATP-gated P2X receptors are trimeric non-selective cation channels important for many physiological events including immune response and neural transmission. These receptors belong to a unique class of ligand-gated ion channels composed of only six transmembrane helices and a relatively small extracellular domain that harbors three ATP-binding pockets. The crystal structures of P2X receptors, including the recent P2X3 structures representing three different stages of the gating cycle, have provided a compelling structural foundation for understanding how this class of ligand-gated ion channels function. These structures, in combination with numerous functional studies ranging from classic mutagenesis and electrophysiology to modern optogenetic pharmacology, have uncovered unique molecular mechanisms of P2X receptor function. This review article summarizes the current knowledge in P2X receptor activation, especially focusing on the mechanisms underlying ATP-binding, conformational changes in the extracellular domain, and channel gating and desensitization.

CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A2BR signaling, forming a positive-feedback loop. A2BR activation, in addition, strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X7R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

Morphine is one of the most widely used drugs for the treatment of pain. However, side effects, including persistent constipation and antinociceptive tolerance, limit its clinical efficacy. Prolonged morphine treatment results in a "leaky" gut, predisposing to colonic inflammation that is facilitated by microbial dysbiosis and associated bacterial translocation. In this study, we examined the role of enteric glia in mediating this secondary inflammatory response to prolonged treatment with morphine. We found that purinergic P2X receptor activity was significantly enhanced in enteric glia that were isolated from mice with long-term morphine treatment (in vivo) but not upon direct exposure of glia to morphine (in vitro). LPS, a major bacterial product, also increased ATP-induced currents, as well as expression of P2X4, P2X7, IL6, IL-1ß mRNA in enteric glia. LPS increased connexin43 (Cx43) expression and enhanced ATP release from enteric glia cells. LPS-induced P2X currents and proinflammatory cytokine mRNA expression were blocked by the Cx43 blockers Gap26 and carbenoxolone. Likewise, colonic inflammation related to prolonged exposure to morphine was significantly attenuated by carbenoxolone (25 mg/kg). Carbenoxolone also prevented gut wall disruption and significantly reduced morphine-induced constipation. These findings imply that enteric glia activation is a significant modulator of morphine-related inflammation and constipation.-Bhave, S., Gade, A., Kang, M., Hauser, K. F., Dewey, W. L., Akbarali, H. I. Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.