Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide

The concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R), triggering ATP release via neuronal pannexin-1 channels that subsequently recruits intracellular calcium ([Ca(2+)]i) responses in the surrounding enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation.We studied neuroinflammation in vivo using the 2,4-dinitrobenzenesulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse intestine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP∷Cre (ERT2+/-)/Cx43(f/f) ] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO) synthase (iNOS (-/-)) were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Ca(2+) and NO imaging were used to monitor glial [Ca(2+)]i and [NO]i.Purinergic activation of enteric glia drove [Ca(2+)]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO.Our results show that the activation of glial cells in the context of neuroinflammation kills enteric neurons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute to the development of motility disorders

Expression of taste receptors in Solitary Chemosensory Cells of rodent airways

<p>Abstract</p> <p>Background</p> <p>Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs.</p> <p>Methods</p> <p>We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP).</p> <p>Results</p> <p>Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways.</p> <p>Conclusions</p> <p>Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.</p

Chemoreception Regulates Chemical Access to Mouse Vomeronasal Organ: Role of Solitary Chemosensory Cells

Controlling stimulus access to sensory organs allows animals to optimize sensory reception and prevent damage. The vomeronasal organ (VNO) detects pheromones and other semiochemicals to regulate innate social and sexual behaviors. This semiochemical detection generally requires the VNO to draw in chemical fluids, such as bodily secretions, which are complex in composition and can be contaminated. Little is known about whether and how chemical constituents are monitored to regulate the fluid access to the VNO. Using transgenic mice and immunolabeling, we found that solitary chemosensory cells (SCCs) reside densely at the entrance duct of the VNO. In this region, most of the intraepithelial trigeminal fibers innervate the SCCs, indicating that SCCs relay sensory information onto the trigeminal fibers. These SCCs express transient receptor potential channel M5 (TRPM5) and the phospholipase C (PLC) β2 signaling pathway. Additionally, the SCCs express choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) for synthesizing and packaging acetylcholine, a potential transmitter. In intracellular Ca2+ imaging, the SCCs responded to various chemical stimuli including high concentrations of odorants and bitter compounds. The responses were suppressed significantly by a PLC inhibitor, suggesting involvement of the PLC pathway. Further, we developed a quantitative dye assay to show that the amount of stimulus fluid that entered the VNOs of behaving mice is inversely correlated to the concentration of odorous and bitter substances in the fluid. Genetic knockout and pharmacological inhibition of TRPM5 resulted in larger amounts of bitter compounds entering the VNOs. Our data uncovered that chemoreception of fluid constituents regulates chemical access to the VNO and plays an important role in limiting the access of non-specific irritating and harmful substances. Our results also provide new insight into the emerging role of SCCs in chemoreception and regulation of physiological actions

Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death

Acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial cell damage, and loss of the alveolar-capillary barrier. We have previously shown that P2X7 receptor (P2X7R), a cell death receptor, is specifically expressed in alveolar epithelial type I cells (AEC I). In this study, we hypothesized that P2X7R-mediated purinergic signaling and its interaction with Wnt/B-catenin signaling contributes to AEC I death. We examined the effect of P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) and Wnt agonist Wnt3a on AEC I death in vitro and in vivo. We also assessed the therapeutic potential of Wnt3a in a clinically relevant ALI model of intratracheal lipopolysaccharide (LPS) exposure in ventilated mice. We found that the activation of P2X7R by BzATP caused the death of AEC I by suppressing Wnt/B-catenin signaling through stimulating glycogen synthase kinase-3B (GSK-3B) and proteasome. On the other hand, the activation of Wnt/B-catenin signaling by Wnt3a, GSK-3B inhibitor, or proteasome inhibitor blocked the P2X7R-mediated cell death. More importantly, Wnt3a attenuated the AEC I damage caused by intratracheal instillation of BzATP in rats or LPS in ventilated mice. Our results suggest that Wnt3a overrides the effect of P2X7R on the Wnt/B-catenin signaling to prevent the AEC I death and restrict the severity of ALI.Peer reviewedPhysiological Science

Neurogenic mechanisms in bladder and bowel ageing

The prevalence of both urinary and faecal incontinence, and also chronic constipation, increases with ageing and these conditions have a major impact on the quality of life of the elderly. Management of bladder and bowel dysfunction in the elderly is currently far from ideal and also carries a significant financial burden. Understanding how these changes occur is thus a major priority in biogerontology. The functions of the bladder and terminal bowel are regulated by complex neuronal networks. In particular neurons of the spinal cord and peripheral ganglia play a key role in regulating micturition and defaecation reflexes as well as promoting continence. In this review we discuss the evidence for ageing-induced neuronal dysfunction that might predispose to neurogenic forms of incontinence in the elderly

Regional heterogeneity of cholecystokinin sensing by enteric glia.

Enteric glia are peripheral glia associated with the enteric nervous system (ENS) that function to orchestrate a variety of integrated ENS functions related to the autonomic control of gastrointestinal homeostasis. Enteric glia are also a key component of a complex gut-brain neuroepithelial circuit by which the brain quickly perceives gut sensory cues. Transcriptomics data show that enteric glia express low levels of mRNA encoding cholecystokinin (CCK) receptors A and B in the colon (35.16% and 19.36%, respectively vs P2RY1 mRNA expression, a known glia-expressed gene) and suggest that enteric glia contribute to gut-brain signalling by sensing CCK. Here, we tested the hypothesis that enteric glia detect CCK and that glial responsiveness to CCK differs among gut regions. We assessed the effects of CCK on enteric glia by using in situ Ca2+ imaging in whole-mount preparations of myenteric plexus from Sox10CreERT2::Polr2atm1[CAG-GCaMP5g,-tdTomato]Tvrd mice that express the optogenetic probe GCaMP5g in enteric glial cells. A comparable percentage of glia responded to 100µM ADP in duodenum and colon (82.4% and 89.2%, respectively; n=120 glial cells in the duodenum and n=130 in the colon), but the percentage of glia responding to 100nM CCK was higher in the colon than in the duodenum (66.4% vs 38.3%, respectively). Interestingly, blocking neuronal activity with 300nM tetrodotoxin increased the percentage of glia responding to CCK in the duodenum, but not in the colon (57.1% in the colon vs 64.8% in the duodenum). Despite higher numbers of glia responding to CCK in the colon than duodenum, CCK resulted a greater peak Ca2+ response in the duodenum than in the colon when it is compared to ADP response peak (24.8% of ADP-induced response in the colon; 33.8% of ADP-induced response in the duodenum). Glial responses to CCK in the duodenum were potentiated by blocking neuronal activity with tetrodotoxin (30% of ADP-induced response in the colon; 93.3% of ADP-induced response in the duodenum). Together, these data show that enteric glia respond to CCK and that glial responses to CCK differ in duodenum and colon. Glial sensitivity to CCK involves signalling with neurons, suggesting a possible region-specific mechanism to locally modulate gut-brain

Clostridium difficile-related postinfectious IBS: a case of enteroglial microbiological stalking and/or the solution of a conundrum?

n/