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Endothelial and epithelial cells form physical barriers that modulate the exchange of fluid and molecules. The integrity of these barriers can be influenced by signaling through G protein-coupled receptors (GPCRs) and ion channels. Serotonin (5-HT) is an important vasoactive mediator of tissue edema and inflammation. However, the mechanisms that drive 5-HT-induced plasma extravasation are poorly defined. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an established enhancer of signaling by GPCRs that promote inflammation and endothelial barrier disruption. Here, we investigated the role of TRPV4 in 5-HT-induced plasma extravasation using pharmacological and genetic approaches. Activation of either TRPV4 or 5-HT receptors promoted significant plasma extravasation in the airway and upper gastrointestinal tract of mice. 5-HT-mediated extravasation was significantly reduced by pharmacological inhibition of the 5-HT2A receptor subtype, or with antagonism or deletion of TRPV4, consistent with functional interaction between 5-HT receptors and TRPV4. Inhibition of receptors for the neuropeptides substance P (SP) or calcitonin gene-related peptide (CGRP) diminished 5-HT-induced plasma extravasation. Supporting studies assessing treatment of HUVEC with 5-HT, CGRP, or SP was associated with ERK phosphorylation. Exposure to the TRPV4 activator GSK1016790A, but not 5-HT, increased intracellular Ca2+ in these cells. However, 5-HT pre-treatment enhanced GSK1016790A-mediated Ca2+ signaling, consistent with sensitization of TRPV4. The functional interaction was further characterized in HEK293 cells expressing 5-HT2A to reveal that TRPV4 enhances the duration of 5-HT-evoked Ca2+ signaling through a PLA2 and PKC-dependent mechanism. In summary, this study demonstrates that TRPV4 contributes to 5-HT2A-induced plasma extravasation in the airways and upper GI tract, with evidence supporting a mechanism of action involving SP and CGRP release., Serotonin (5-HT) is an important mediator of tissue edema and inflammation. The authors used mouse models and cell-based signaling assays to provide greater understanding of the mechanisms involved. They demonstrate that effects of 5-HT are mediated through the 5-HT2A receptor and involve activation of the mechanosensitive ion channel TRPV4 and neuropeptide release.

Background & Aims: Functional interactions between the mu opioid receptor (MOR) and delta opioid receptor (DOR) represent a potential target for novel analgesics and may drive the effects of the clinically approved drug eluxadoline for the treatment of diarrhea-predominant irritable bowel syndrome. Although the enteric nervous system (ENS) is a likely site of action, the coexpression and potential interaction between MOR and DOR in the ENS are largely undefined. In the present study, we have characterized the distribution of MOR in the mouse ENS and examined MOR-DOR interactions by using pharmacologic and cell biology techniques. Methods: MOR and DOR expression was defined by using MORmCherry and MORmCherry–DOR-eGFP knockin mice. MOR-DOR interactions were assessed by using DOR-eGFP internalization assays and by pharmacologic analysis of neurogenic contractions of the colon. Results: Although MOR was expressed by approximately half of all myenteric neurons, MOR-positive submucosal neurons were rarely observed. There was extensive overlap between MOR and DOR in both excitatory and inhibitory pathways involved in the coordination of intestinal motility. MOR and DOR can functionally interact, as shown through heterologous desensitization of MOR-dependent responses by DOR agonists. Functional evidence suggests that MOR and DOR may not exist as heteromers in the ENS. Pharmacologic studies show no evidence of cooperativity between MOR and DOR. DOR internalizes independently of MOR in myenteric neurons, and MOR-evoked contractions are unaffected by the sequestration of DOR. Conclusions: Collectively, these findings demonstrate that although MOR and DOR are coexpressed in the ENS and functionally interact, they are unlikely to exist as heteromers under physiological conditions. Keywords: G Protein-Coupled Receptor, Opioid Receptor, Heteromer, Heterologous Desensitization

The development of nanoparticle-based targeted therapeutics for the treatment of cancer requires a well-defined understanding of the tumor microenvironment, which is challenging due to tumor complexity and heterogeneity. Recent advancements in three-dimensional (3D) cell models such as tumor-on-a-chip devices can overcome some of these challenges by providing coculture in vitro systems (tumor surrounded by tubular endothelial cells) that mimic native cellular environments to accurately study the enhanced permeability and retention (EPR) potential of drug delivery systems under flow conditions. However, inducing “leaky” vasculature in endothelial cells surrounding solid tumors in microfluidic devices is not readily controllable and highly dependent on tumor cell identity. Utilizing a microfluidic tumor model (MTM) consisting of a tumor region surrounded by a 3D microvascular network, we have simulated the EPR effect by activating a known regulator of endothelial junction formation and edema: the transient ...

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.

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: https://clij.github.io/assistant/

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gα(i/o) and β-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain.

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 STATEMENTThe 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.

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. Ca2+ 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.

BACKGROUND: Endogenous opioids released from CD4+T cells are increased in inflammatory bowel disease patients (IBD) and pre-clinical models of IBD, and have an important analgesic effect. We have shown that endogenous opioids in supernatant from cDSS mice have an antinociceptive action on dorsal root ganglia (DRG) neurons that is mediated by δ (DOR) and μ (MOR) opioid receptors. We also found that acute application of opioids for minutes had an unexpectedly sustained analgesic effect but the mechanism was unclear. AIMS: We hypothesized that endogenous opioids have a sustained antinociceptive effect on nociceptors via endosomal signaling. METHODS: Chronic colitis was induced with 2% dextran sulfate sodium (DSS; three cycles: 5 days of DSS/5 days of water) in C57BL/6 mice and colonic supernatants were collected. DRG neurons were exposed to control or cDSS supernatants (1h), or opioid agonists (10nM, 15min); DAMGO (MOR-selective) or DADLE (DOR-selective). We recorded changes in rheobase (minimum current to fire action potential) by perforated patch-clamp, immediately after washout of drugs (T0) or 30 min later (T30). We used Förster resonance energy transfer (FRET) biosensors to examine signaling (protein kinase C; PKC and extracellular signal-regulated kinase; ERK) in subcellular compartments mediated by DOR agonists: DADLE and SNC80 (100nM). DRG neurons from DOR-eGFP mice were nucleofected with FRET sensors. Neurons were pre-treated with dynamin GTPase (50 μM dyngo4a) or clathrin inhibitor, (15 μM pistop2) for 30 min prior to agonists. One or Two way ANOVA and post hoc Tukey’s tests were used to analyze the data. RESULTS: Supernatant from cDSS mice had a sustained antinociceptive effect on neuronal excitability (increased rheobase 39.6%, p

SUMMARY Mechanisms resulting in abdominal pain include altered neuro-immune interactions in the gastrointestinal tract, but the signaling processes that link immune activation with visceral hypersensitivity are unresolved. We hypothesized that enteric glia link the neural and immune systems of the gut and that communication between enteric glia and immune cells modulates the development of visceral hypersensitivity. To this end, we manipulated a major mechanism of glial intercellular communication that requires connexin-43 and assessed the effects on acute and chronic inflammation, visceral hypersensitivity, and immune responses. Deleting connexin-43 in glia protected against the development of visceral hypersensitivity following chronic colitis. Mechanistically, the protective effects of glial manipulation were mediated by disrupting the glial-mediated activation of macrophages through the macrophage colony-stimulating factor. Collectively, our data identified enteric glia as a critical link between gastrointestinal neural and immune systems that could be harnessed by therapies to ameliorate abdominal pain., Graphical Abstract, In Brief Grubišić et al. report that enteric glia regulate macrophage activation and visceral sensitivity following intestinal inflammation through mechanisms that require glial connexin-43 (Cx43) and macrophage colony-stimulating factor (M-CSF) production. Proinflammatory signals induce glial Cx43-dependent M-CSF production through protein kinase C (PKC) and tumor necrosis factor (TNF)-alpha converting enzyme (TACE).

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.

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca(2+) ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4(-/-) mice. The P2Y1-selective agonist 2-methylthio-ADP increased [Ca(2+)]i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y1 receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y1 couples to and activates TRPV4. PKC inhibitors prevented P2Y1 receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.

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 diverse and promiscuous protein.

Transient Receptor Potential (TRP) channels are expressed throughout the gastrointestinal tract and have functional roles as detectors of taste and visceral sensation, as well as mediators of motility and secretion. Altered TRP expression or dysregulation of TRP channel function is associated with the etiology of gastrointestinal disease, including visceral pain, inflammatory bowel disease, pancreatitis and gastroesophageal reflux disease. This chapter summarizes the expression and functional roles of TRP channels in the gastrointestinal tract. The role of TRP channels in the development and maintenance of digestive diseases and the therapeutic potential of TRP channel modulators is also provided.