ORCID Profile
0000-0002-6168-8422
Current Organisation
Monash University
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Nanotechnology | Nanobiotechnology
Publisher: European Respiratory Society (ERS)
Date: 2018
DOI: 10.1183/23120541.00127-2017
Abstract: Ivacaftor–lumacaftor and ivacaftor are two new breakthrough cystic fibrosis transmembrane conductance modulators. The interactions of ivacaftor and its two metabolites hydroxymethylivacaftor (iva-M1) and ivacaftorcarboxylate (iva-M6) with neurotransmitter receptors were investigated in radioligand binding assays. Ivacaftor displayed significant affinity to the 5-hydroxytryptamine (5-HT serotonin) 5-HT 2C receptor (p K i =6.06±0.03), β 3 -adrenergic receptor (p K i =5.71±0.07), δ-opioid receptor (p K i =5.59±0.06) and the dopamine transporter (p K i =5.50±0.20) iva-M1 displayed significant affinity to the 5-HT 2C receptor (p K i =5.81±0.04) and the muscarinic M3 receptor (p K i =5.70±0.10) iva-M6 displayed significant affinity to the 5-HT 2A receptor (p K i =7.33±0.05). The in vivo central nervous system activity of ivacaftor (40 mg·kg −1 intraperitoneally for 21 days) was assessed in a chronic mouse model of depression. In the forced swim test, the ivacaftor-treated group displayed decreased immobility (52.8±7.6 s), similarly to fluoxetine (33.8±11.0 s), and increased climbing/swimming activity (181.5±9.2 s). In the open field test, ivacaftor produced higher locomotor activity than the fluoxetine group, measured both as mean number of paw touches (ivacaftor 81.1±9.6 versus fluoxetine 57.9±9.5) and total distance travelled (ivacaftor 120.6±16.8 cm versus fluoxetine 84.5±16.0 cm) in 600 s. Treatment of 23 cystic fibrosis patients with ivacaftor–lumacaftor resulted in significant improvements in quality of life (including anxiety) in all five domains of the AweScoreCF questionnaire (p=0.092–0.096). Our findings suggest ivacaftor displays potential clinical anxiolytic and stimulating properties, and may have beneficial effects on mood.
Publisher: Elsevier BV
Date: 09-2020
Publisher: American Physiological Society
Date: 09-2010
Abstract: The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca 2+ concentration ([Ca 2+ ] i ) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca 2+ ] i , pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.
Publisher: Elsevier BV
Date: 08-2009
Publisher: American Physiological Society
Date: 15-08-2015
Abstract: Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK 1 R) in enteric neurons. We studied NK 1 R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10 −/− -piroxicam and trinitrobenzenesulfonic acid colitis models. NK 1 R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK 1 R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK 1 R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK 1 R endocytosis in myenteric but not submucosal neurons. Whereas the NK 1 R in uninflamed neurons recycled within 60 min, NK 1 R recycling in inflamed neurons was delayed for min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK 1 R recycling. We conclude that inflammation evokes redistribution of NK 1 R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK 1 R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK 1 R may generate sustained proinflammatory signals that disrupt normal neuronal functions.
Publisher: Proceedings of the National Academy of Sciences
Date: 02-02-2022
Abstract: G protein–coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR 2 ) in colitis. To localize PAR 2 and assess redistribution during disease, we generated knockin mice expressing PAR 2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR 2 -muGFP signaled and trafficked normally. PAR 2 messenger RNA was detected at similar levels in Par 2 -mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR 2 ) and Gfp probes revealed that PAR 2 -muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR 2 -muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR 2 -muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR 2 . PAR 2 agonists stimulated endocytosis of PAR 2 and recruitment of Gα q , Gα i , and β-arrestin to early endosomes of T84 colon carcinoma cells. PAR 2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 ( Dnm2 ), the major colonocyte isoform, and Dnm inhibition attenuated PAR 2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR 2 endocytosis sustains protease-evoked inflammation and nociception and PAR 2 in endosomes is a potential therapeutic target for colitis.
Publisher: Frontiers Media SA
Date: 20-01-2022
DOI: 10.3389/FIMMU.2021.828115
Abstract: Transient receptor potential vanilloid 4 (TRPV4) is a non-selective mechanosensitive ion channel expressed by various macrophage populations. Recent reports have characterized the role of TRPV4 in shaping the activity and phenotype of macrophages to influence the innate immune response to pathogen exposure and inflammation. TRPV4 has been studied extensively in the context of inflammation and inflammatory pain. Although TRPV4 activity has been generally described as pro-inflammatory, emerging evidence suggests a more complex role where this channel may also contribute to anti-inflammatory activities. However, detailed understanding of how TRPV4 may influence the initiation, maintenance, and resolution of inflammatory disease remains limited. This review highlights recent insights into the cellular processes through which TRPV4 contributes to pathological conditions and immune processes, with a focus on macrophage biology. The potential use of high-throughput and omics methods as an unbiased approach for studying the functional outcomes of TRPV4 activation is also discussed.
Publisher: American Physiological Society
Date: 2021
Abstract: This study assesses the use of positive allosteric modulation as a pharmacological approach to enhance opioid receptor signaling in the enteric nervous system. We demonstrate that selective modulation of endogenous delta opioid receptor signaling can suppress colonic motility without causing constipation. We propose that allosteric modulation of opioid receptor signaling may be a therapeutic strategy to normalize gastrointestinal motility in conditions such as irritable bowel syndrome.
Publisher: Elsevier BV
Date: 10-2002
DOI: 10.1016/S1566-0702(02)00179-0
Abstract: Adenosine 5'-triphosphate (ATP) excites 70-90% of enteric neurons through P2X type purine receptors, and is likely to be an enteric neurotransmitter. Recent studies indicate that the P2X2 subunit is expressed by specific subgroups of enteric neurons, and that there are enteric neurons that are responsive to ATP but lack this subunit. In the present work, we have investigated whether the P2X3 subunit is similarly localised to specific subgroups of neurons, and whether these are different from the P2X2 subunit-expressing neurons. The P2X3 subunit was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines, and nerve cells of the submucosal ganglia in the small and large intestines of the guinea pig. All immunoreactivity was absorbed with the P2X3 receptor peptide against which the antiserum was raised. In myenteric ganglia of the ileum, P2X3 receptor immunoreactivity was in calretinin, enkephalin and nitric oxide synthase (NOS)-immunoreactive neurons. In submucosal ganglia, all calretinin-immunoreactive nerve cells were P2X3 receptor immunoreactive. In the submucosal ganglia of the ileum, 13 +/- 3% of neuropeptide Y (NPY)-immunoreactive neurons were also P2X3 receptor immunoreactive, whereas in the distal colon, almost all NPY-expressing nerve cells were P2X3 receptor immunoreactive. The localisation of the P2X3 subunit was largely distinct from that of the P2X2 subunit, although both subunits occur in some NOS neurons, where P2X2 and P2X3 subunits may form heteromeric receptors. Unlike the P2X2 subunit, the P2X3 subunit is not expressed in intrinsic sensory neurons in the ileum. It is concluded that the P2X3 receptor subunit is expressed in specific functional groups of neurons the major types are excitatory and inhibitory muscle motor neurons, ascending interneurons and cholinergic secretomotor neurons.
Publisher: Elsevier BV
Date: 02-2013
Publisher: Wiley
Date: 12-03-2010
Publisher: Cold Spring Harbor Laboratory
Date: 20-11-2020
DOI: 10.1101/2020.11.19.386565
Abstract: Modern life science relies heavily on fluorescent microscopy and subsequent quantitative bio-image analysis. The current rise of graphics processing units (GPUs) in the context of image processing enables batch processing large amounts of image data at unprecedented speed. In order to facilitate adoption of this technology in daily practice, we present an expert system based on the GPU-accelerated image processing library CLIJ: The CLIJ-assistant keeps track of which operations formed an image and suggests subsequent operations. It enables new ways of interaction with image data and image processing operations because its underlying GPU-accelerated image data flow graphs (IDFGs) allow changes to parameters of early processing steps and instantaneous visualization of their final results. Operations, their parameters and connections in the IDFG are stored at any point in time enabling the CLIJ-assistant to offer an undo-function for virtually unlimited rewinding parameter changes. Furthermore, to improve reproducibility of image data analysis workflows and interoperability with established image analysis platforms, the CLIJ-assistant can generate code from IDFGs in programming languages such as ImageJ Macro, Java, Jython, JavaScipt, Groovy, Python and C++ for later use in ImageJ, Fiji, Icy, Matlab, QuPath, Jupyter Notebooks and Napari. We demonstrate the CLIJ-assistant for processing image data in multiple scenarios to highlight its general applicability. The CLIJ-assistant is open source and available online: clij.github.io/assistant/
Publisher: Elsevier BV
Date: 06-2022
DOI: 10.1016/J.BIOMATERIALS.2022.121536
Abstract: Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NK
Publisher: Elsevier BV
Date: 07-2014
Publisher: Springer Science and Business Media LLC
Date: 02-09-2015
DOI: 10.1007/S00109-015-1336-5
Abstract: Recent studies implicate TRPV4 receptors in visceral pain signaling and intestinal inflammation. Our aim was to evaluate the role of TRPV4 in the control of gastrointestinal (GI) motility and to establish the underlying mechanisms. We used immunohistochemistry and PCR to study TRPV4 expression in the GI tract. The effect of TRPV4 activation on GI motility was characterized using in vitro and in vivo motility assays. Calcium and nitric oxide (NO) imaging were performed to study the intracellular signaling pathways. Finally, TRPV4 expression was examined in the colon of healthy human subjects. We demonstrated that TRPV4 can be found on myenteric neurons of the colon and is co-localized with NO synthase (NOS-1). In vitro, the TRPV4 agonist GSK1016790A reduced colonic contractility and increased inhibitory neurotransmission. In vivo, TRPV4 activation slowed GI motility and reduced stool production in mouse models mimicking pathophysiological conditions. We also showed that TRPV4 activation inhibited GI motility by reducing NO-dependent Ca(2+) release from enteric neurons. In conclusion, TRPV4 is involved in the regulation of GI motility in health and disease. • Recent studies implicate TRPV4 in pain signaling and intestinal inflammation. • Our aim was to characterize the role of TRPV4 in the control of GI motility. • We found that TRPV4 activation reduced colonic contractility. • Our studies also showed altered TRPV4 mRNA expression in IBS-C patients. • TRPV4 may be a novel pharmacological target in functional GI diseases.
Publisher: Proceedings of the National Academy of Sciences
Date: 30-10-2017
Abstract: G protein-coupled receptors (GPCRs) have long been considered to function primarily at the plasma membrane. Consequently, most drugs are designed to target GPCRs at the cell surface. Ligand-bound GPCRs undergo clathrin- and dynamin-dependent endocytosis. It is uncertain whether GPCRs in endosomes control complex pathophysiological processes in vivo and are a viable therapeutic target. We report that the CGRP receptor signals from endosomes to regulate activity of pain-transmitting neurons in the spinal cord. Lipid-conjugated CGRP receptor antagonists accumulate in endosomes, selectively inhibit endosomal signals, and block sustained excitation of spinal neurons and persistent nociception. The results suggest that GPCRs in endosomes, in addition to those at the cell surface, control ongoing pathophysiological processes in vivo and identify GPCRs in endosomes as a new target for therapy.
Publisher: Springer Science and Business Media LLC
Date: 16-09-2019
DOI: 10.1038/S41598-019-49840-4
Abstract: Neutrophil elastase is a serine protease that has been implicated in the pathogenesis of inflammatory bowel disease. Due to post-translational control of its activation and high expression of its inhibitors in the gut, measurements of total expression poorly reflect the pool of active, functional neutrophil elastase. Fluorogenic substrate probes have been used to measure neutrophil elastase activity, though these tools lack specificity and traceability. PK105 is a recently described fluorescent activity-based probe, which binds to neutrophil elastase in an activity-dependent manner. The irreversible nature of this probe allows for accurate identification of its targets in complex protein mixtures. We describe the reactivity profile of PK105b, a new analogue of PK105, against recombinant serine proteases and in tissue extracts from healthy mice and from models of inflammation induced by oral cancer and Legionella pneumophila infection. We apply PK105b to measure neutrophil elastase activation in an acute model of experimental colitis. Neutrophil elastase activity is detected in inflamed, but not healthy, colons. We corroborate this finding in mucosal biopsies from patients with ulcerative colitis. Thus, PK105b facilitates detection of neutrophil elastase activity in tissue lysates, and we have applied it to demonstrate that this protease is unequivocally activated during colitis.
Publisher: Springer Science and Business Media LLC
Date: 07-2003
DOI: 10.1007/S00418-003-0541-4
Abstract: C kinases (PKCs) are a family of enzymes essential for the transduction of signals in a erse range of cell types, including neurons. The different isoforms vary in their activation requirements. Therefore, cell-specific expression of different isoforms has implications for PKC-mediated control of organ function. This study has investigated the types and distributions of PKC isoforms in the small intestine of the guinea-pig, with particular emphasis on their localisation in myenteric neurons, using immunohistochemistry and western blotting techniques. Three PKC isoforms, gamma, eta and theta, were detected in the calbindin-immunoreactive subset of intrinsic primary afferent neurons, but not in other myenteric neurons. Both gamma and theta immunoreactivities were also located in interstitial cells of Cajal. In contrast to these isoforms, immunoreactivity for PKCs lambda and epsilon was present in all myenteric neurons of the ileum. PKCalpha immunoreactivity was detected primarily in the glial network, as shown through double labelling with antibodies to the glial filament protein, S100b. Myenteric neurons were also weakly immunoreactive for this isoform. PKCdelta immunoreactivity was very highly expressed in smooth muscle, but was largely absent from neurons. Immunoreactivity for RACK1, a binding protein for PKCbeta, was detected in both calbindin-immunoreactive neurons and in smooth muscle cells. This study indicates a selective distribution of PKC isoforms to specific cell types. Isoform-specific activity of these enzymes could provide a means through which targeted modulation of intestinal function is achieved.
Publisher: Proceedings of the National Academy of Sciences
Date: 16-06-2020
Abstract: G protein-coupled receptors are considered to function principally at the cell surface. We present evidence that the δ-opioid receptor (DOPr) signals from endosomes to cause a sustained inhibition of pain. Opioids from the inflamed human and mouse colon, along with selective agonists that evoked DOPr internalization, inhibited the excitability of nociceptors by a mechanism requiring DOPr endocytosis. DOPr in endosomes generated a subset of signals in subcellular compartments that inhibited neuronal excitability. A DOPr agonist that was encapsulated into nanoparticles designed to selectively activate DOPr in endosomes of nociceptors caused a long-lasting inhibition of neuronal excitability and pain. Our results support the hypothesis that endosomal signaling of DOPr is an endogenous mechanism and therapeutic target for relief from inflammatory pain.
Publisher: Wiley
Date: 02-2005
DOI: 10.1111/J.1460-9568.2005.03931.X
Abstract: AH neurons in the enteric nervous system play an essential role in initiating intestinal reflexes and factors that control AH neuron excitability therefore influence the state of the digestive system. Prominent afterhyperpolarizations that follow action potentials in these neurons strongly affect their excitability. In the present work, we have investigated the regulation of the afterhyperpolarizing current (I(AHP)) by protein kinase C (PKC). Electrophysiological responses and protein translocation were investigated in AH neurons of freshly dissected preparations of myenteric ganglia from the guinea-pig ileum. The activator of conventional and novel PKCs, phorbol dibutyrate, but not the activator of novel PKCs, ingenol, blocked the I(AHP). Phorbol dibutyrate had no effect on the hyperpolarization-activated current (I(h)) or on the A current (I(A)). Stimulation of synaptic inputs to the neurons also reduced the I(AHP), and had no effect on I(h) or I(A). Phorbol dibutyrate also reduced a background outward current that was present after the I(AHP) current had been blocked by clotrimazole. Both phorbol dibutyrate and ingenol caused translocation of the novel PKC, PKCepsilon, in these neurons. Only phorbol dibutyrate caused translocation of PKCgamma, a conventional PKC. The studies thus indicate that the activation of PKC by phorbol esters and by nerve stimulation affects AH neurons in a similar way, and that PKC activation targets both the I(AHP) and another background K(+) current. The I(AHP) is targeted by a conventional PKC, suggested to be PKCgamma, as this is the only conventional PKC that is prominent in AH neurons.
Publisher: Springer International Publishing
Date: 2016
DOI: 10.1007/978-3-319-27592-5_14
Abstract: G protein-coupled receptors (GPCRs) enable cells to detect and respond to changes in their extracellular environment. With over 800 members, the GPCR family includes receptors for a erse range of agonists including olfactants, neurotransmitters and hormones. Importantly, GPCRs represent a major therapeutic target, with approximately 50 % of all current drugs acting at some aspect of GPCR signalling (Audet and Bouvier 2008). GPCRs are widely expressed by all cell types in the gastrointestinal (GI) tract and are major regulators of every aspect of gut function. Many GPCRs are internalised upon activation, and this represents one of the mechanisms through which G protein-signalling is terminated. The latency between the endocytosis of GPCRs and their recycling and resensitization is a major determinant of the cell's ability to respond to subsequent exposure to agonists.
Publisher: Proceedings of the National Academy of Sciences
Date: 16-07-2018
Abstract: Activated G protein-coupled receptors (GPCRs) internalize and can continue to signal from endosomes. The contribution of endosomal signaling to human disease is unknown. Proteases that are generated in the colon of patients with irritable bowel syndrome (IBS) can cleave protease-activated receptor-2 (PAR 2 ) on nociceptors to cause pain. We evaluated whether PAR 2 generates signals in endosomes of nociceptors that mediate persistent hyperexcitability and pain. Biopsies of colonic mucosa from IBS patients released proteases that induced PAR 2 endocytosis, endosomal signaling, and persistent hyperexcitability of nociceptors. When conjugated to the transmembrane lipid cholestanol, PAR 2 antagonists accumulated in endosomes and suppressed persistent hyperexcitability. The results reveal the therapeutic potential of endosomally targeted PAR 2 antagonists for IBS pain, and expand the contribution of endosomal GPCR signaling to encompass processes that are relevant to disease.
Publisher: Elsevier
Date: 2012
Publisher: Portland Press Ltd.
Date: 29-01-2013
DOI: 10.1042/BST20120343
Abstract: GPCR (G-protein-coupled receptor) signalling at the plasma membrane is under tight control. In the case of neuropeptides such as SP (substance P), plasma membrane signalling is regulated by cell-surface endopeptidases (e.g. neprilysin) that degrade extracellular neuropeptides, and receptor interaction with β-arrestins, which uncouple receptors from heterotrimeric G-proteins and mediate receptor endocytosis. By recruiting GPCRs, kinases and phosphatases to endocytosed GPCRs, β-arrestins assemble signalosomes that can mediate a second wave of signalling by internalized receptors. Endosomal peptidases, such as ECE-1 (endothelin-converting enzyme-1), can degrade SP in acidified endosomes, which destabilizes signalosomes and allows receptors, freed from β-arrestins, to recycle and resensitize. By disassembling signalosomes, ECE-1 terminates β-arrestin-mediated endosomal signalling. These mechanisms have been studied in model cell systems, and the relative importance of plasma membrane and endosomal signalling to complex pathophysiological processes, such as inflammation, pain and proliferation, is unclear. However, deletion or inhibition of metalloendopeptidases that control neuropeptide signalling at the plasma membrane and in endosomes has marked effects on inflammation. Neprilysin deletion exacerbates inflammation because of diminished degradation of pro-inflammatory SP. Conversely, inhibition of ECE-1 attenuates inflammation by preventing receptor recycling/resensitization, which is required for sustained pro-inflammatory signals from the plasma membrane. β-Arrestin deletion also affects inflammation because of the involvement of β-arrestins in pro-inflammatory signalling and migration of inflammatory cells. Knowledge of GPCR signalling in specific subcellular locations provides insights into pathophysiological processes, and can provide new opportunities for therapy. Selective targeting of β-arrestin-mediated endosomal signalling or of mechanisms of receptor recycling/resensitization may offer more effective and selective treatments than global targeting of cell-surface signalling.
Publisher: American Physiological Society
Date: 10-2018
Abstract: Endogenous opioids activate opioid receptors (ORs) in the enteric nervous system to control intestinal motility and secretion. The μ-OR mediates the deleterious side effects of opioid analgesics, including constipation, respiratory depression, and addiction. Although the δ-OR (DOR) is a promising target for analgesia, the function and regulation of DOR in the colon are poorly understood. This study provides evidence that endogenous opioids activate DOR in myenteric neurons that may regulate colonic motility. The DOR agonists DADLE, deltorphin II, and SNC80 inhibited electrically evoked contractions and induced neurogenic contractions in the mouse colon. Electrical, chemical, and mechanical stimulation of the colon evoked the release of endogenous opioids, which stimulated endocytosis of DOR in the soma and proximal neurites of myenteric neurons of transgenic mice expressing DOR fused to enhanced green fluorescent protein. In contrast, DOR was not internalized in nerve fibers within the circular muscle. Administration of dextran sulfate sodium induced acute colitis, which was accompanied by DOR endocytosis and an increased density of DOR-positive nerve fibers within the circular muscle. The potency with which SNC80 inhibited neurogenic contractions was significantly enhanced in the inflamed colon. This study demonstrates that DOR-expressing neurons in the mouse colon can be activated by exogenous and endogenous opioids. Activated DOR traffics to endosomes and inhibits neurogenic motility of the colon. DOR signaling is enhanced during intestinal inflammation. This study demonstrates functional expression of DOR by myenteric neurons and supports the therapeutic targeting of DOR in the enteric nervous system. NEW & NOTEWORTHY DOR is activated during physiologically relevant reflex stimulation. Agonist-evoked DOR endocytosis is spatially and temporally regulated. A significant proportion of DOR is internalized in myenteric neurons during inflammation. The relative proportion of all myenteric neurons that expressed DOR and the overlap with the nNOS-positive population are increased in inflammation. DOR-specific innervation of the circular muscle is increased in inflammation, and this is consistent with enhanced responsiveness to the DOR agonist SNC80.
Publisher: American Society for Clinical Investigation
Date: 17-10-2019
Publisher: Wiley
Date: 07-07-2009
DOI: 10.1002/CNE.22104
Publisher: The American Association of Immunologists
Date: 05-2017
Abstract: G-CSF or CSF-3, originally defined as a regulator of granulocyte lineage development via its cell surface receptor (G-CSFR), can play a role in inflammation, and hence in many pathologies, due to its effects on mature lineage populations. Given this, and because pain is an extremely important arthritis symptom, the efficacy of an anti–G-CSFR mAb for arthritic pain and disease was compared with that of a neutrophil-depleting mAb, anti-Ly6G, in both adaptive and innate immune-mediated murine models. Pain and disease were ameliorated in Ag-induced arthritis, zymosan-induced arthritis, and methylated BSA/IL-1 arthritis by both prophylactic and therapeutic anti–G-CSFR mAb treatment, whereas only prophylactic anti-Ly6G mAb treatment was effective. Efficacy for pain and disease correlated with reduced joint neutrophil numbers and, importantly, benefits were noted without necessarily the concomitant reduction in circulating neutrophils. Anti–G-CSFR mAb also suppressed zymosan-induced inflammatory pain. A new G-CSF–driven (methylated BSA/G-CSF) arthritis model was established enabling us to demonstrate that pain was blocked by a cyclooxygenase-2 inhibitor, suggesting an indirect effect on neurons. Correspondingly, dorsal root ganglion neurons cultured in G-CSF failed to respond to G-CSF in vitro, and Csf3r gene expression could not be detected in dorsal root ganglion neurons by single-cell RT-PCR. These data suggest that G-CSFR/G-CSF targeting may be a safe therapeutic strategy for arthritis and other inflammatory conditions, particularly those in which pain is important, as well as for inflammatory pain per se.
Publisher: American Physiological Society
Date: 15-04-2014
Abstract: Lymphatic fluid is a plasma filtrate that can be viewed as having biological activity through the passive accumulation of molecules from the interstitial fluid. The possibility that lymphatic fluid is part of an active self-contained signaling process that parallels the endocrine system, through the activation of G-protein coupled receptors (GPCR), has remained unexplored. We show that the GPCR lysophosphatidic acid 5 (LPA5) is found in sensory nerve fibers expressing calcitonin gene-related peptide (CGRP) that innervate the lumen of lymphatic lacteals and enteric nerves. Using LPA5 as a model for nutrient-responsive GPCRs present on sensory nerves, we demonstrate that dietary protein hydrolysate (peptone) can induce c-Fos expression in enterocytes and nerves that express LPA5. Mesenteric lymphatic fluid (MLF) mobilizes intracellular calcium in cell models expressing LPA5 upon feeding in a time- and dose-dependent manner. Primary cultured neurons of the dorsal root ganglia expressing CGRP are activated by MLF, which is enhanced upon LPA5 overexpression. Activation is independent of the known LPA5 agonists, lysophosphatidic acid and farnesyl pyrophosphate. These data bring forth a pathway for the direct stimulation of sensory nerves by luminal contents and interstitial fluid. Thus, by activating LPA5 on sensory nerves, MLF provides a means for known and yet to be identified constituents of the interstitial fluid to act as signals to comprise a “neurolymphocrine” system.
Publisher: Elsevier BV
Date: 06-2012
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2914-6_10
Abstract: Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons.
Publisher: Society for Neuroscience
Date: 04-02-2020
DOI: 10.1523/JNEUROSCI.2268-19.2020
Abstract: The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk. SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.
Publisher: Elsevier BV
Date: 07-2019
Publisher: American Physiological Society
Date: 04-2019
Abstract: G protein-coupled receptors (GPCRs) are essential for the neurogenic control of gastrointestinal (GI) function and are important and emerging therapeutic targets in the gut. Detailed knowledge of both the distribution and functional expression of GPCRs in the enteric nervous system (ENS) is critical toward advancing our understanding of how these receptors contribute to GI function during physiological and pathophysiological states. Equally important, but less well defined, is the complex relationship between receptor expression, ligand binding, signaling, and trafficking within enteric neurons. Neuronal GPCRs are internalized following exposure to agonists and under pathological conditions, such as intestinal inflammation. However, the relationship between the intracellular distribution of GPCRs and their signaling outputs in this setting remains a “black box”. This review will briefly summarize current knowledge of agonist-evoked GPCR trafficking and location-specific signaling in the ENS and identifies key areas where future research could be focused. Greater understanding of the cellular and molecular mechanisms involved in regulating GPCR signaling in the ENS will provide new insights into GI function and may open novel avenues for therapeutic targeting of GPCRs for the treatment of digestive disorders.
Publisher: Springer Science and Business Media LLC
Date: 21-04-2006
DOI: 10.1007/S00441-006-0181-9
Abstract: 5-Hydroxytryptamine (5-HT) is an endogenous stimulant of intestinal propulsive reflexes. It exerts its effects partly through 5-HT4 receptors 5-HT4 receptor agonists that are stimulants of intestinal transit are in clinical use. Both pharmacological and recent immunohistochemical studies indicate that 5-HT4 receptors are present on enteric neurons but the specific neurons that express the receptors have not been determined. In the present work, we describe the characterization of an anti-5-HT4 receptor antiserum that reveals immunoreactivity for enteric neurons and other cell types in the gastrointestinal tract. With this antiserum, 5-HT4 receptor immunoreactivity has been found in the muscularis mucosae of the rat oesophagus, a standard assay tissue for 5-HT4 receptors. It is also present in the muscularis mucosae of the guinea-pig and mouse oesophagus. In guinea-pig small intestine and rat and mouse colon, 5-HT4 receptor immunoreactivity occurs in subpopulations of enteric neurons, including prominent large neurons. Double-staining has shown that these large neurons in the guinea-pig small intestine are also immunoreactive for two markers of intrinsic primary afferent neurons, cytoplasmic NeuN and calbindin. Some muscle motor neurons in the myenteric ganglia are immunoreactive for this receptor, whereas it is rarely expressed by secretomotor neurons. Immunoreactivity also occurs in the interstitial cells of Cajal but is faint in the external muscle. Expression of the protein and mRNA has been confirmed in extracts containing enteric neurons. The observations suggest that one site of action of 5-HT4 receptor agonists is the intrinsic primary afferent neurons.
Publisher: Elsevier BV
Date: 10-2007
DOI: 10.1053/J.GASTRO.2007.07.002
Abstract: Properties of enteric neurons are transformed by inflammation and protein kinase C (PKC) isoforms are involved both in long-term changes in enteric neurons, and in transducing the effects of substances released during inflammation. We investigated roles of PKCepsilon in submucosal neurons by studying translocation in response to inflammatory mediators, effects on neuron excitability, and the changes in PKCepsilon distribution in a trinitrobenzene sulphonate model of ileitis. Immunohistochemical detection and analysis of association with membrane and cytosolic fractions, and Western blot analysis of cytosolic and particulate fractions were used to quantify translocation. Electrophysiology methods were used to measure effects on neuron excitability. All submucosal neurons were immunoreactive for the novel PKC, PKCepsilon, and direct PKC activators, phorbol 12,13-dibutyrate, ingenol 3,20-dibenzoate, and the PKCepsilon-specific activator, transactivator of transduction-Psiepsilon receptor for activated C kinase, all caused PKCepsilon translocation from cytoplasm to surfaces of the neurons. Electrophysiologic studies showed that the stimulant of novel PKCs, ingenol (1 micromol/L), increased excitability of all neurons. Stimulation of protease-activated receptors caused PKCepsilon translocation selectively in vasoactive intestinal peptide secretomotor neurons, whereas a neurokinin 3 tachykinin receptor agonist caused translocation in neuropeptide Y and calretinin neurons. In all cases translocation was reduced significantly by a PKCepsilon-specific translocation inhibitor peptide. Increased PKCepsilon at the plasma membrane occurred in all neurons 6-7 days after an inflammatory stimulus. Major targets for PKCepsilon include ion channels near the plasma membrane. PKCepsilon is likely to have a significant role in controlling the excitability of submucosal neurons and is probably an intermediate in causing hyperexcitability after inflammation.
Publisher: Wiley
Date: 18-01-2023
DOI: 10.1111/BPH.16023
Abstract: Gastrointestinal motility is tightly regulated by the enteric nervous system (ENS). Disruption of coordinated enteric nervous system activity can result in dysmotility. Pharmacological treatment options for dysmotility include targeting of G protein‐coupled receptors (GPCRs) expressed by neurons of the enteric nervous system. Current GPCR‐targeting drugs for motility disorders bind to the highly conserved endogenous ligand‐binding site and promote indiscriminate activation or inhibition of the target receptor throughout the body. This can be associated with significant side‐effect liability and a loss of physiological tone. Allosteric modulators of GPCRs bind to a distinct site from the endogenous ligand, which is typically less conserved across multiple receptor subtypes and can modulate endogenous ligand signalling. Allosteric modulation of GPCRs that are important for enteric nervous system function may provide effective relief from motility disorders while limiting side‐effects. This review will focus on how allosteric modulators of GPCRs may influence gastrointestinal motility, using 5‐hydroxytryptamine (5‐HT), acetylcholine (ACh) and opioid receptors as ex les.
Publisher: Springer Science and Business Media LLC
Date: 15-06-2017
Publisher: Springer Science and Business Media LLC
Date: 30-05-2006
DOI: 10.1007/S00418-006-0190-5
Abstract: In many organs, different protein kinase C (PKC) isoforms are expressed in specific cell types, suggesting that the different PKCs have cell-specific roles, and also that drugs acting on a particular PKC may have effects on the whole organ that are distinguishable from drugs that target other isoforms. Previous studies of the guinea-pig and mouse intestine indicate that there are cell-specific expressions of PKC isoforms in neurons, muscle and the interstitial cells of Cajal. In the present study we have investigated the expression of different PKCs in human intestine. Immunohistochemical studies showed that the forms that are prominent in human enteric neurons are PKCs gamma and epsilon and in muscle the dominant form is PKCdelta. Neurons were weakly stained for PKCbetaI. These observations parallel findings in guinea-pig and mouse, except that in human PKCgamma-IR was not present in the same types of neurons that express it in the guinea-pig. Enteric glial cells were strongly immunoreactive for PKCalpha, which is also the major isoform in enteric glial cells of guinea-pig. In human and guinea-pig, glial cells also express PKCbetaI. Spindle-shaped cells in the mucosa were immunoreactive for PKCalpha and PKCgamma and in the muscle layers similar cells had PKCgamma-IR and PKCtheta-IR. The spindle-shaped cells were similar in morphology to interstitial cells of Cajal. Western analysis and RT-PCR confirmed the presence of the PKC isoform proteins and mRNA in the tissue. We conclude that there is cell-type specific expression of different PKCs in enteric neurons and intestinal muscle in human tissue, and that there are strong similarities in patterns of expression between laboratory animals and human, but some clear differences are also observed.
Publisher: Springer Science and Business Media LLC
Date: 04-11-2019
Publisher: Wiley
Date: 03-08-2016
DOI: 10.1111/BPH.13554
Publisher: Proceedings of the National Academy of Sciences
Date: 22-05-2023
Abstract: The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK 1 R), Gα q/i , and βarrestin-2. Whereas the FDA-approved NK 1 R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK 1 R+ve neurons in knockin mice expressing human NK 1 R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK 1 R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK 1 R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK 1 R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of erse diseases.
Publisher: Frontiers Media SA
Date: 14-10-2020
Publisher: Wiley
Date: 04-09-2011
DOI: 10.1111/J.1365-2982.2011.01780.X
Abstract: Nitric oxide (NO), produced by the neural nitric oxide synthase enzyme (nNOS) is a transmitter of inhibitory neurons supplying the muscle of the gastrointestinal tract. Transmission from these neurons is necessary for sphincter relaxation that allows the passage of gut contents, and also for relaxation of muscle during propulsive activity in the colon. There are deficiencies of transmission from NOS neurons to the lower esophageal sphincter in esophageal achalasia, to the pyloric sphincter in hypertrophic pyloric stenosis and to the internal anal sphincter in colonic achalasia. Deficits in NOS neurons are observed in two disorders in which colonic propulsion fails, Hirschsprung's disease and Chagas' disease. In addition, damage to NOS neurons occurs when there is stress to cells, in diabetes, resulting in gastroparesis, and following ischemia and reperfusion. A number of factors may contribute to the propensity of NOS neurons to be involved in enteric neuropathies. One of these is the failure of the neurons to maintain Ca(2+) homeostasis. In neurons in general, stress can increase cytoplasmic Ca(2+), causing a Ca(2+) toxicity. NOS neurons face the additional problem that NOS is activated by Ca(2+). This is hypothesized to produce an excess of NO, whose free radical properties can cause cell damage, which is exacerbated by peroxynitrite formed when NO reacts with oxygen free radicals.
Publisher: Elsevier BV
Date: 2019
Publisher: Springer Science and Business Media LLC
Date: 2004
DOI: 10.1007/S00418-003-0602-8
Abstract: Interstitial cells of Cajal (ICC) are involved in the generation of electrical rhythmicity of intestinal muscle and in the transduction of neural inputs in the gut. Although the expression of receptors for neurotransmitters and hormones and some second messengers have been investigated in ICC, the protein kinases present in these cells have not been well documented. This study has demonstrated the immunohistochemical localisation of PKA, PKC gamma and PKC theta in ICC that were identified by the known ICC marker, c-Kit, in the guinea-pig gut. Other PKCs, PKC alpha, beta, delta, epsilon, eta, iota and lambda, and Ca(2+)-calmodulin-dependent protein kinase II were not localised in ICC. Double labelling studies were conducted on longitudinal muscle-myenteric plexus and external muscle-myenteric plexus preparations of the oesophagus, stomach (fundus, corpus and antrum), duodenum, distal ileum, caecum, proximal and distal colon, and rectum. The three protein kinases were detected in c-Kit-immunoreactive ICC at the level of the myenteric plexus (IC-MY), in the muscle (IC-IM) and at the level of the deep muscular plexus (IC-DMP) in the small intestine. PKA was found in over 90% of IC-IM in all regions examined, and in over 90% of IC-MY in the gastric body and antrum and throughout the small and large intestines. PKC gamma was in the majority of ICC in the gastric body and antrum and in the small intestine, but was largely absent from ICC in the oesophagus, proximal stomach and large intestine. PKC theta occurred in the majority of ICC in all regions except the rectum. The intensity of staining was greatest for PKA, with PKC gamma giving comparatively weak labelling of ICC. PKA was also detected in myenteric neurons, smooth muscle, macrophages and fibroblast-like cells. PKC gamma labelling occurred in large, multipolar neurons throughout the small and large intestine, as well as in lymph vessels and in capillaries. It is concluded that PKA, PKC gamma and PKC theta are all present in ICC, with the differences in their localisations suggesting specific roles for each in ICC function.
Publisher: Springer Science and Business Media LLC
Date: 24-03-2007
DOI: 10.1007/S10620-006-9680-5
Abstract: Neuronal destruction has been considered the hallmark of pathogenic mechanisms in chagasic megacolon. Characterization of neuropeptides in the enteric nervous system from chagasic patients with megacolon could elucidate some aspects of the development of this syndrome. In the present work we demonstrate the changes in expression of neuropeptides and neurochemical markers present in neuronal plexuses from the colons of chagasic patients with megacolon. Sections of frozen tissue s les were immunohistochemically labeled for anticalretinin, cChaT, substance P, VIP, NOS, and NPY. Immunoreactivity was observed using a confocal microscope. Our results demonstrate that in chagasic patients with megacolon, inhibitory motor neurons (VIP and NOS immunoreactive) are preferentially destroyed by Trypanosoma cruzi and/or the inflammatory process. These results suggest a selective destruction of enteric neurons in the colon of chagasic patients with megacolon, pointing to an important discovery in the mechanism of pathogenesis of Chagas' disease.
Publisher: Wiley
Date: 24-08-2015
DOI: 10.1111/NMO.12656
Abstract: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue s les. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.
Publisher: Wiley
Date: 30-01-2020
DOI: 10.1111/NMO.13787
Abstract: Visceral pain is commonly associated with acute or remitting inflammatory bowel disease (IBD). In marked contrast, chronic IBD is often painless, even in the presence of active inflammation. This suggests that inflammation in itself is insufficient to sustain altered nociceptive signaling and raises the possibility that there is an endogenous analgesic system in effect in chronic disease. A new study by Basso et al. published in this issue of Neurogastroenterology & Motility provides additional support for an immune-mediated mechanism that suppresses visceral hypersensitivity. The authors examined visceral pain in the IL-10-piroxicam model of chronic colitis, which differs from other experimental IBD models in that it involves immune suppression. During active inflammation, responses by these mice to graded increases in colorectal distension were equivalent to healthy controls, consistent with normal afferent signaling. However, treatment with a peripherally restricted opioid receptor antagonist resulted in marked visceral hypersensitivity to the same stimuli. This effect was attributed to the production of endogenous opioids by colitogenic CD4
Publisher: Elsevier BV
Date: 11-2015
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 31-10-2014
Abstract: Sensory nerves are equipped with receptors and ion channels that allow them to detect and respond to erse chemical, mechanical, and thermal stimuli. These sensory proteins include G protein-coupled receptors (GPCRs) and transient receptor potential (TRP) ion channels. A subclass of peptidergic sensory nerves express GPCRs and TRP channels that detect noxious, irritant, and inflammatory stimuli. Activation of these nerves triggers protective mechanisms that lead to withdrawal from danger (pain), removal of irritants (itch, cough), and resolution of infection (neurogenic inflammation). The GPCR-TRP axis is central to these mechanisms. Signals that emanate from the GPCR superfamily converge on the small TRP family, leading to channel sensitization and activation, which lify pain, itch, cough, and neurogenic inflammation. Herein we discuss how GPCRs and TRP channels function independently and synergistically to excite sensory nerves that mediate noxious and irritant responses and inflammation in the skin and the gastrointestinal and respiratory systems. We discuss the signaling mechanisms that underlie the GPCR-TRP axis and evaluate how new information about the structure of GPCRs and TRP channels provides insights into their functional interactions. We propose that a deeper understanding of the GPCR-TRP axis may facilitate the development of more selective and effective therapies to treat dysregulated processes that underlie chronic pain, itch, cough, and inflammation.
Publisher: Elsevier
Date: 2015
Publisher: Wiley
Date: 27-11-2015
Publisher: American Society for Clinical Investigation
Date: 06-2008
DOI: 10.1172/JCI34886
Publisher: Springer Science and Business Media LLC
Date: 2003
DOI: 10.1007/S00441-002-0652-6
Abstract: Cholinergic neurons have been revealed in the enteric nervous system by functional and biochemical studies but not by antibodies that provide excellent localisation of the synthesising enzyme, choline acetyltransferase (ChAT), in the central nervous system. In order to determine whether a newly described peripheral form of ChAT (pChAT) is a ChAT enzyme of enteric neurons, we have compared pChAT distribution with that of the common form of ChAT, cChAT, by quantitative analysis of the co-localisation of pChAT and cChAT with other neurochemical markers in enteric neurons of the guinea-pig ileum. We found classes of neuron with strong pChAT immunoreactivity (IR) and others with strong cChAT-IR. In myenteric ganglia, strong pChAT-IR was in calbindin-positive intrinsic primary afferent neurons (IPANs), whereas cChAT-IR of these neurons was weak. Calretinin neurons were immunoreactive for cChAT, but not pChAT. Only 4% of nitric oxide synthase (NOS) neurons (possibly interneurons) were pChAT-immunoreactive, similar to observations with cChAT. NOS-immunoreactive inhibitory motor neurons stained with neither cChAT nor pChAT antisera. In the submucosal ganglia, pChAT-IR was strongly expressed in IPANs (identified by cytoplasmic staining for the neuronal nuclear marker, NeuN) and in neuropeptide Y (NPY)-immunoreactive secretomotor neurons, but not in calretinin-immunoreactive neurons. cChAT-IR occurred weakly in submucosal IPANs and also labelled NPY- and calretinin-immunoreactive neurons. Submucosal vasoactive-intestinal-peptide-immunoreactive neurons (non-cholinergic secretomotor neurons) were not reactive for either form of ChAT.
Publisher: Zenodo
Date: 2022
Publisher: Elsevier BV
Date: 08-2020
Publisher: Springer Science and Business Media LLC
Date: 26-04-2002
DOI: 10.1007/S00418-002-0404-4
Abstract: The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.
Publisher: American Chemical Society (ACS)
Date: 29-03-2019
Publisher: Elsevier BV
Date: 08-2013
Publisher: American Physiological Society
Date: 03-2007
Abstract: PKC is involved in mediating the tonic component of gastrointestinal smooth muscle contraction in response to stimulation by agonists for G protein-coupled receptors. Here, we present pharmacological and immunohistochemical evidence indicating that a member of the novel PKC isoforms, PKC-δ, is involved in maintaining muscarinic receptor-coupled tonic contractions of the guinea pig ileum. The tonic component of carbachol-evoked contractions was enhanced by an activator of conventional and novel PKCs, phorbol 12,13-dibutyrate (PDBu 200 nM or 1 μM), and by an activator of novel PKCs, ingenol 3,20-dibenzoate (IDB 100 or 500 nM). Enhancement was unaffected by concentrations of bisindolylmaleimide I (BIM-I 22 nM) that block conventional PKCs or by a PKC-ε-specific inhibitor peptide but was attenuated by higher doses of BIM-I (2.2 μM). Relevant proteins were localized at a cellular and subcellular level using confocal analysis. Immunohistochemical staining of the ileum showed that PKC-δ was exclusively expressed in smooth muscles distributed throughout the layers of the gut wall. PKC-ε immunoreactivity was prominent in enteric neurons but was largely absent from smooth muscle of the muscularis externa. Treatment with PDBu, IDB, or carbachol resulted in a time- and concentration-dependent translocation of PKC-δ from the cytoplasm to filamentous structures within smooth muscle cells. These were parallel to, but distinct from, actin filaments. The translocation of PKC-δ in response to carbachol was significantly reduced by scopolamine or calphostin C. The present study indicates that the tonic carbachol-induced contraction of the guinea pig ileum is mediated through a novel PKC, probably PKC-δ.
Publisher: Springer Science and Business Media LLC
Date: 23-03-2019
DOI: 10.1007/S00018-019-03077-6
Abstract: The use of opioid analgesics is severely limited due to the development of intractable constipation, mediated through activation of mu opioid receptors (MOR) expressed by enteric neurons. The related delta opioid receptor (DOR) is an emerging therapeutic target for chronic pain, depression and anxiety. Whether DOR agonists also promote sustained inhibition of colonic transit is unknown. This study examined acute and chronic tolerance to SNC80 and ARM390, which were full and partial DOR agonists in neural pathways controlling colonic motility, respectively. Excitatory pathways developed acute and chronic tolerance to SNC80, whereas only chronic tolerance developed in inhibitory pathways. Both pathways remained functional after acute or chronic ARM390 exposure. Propagating colonic motor patterns were significantly reduced after acute or chronic SNC80 treatment, but not by ARM390 pre-treatment. These findings demonstrate that SNC80 has a prolonged inhibitory effect on propagating colonic motility. ARM390 had no effect on motor patterns and thus may have fewer gastrointestinal side-effects.
Publisher: Elsevier BV
Date: 2022
DOI: 10.1016/J.NEULET.2021.136377
Abstract: Transient Receptor Potential Vanilloid 4 (TRPV4) is a polymodal, non-selective cation channel that detects thermal, mechanical, and environmental cues and contributes to a range of erse physiological processes. The effects of chronic TRPV4 stimulation and gain-of-function genetic mutations suggest that TRPV4 may also be a valuable therapeutic target for pathophysiological events including neurogenic inflammation, peripheral neuropathies, and impaired wound healing. There has been significant interest in defining how and where TRPV4 may promote inflammation and pain. Endogenous stimuli such as osmotic stress and lipid binding are established TRPV4 activators. The TRP channel family is also well-known to be controlled by 'receptor-operated' pathways. For ex le, G protein-coupled receptors (GPCRs) expressed by primary afferent neurons or other cells in inflammatory pathways utilize TRPV4 as an effector protein to lify nociceptive and inflammatory signaling. Contributing to disorders including arthritis, neuropathies, and pulmonary edema, GPCRs such as the protease-activated receptor PAR2 mediate activation of kinase signaling cascades to increase TRPV4 phosphorylation, resulting in sensitization and enhanced neuronal excitability. Phospholipase activity also leads to production of polyunsaturated fatty acid lipid mediators that directly activate TRPV4. Consistent with the contribution of TRPV4 to disease, pharmacological inhibition or genetic ablation of TRPV4 can diminish receptor-mediated inflammatory events. This review outlines how receptor-mediated signaling is a major endogenous driver of TRPV4 gating and discusses key signaling pathways and emerging TRPV4 modulators such as the mechanosensitive Piezo1 ion channel. A collective understanding of how endogenous stimuli can influence TRPV4 function is critical for future therapeutic endeavors to modulate this channel.
Publisher: Elsevier BV
Date: 08-2011
DOI: 10.1053/J.GASTRO.2011.04.049
Abstract: Transient receptor potential ankyrin (TRPA) 1, an excitatory ion channel expressed by sensory neurons, mediates somatic and visceral pain in response to direct activation or noxious mechanical stimulation. Although the intestine is routinely exposed to irritant alimentary compounds and inflammatory mediators that activate TRPA1, there is no direct evidence for functional TRPA1 receptors on enteric neurons, and the effects of TRPA1 activation on intestinal function have not been determined. We characterized expression of TRPA1 by enteric neurons and determined its involvement in the control of intestinal contractility and transit. TRPA1 expression was characterized by reverse-transcription polymerase chain reaction and immunofluorescence analyses. TRPA1 function was examined by Ca(2+) imaging and by assays of contractile activity and transit. We detected TRPA1 messenger RNA in the mouse intestine and TRPA1 immunoreactivity in enteric neurons. The cecum and colon had immunoreactivity for neuronal TRPA1, but the duodenum did not. TRPA1 immunoreactivity was also detected in inhibitory motoneurons and descending interneurons, cholinergic neurons, and intrinsic primary afferent neurons. TRPA1 activators, including cinnamaldehyde, allyl isothiocyanate (AITC), and 4-hydroxynonenal, increased [Ca(2+)](i) in myenteric neurons. These were reduced by a TRPA1 antagonist (HC-030031) or deletion of Trpa1. TRPA1 activation inhibited contractility of the segments of colon but not stomach or small intestine of Trpa1(+/+) but not Trpa1(-/-) mice this effect was reduced by tetrodotoxin or N(G)-nitro-l-arginine methyl ester. Administration of AITC by gavage did not alter gastric emptying or small intestinal transit, but luminal AITC inhibited colonic transit via TRPA1. Functional TRPA1 is expressed by enteric neurons, and activation of neuronal TRPA1 inhibits spontaneous neurogenic contractions and transit of the colon.
Publisher: American Physiological Society
Date: 08-2019
Abstract: Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca 2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.
Publisher: Springer Science and Business Media LLC
Date: 12-06-2012
DOI: 10.1007/S00441-012-1451-3
Abstract: Recent investigation of the intestine following ischemia and reperfusion (I/R) has revealed that nitric oxide synthase (NOS) neurons are more strongly affected than other neuron types. This implies that NO originating from NOS neurons contributes to neuronal damage. However, there is also evidence of the neuroprotective effects of NO. In this study, we compared the effects of I/R on the intestines of neuronal NOS knockout (nNOS(-/-)) mice and wild-type mice. I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the external muscle layers. Damage to the mucosa and muscle was more severe and greater infiltration by neutrophils occurred in the first 24 h in nNOS(-/-) mice. Immunohistochemistry for the contractile protein, α-smooth muscle actin, was used to evaluate muscle damage. Smooth muscle actin occurred in the majority of smooth muscle cells in the external musculature of normal mice but was absent from most cells and was reduced in the cytoplasm of other cells following I/R. The loss was greater in nNOS(-/-) mice. Basal contractile activity of the longitudinal muscle and contractile responses to nerve stimulation or a muscarinic agonist were reduced in regions subjected to I/R and the effects were greater in nNOS(-/-) mice. Reductions in responsiveness also occurred in regions of operated mice not subjected to I/R. This is attributed to post-operative ileus that is not significantly affected by knockout of nNOS. The results indicate that deleterious effects are greater in regions subjected to I/R in mice lacking nNOS compared with normal mice, implying that NO produced by nNOS has protective effects that outweigh any damaging effect of this free radical produced by enteric neurons.
Publisher: Springer Science and Business Media LLC
Date: 12-04-2014
DOI: 10.1007/S00441-014-1822-Z
Abstract: The substance P neurokinin 1 receptor (NK1R) regulates motility, secretion, inflammation and pain in the intestine. The distribution of the NK1R is a key determinant of the functional effects of substance P in the gut. Information regarding the distribution of NK1R in subtypes of mouse enteric neurons is lacking and is the focus of the present study. NK1R immunoreactivity (NK1R-IR) is examined in whole-mount preparations of the mouse distal colon by indirect immunofluorescence and confocal microscopy. The distribution of NK1R-IR within key functional neuronal subclasses was determined by using established neurochemical markers. NK1R-IR was expressed by a subpopulation of myenteric and submucosal neurons it was mainly detected in large multipolar myenteric neurons and was colocalized with calcitonin gene-related peptide, neurofilament M, choline acetyltransferase and calretinin. The remaining NK1R-immunoreactive neurons were positive for nitric oxide synthase. NK1R was expressed by most of the submucosal neurons and was exclusively co-expressed with vasoactive intestinal peptide, with no overlap with choline acetyltransferase. Treatment with substance P resulted in the concentration-dependent internalisation of NK1R from the cell surface into endosome-like structures. Myenteric NK1R was mainly expressed by intrinsic primary afferent neurons, with minor expression by descending interneurons and inhibitory motor neurons. Submucosal NK1R was restricted to non-cholinergic secretomotor neurons. These findings highlight key differences in the neuronal distribution of NK1R-IR between the mouse, rat and guinea-pig, with important implications for the functional role of NK1R in regulating intestinal motility and secretion.
Publisher: Elsevier BV
Date: 2019
Publisher: Oxford University Press (OUP)
Date: 04-2012
Abstract: The functioning of the gastrointestinal tract is under the control of the most extensive system of peripheral neurons in the body, the enteric nervous system, and the largest endocrine system of the body, the GEP endocrine system. The enteric nervous system in large mammals contains 500 million neurons, and the GEP endocrine system produces more than 30 hormones. Numerous enteric neuropathies affecting both humans and animals have been described and digestive disorders affect commercially important species, such as horses and cattle. The most severe enteric neuropathies (e.g., lethal white syndrome in horses or Hirschsprung's disease in humans) can be fatal. Also, horses with ileus or other digestive disorders are commonly euthanized. In this review we discuss ex les of enteric neuropathies that affect agricultural animals and humans: prion disease, postoperative ileus, distal enteric aganglionosis, and infective diarrhea. Enteric neurons and glia are a location of prion proteins and are involved in transmission of the infection from gut to brain and brain to gut. Postoperative ileus is a complex disorder involving the local inhibitory effects of sympathetic nervous system activation and the release of opioids, presumably from enteric neurons. Intestinal inflammation, especially of the external muscle that includes enteric ganglia, also occurs in ileus. Congenital distal bowel aganglionosis, responsible for lethal white syndrome in horses, Hirschsprung's disease in humans, and similar conditions in mice and rats, is a fatal condition if untreated. Mutations of the same genes can cause the condition in each of these species. The only effective current treatment is surgical removal of the aganglionic bowel. Infectious diarrheas involve activation of enteric secretomotor neurons by pathogens and the toxins they produce, which causes substantial fluid loss. Strategies to target enteric neurons in the treatment of secretory diarrheas have not been developed. Disorders of enteroendocrine cells, other than GEP endocrine tumors, are less well documented. However, evidence for the involvement of gut endocrine cells in a subset of patients with irritable bowel syndrome, and in the symptomology of celiac disease, has been demonstrated. Further investigation of the involvement of enteric neural and endocrine signaling systems in digestive disorders, especially in agricultural and companion animals, may lead to diagnostic and therapeutic advances.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 31-05-2017
DOI: 10.1126/SCITRANSLMED.AAL3447
Abstract: Therapeutic targeting of the neurokinin 1 receptor in endosomes provides efficacious and prolonged pain relief.
Publisher: Elsevier BV
Date: 06-2008
DOI: 10.1016/J.PEPTIDES.2008.01.025
Abstract: In this study we evaluate the uptake by murine dendritic cells (DCs) of different synthetic, branched cationic peptide structures with a view to facilitating peptide epitope delivery. The level of cell uptake by fluorescenated peptides was measured by flow cytometry following quenching of extracellular fluorescence with trypan blue. Branched peptides containing either N-terminal arginine or N-terminal lysine residues were able to mediate cell entry but the peptide containing four arginine residues in a branching configuration (R4) was found to be superior not only to other branched peptides in translocating to the cell interior and also to a peptide containing four arginine residues arranged linearly. Fluorescenated R4 was found to be localized within intracellular vesicle-like compartments as well as being distributed throughout the cell cytoplasm. Uptake of R4 utilized an energy-dependent process that appeared to involve phosphatidylinositol-3-kinase and could induce intermediate levels of DC maturation. R4 when conjugated to a T-helper cell and CTL epitope construct was able to induce antigen-specific CD8+ T-cell mediated immune responses in mice when administered in adjuvant as were DCs that were pulsed with this construct and then matured with LPS. Fluorescenated R4 was also found to translocate into the interior of other cell types indicating that it may be useful for the delivery of peptide cargo into other specialized cells.
Publisher: Elsevier BV
Date: 09-2011
Publisher: Future Science Ltd
Date: 2021
Abstract: G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure–activity relationship considerations, and discusses advantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.
Publisher: Elsevier
Date: 2022
Publisher: American Society for Clinical Investigation
Date: 25-03-2013
DOI: 10.1172/JCI64551
Publisher: Elsevier
Date: 2017
DOI: 10.1016/BS.APHA.2017.03.002
Abstract: Transient receptor potential (TRP) ion channels are important signaling components in nociceptive and inflammatory pathways. This is attributed to their ability to function as polymodal sensors of environmental stimuli (chemical and mechanical) and as effector molecules in receptor signaling pathways. TRP vanilloid 4 (TRPV4) is a nonselective cation channel that is activated by multiple endogenous stimuli including shear stress, membrane stretch, and arachidonic acid metabolites. TRPV4 contributes to many important physiological processes and dysregulation of its activity is associated with chronic conditions of metabolism, inflammation, peripheral neuropathies, musculoskeletal development, and cardiovascular regulation. Mechanosensory and receptor- or lipid-mediated signaling functions of TRPV4 have historically been attributed to central and peripheral neurons. However, with the development of potent and selective pharmacological tools, transgenic mice and improved molecular and imaging techniques, many new roles for TRPV4 have been revealed in nonneuronal cells. In this chapter, we discuss these recent findings and highlight the need for greater characterization of TRPV4-mediated signaling in nonneuronal cell types that are either directly associated with neurons or indirectly control their excitability through release of sensitizing cellular factors. We address the integral role of these cells in sensory and inflammatory processes as well as their importance when considering undesirable on-target effects that may be caused by systemic delivery of TRPV4-selective pharmaceutical agents for treatment of chronic diseases. In future, this will drive a need for targeted drug delivery strategies to regulate such a erse and promiscuous protein.
Publisher: Elsevier BV
Date: 2013
Publisher: Bioscientifica
Date: 04-2018
DOI: 10.1530/JME-17-0152
Abstract: Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and in situ hybridisation that Insl5 mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr 202 /Tyr 204 ), AKT (Thr 308 and Ser 473 ) and S6RP (Ser 235/236 ) and inhibited cAMP production but did not stimulate Ca 2+ release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing RXFP4 . Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine aracrine roles of INSL5 in the intestinal tract.
Publisher: American Physiological Society
Date: 08-2016
Abstract: The μ-opioid receptor (MOR) is a major regulator of gastrointestinal motility and secretion and mediates opiate-induced bowel dysfunction. Although MOR is of physiological and therapeutic importance to gut function, the cellular and subcellular distribution and regulation of MOR within the enteric nervous system are largely undefined. Herein, we defined the neurochemical coding of MOR-expressing neurons in the guinea pig gut and examined the effects of opioids on MOR trafficking and regulation. MOR expression was restricted to subsets of enteric neurons. In the stomach MOR was mainly localized to nitrergic neurons (∼88%), with some overlap with neuropeptide Y (NPY) and no expression by cholinergic neurons. These neurons are likely to have inhibitory motor and secretomotor functions. MOR was restricted to noncholinergic secretomotor neurons (VIP-positive) of the ileum and distal colon submucosal plexus. MOR was mainly detected in nitrergic neurons of the colon (nitric oxide synthase positive, 87%), with some overlap with choline acetyltransferase (ChAT). No expression of MOR by intrinsic sensory neurons was detected. [d-Ala 2 , MePhe 4 , Gly(ol) 5 ]enkephalin (DAMGO), morphiceptin, and loperamide induced MOR endocytosis in myenteric neurons. After stimulation with DAMGO and morphiceptin, MOR recycled, whereas MOR was retained within endosomes following loperamide treatment. Herkinorin or the δ-opioid receptor agonist [d-Ala 2 , d-Leu 5 ]enkephalin (DADLE) did not evoke MOR endocytosis. In summary, we have identified the neurochemical coding of MOR-positive enteric neurons and have demonstrated differential trafficking of MOR in these neurons in response to established and putative MOR agonists.
Publisher: American Physiological Society
Date: 02-2022
Abstract: Bile acids (BAs) are known to be important regulators of intestinal motility and epithelial fluid and electrolyte transport. Over the past two decades, significant advances in identifying and characterizing the receptors, transporters, and ion channels targeted by BAs have led to exciting new insights into the molecular mechanisms involved in these processes. Our appreciation of BAs, their receptors, and BA-modulated ion channels as potential targets for the development of new approaches to treat intestinal motility and transport disorders is increasing. In the current review, we aim to summarize recent advances in our knowledge of the different BA receptors and BA-modulated ion channels present in the gastrointestinal system. We discuss how they regulate motility and epithelial transport, their roles in pathogenesis, and their therapeutic potential in a range of gastrointestinal diseases.
Publisher: Frontiers Media SA
Date: 12-11-2019
Publisher: Elsevier BV
Date: 09-2013
Publisher: Cold Spring Harbor Laboratory
Date: 05-05-2020
DOI: 10.1101/2020.05.05.078717
Abstract: γδ T cells play an essential role in the immune response to malaria infection. However, long-lasting effects of malaria infection on the γδ T cell population still remain inadequately understood. This study investigated transcriptional changes and memory-like functional capacity of malaria pre-exposed γδ T cells using a Plasmodium chabaudi infection model. We show that multiple genes associated with effector function (chemokines, cytokines and cytotoxicity) and antigen-presentation were upregulated in P. chabaudi -exposed γδ T cells compared to γδ T cells from naïve mice. This transcriptional profile was positively correlated with profiles observed in conventional memory CD8 + T cells and was accompanied by enhanced reactivation upon secondary encounter with Plasmodium -infected red blood cells in vitro . Collectively our data demonstrate that Plasmodium exposure result in “memory-like imprints” in the γδ T cell population and also promotes γδ T cells that can support antigen-presentation during subsequent infections.
Publisher: Wiley
Date: 28-10-2011
Publisher: American Physiological Society
Date: 05-2008
Abstract: Tachykinins, acting through NK 3 receptors (NK 3 R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is dependent on protein kinase C (PKC), its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in IPANs of the guinea pig ileum, demonstrated by double staining with the IPAN marker NeuN. PKCε was also colocalized with PKD1/2 in IPANs. PKCε and PKD1/2 trafficking was studied in enteric neurons within whole mounts of the ileal wall. In untreated preparations, PKCε and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK 3 R agonist senktide evoked a transient translocation of PKCε and PKD1/2 from the cytosol to the plasma membrane and induced PKD1/2 phosphorylation at the plasma membrane. PKCε translocation was maximal at 10 s and returned to the cytosol within 2 min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15 s and translocated to the cytosol by 2 min, where it remained active up to 30 min after NK 3 R stimulation. PKD1/2 activation was reduced by a PKCε inhibitor and prevented by NK 3 R inhibition. NK 3 R-mediated PKCε and PKD activation was confirmed in HEK293 cells transiently expressing NK 3 R and green fluorescent protein-tagged PKCε, PKD1, PKD2, or PKD3. Senktide caused membrane translocation and activation of kinases within 30 s. After 15 min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus stimulation of NK 3 R activates PKCε and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.
Publisher: The Endocrine Society
Date: 09-10-2009
DOI: 10.1210/EN.2009-0489
Abstract: Urocortin 1 (Ucn1) is a neuropeptide that regulates vascular tone and is implicated in both the vascular and immune cell-mediated responses to inflammation. The role of Ucn1 in regulating microvascular permeability has not been determined. We hypothesized that local Ucn1 release promotes microvascular permeability and that this effect augments the local gastrointestinal vascular response to lipopolysaccharide (LPS)-induced systemic inflammation. We measured hydraulic (Lp) and macromolecule permeability in mesenteric venules. We show that a continuous infusion of 10−7m Ucn1 in a postcapillary venule increased Lp 2-fold over baseline, as did LPS-induced inflammation. However, simultaneous infusion of Ucn1 and LPS markedly increased Lp by 7-fold. After local knockdown of Ucn1 using RNA interference, infusion of Ucn1 with LPS resulted in return to 2-fold increase, confirming that Ucn1 synergistically augments hydraulic permeability during inflammation. LPS and Ucn1 treatment also resulted in increased numbers of interstitial microspheres, which colocalized with CD31+ immune cells. Ucn1 activity is mediated through two receptor subtypes, CRH-R1 and CRH-R2. CRH-R1 receptor blockade exacerbated, whereas CRH-R2 receptor blockade decreased the LPS-induced increase in Lp. Finally, treatment with the c-JUN N-terminal kinase (JNK) antagonist SP600125 during infusion of LPS, but not Ucn1, decreased Lp. These findings suggest that Ucn1 increases microvascular permeability and acts synergistically with LPS to increase fluid and macromolecule losses during inflammation. Knockdown of endogenous Ucn1 during inflammation attenuates synergistic increases in Lp. Ucn1’s effect on Lp is partially mediated by the CRH-R2 receptor and acts independently of the c-JUN N-terminal kinase signal transduction pathway.
Publisher: Wiley
Date: 22-05-2018
DOI: 10.1111/BPH.14222
Publisher: Frontiers Media SA
Date: 28-11-2014
Publisher: American Veterinary Medical Association (AVMA)
Date: 02-2009
Abstract: Objective —To investigate effects and mechanisms of ergotamine and ergovaline and effects of peramine on reticulum motility of sheep. S le Population —3 sheep with indwelling electrodes in the reticulum and s les of reticulum collected from 126 sheep at an abattoir. Procedures —In conscious sheep, motility was recorded as integrated electromyograms from the reticulum. Ergotamine was administered IV alone or in combination with the cholinergic muscarinic receptor antagonist atropine to sheep, and motility of the reticulum was assessed. In vitro, whole wall strips of the reticulum, cut in a direction to record longitudinal muscle activity via force transducers, were placed in 10-mL organ baths and superfused with Tyrode Ringer's solution at 37°C and oxygenated with 95% oxygen and 5% carbon dioxide. Testing involved incubation of reticulum strips with ergotamine, ergovaline, and peramine and measurement of motility of the reticulum tissues. Results —Administration of ergotamine to sheep reduced the frequency of reticulum contractions and increased baseline electromyographic activity (tonus). Frequency was unaffected by atropine, whereas tonus was significantly reduced. In vitro, ergotamine and ergovaline increased tonic contractions and stimulated phasic contractions of reticulum tissues and potentiated electrically stimulated contractions. Atropine and tetrodotoxin re-duced tonic contractions, but stimulation of large- litude phasic contractions remained. Peramine had no effect on motility of reticulum tissues. Conclusions and Clinical Relevance —Results of the study indicated that peripheral excitatory effects of the ergopeptides on motility of the reticulum appear to be mediated partly through myenteric neurons and muscarinic receptors and also through direct effects on the muscles.
Publisher: Elsevier BV
Date: 2021
Publisher: Elsevier BV
Date: 07-2021
Start Date: 2021
End Date: 2025
Funder: Department of Health
View Funded ActivityStart Date: 2020
End Date: 2022
Funder: Australian Research Council
View Funded ActivityStart Date: 2022
End Date: 2025
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: 2015
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2012
End Date: 2014
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2020
End Date: 09-2023
Amount: $506,000.00
Funder: Australian Research Council
View Funded Activity