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BACKGROUND & AIMS: Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. METHODS: In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 µg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition. RESULTS: Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 µmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2. CONCLUSIONS: Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect.
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Nerve growth factor (NGF) monoclonal antibodies (mAb) are one of the few patient-validated non-opioid treatments for chronic pain, despite failing to gain FDA approval due to worsened joint damage in some osteoarthritis patients. Herein, we demonstrate that neuropilin-1 (NRP1) is a nociceptor-enriched co-receptor for NGF that is necessary for tropomyosin-related kinase A (TrkA) signaling of pain. NGF binds NRP1 with nanomolar affinity. NRP1 and G Alpha Interacting Protein C-terminus 1 (GIPC1), a NRP1/TrkA adaptor, are coexpressed with TrkA in human and mouse nociceptors. NRP1 small molecule inhibitors and blocking mAb prevent NGF-stimulated action potential firing and activation of Na+ and Ca2+ channels in human and mouse nociceptors and abrogate NGF-evoked and inflammatory nociception in mice. NRP1 knockdown blunts NGF-stimulated TrkA phosphorylation, kinase signaling and transcription, whereas NRP1 overexpression enhances NGF and TrkA signaling. As well as interacting with NGF, NRP1 forms a heteromeric complex with TrkA. NRP1 thereby chaperones TrkA from the biosynthetic pathway to the plasma membrane and then to signaling endosomes, which enhances NGF-induced TrkA dimerization, endocytosis and signaling. Knockdown of GIPC1, a PDZ-binding protein that scaffolds NRP1 and TrkA to myosin VI, abrogates NGF-evoked excitation of nociceptors and pain-like behavior in mice. We identify NRP1 as a previously unrecognized co-receptor necessary for NGF/TrkA pain signaling by direct NGF binding and by chaperoning TrkA to the plasma membrane and signaling endosomes via the adaptor protein GIPC1. Antagonism of NRP1 and GIPC1 in nociceptors offers a long-awaited alternative to systemic sequestration of NGF with mAbs for the treatment of pain.
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Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.
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Dor do Câncer , Neoplasias Bucais , Hormônios Peptídicos , Humanos , Camundongos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Calcitonina , Pró-Calcitonina , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Dor do Câncer/tratamento farmacológico , Células Endoteliais/metabolismo , Neoplasias Bucais/tratamento farmacológico , Microambiente TumoralRESUMO
ABSTRACT: Targeting the acidified inflammatory microenvironment with pH-sensitive opioids is a novel approach for managing visceral pain while mitigating side effects. The analgesic efficacy of pH-dependent opioids has not been studied during the evolution of inflammation, where fluctuating tissue pH and repeated therapeutic dosing could influence analgesia and side effects. Whether pH-dependent opioids can inhibit human nociceptors during extracellular acidification is unexplored. We studied the analgesic efficacy and side-effect profile of a pH-sensitive fentanyl analog, (±)- N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide (NFEPP), during the evolution of colitis induced in mice with dextran sulphate sodium. Colitis was characterized by granulocyte infiltration, histological damage, and acidification of the mucosa and submucosa at sites of immune cell infiltration. Changes in nociception were determined by measuring visceromotor responses to noxious colorectal distension in conscious mice. Repeated doses of NFEPP inhibited nociception throughout the course of disease, with maximal efficacy at the peak of inflammation. Fentanyl was antinociceptive regardless of the stage of inflammation. Fentanyl inhibited gastrointestinal transit, blocked defaecation, and induced hypoxemia, whereas NFEPP had no such side effects. In proof-of-principle experiments, NFEPP inhibited mechanically provoked activation of human colonic nociceptors under acidic conditions mimicking the inflamed state. Thus, NFEPP provides analgesia throughout the evolution of colitis with maximal activity at peak inflammation. The actions of NFEPP are restricted to acidified layers of the colon, without common side effects in normal tissues. N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide could provide safe and effective analgesia during acute colitis, such as flares of ulcerative colitis.
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Colite , Dor Visceral , Camundongos , Humanos , Animais , Analgésicos Opioides/farmacologia , Analgésicos Opioides/uso terapêutico , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colo , Analgésicos/farmacologia , Inflamação/patologia , Dor Visceral/patologia , Fentanila/farmacologia , Fentanila/uso terapêutico , Concentração de Íons de HidrogênioRESUMO
ABSTRACT: Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that binds numerous ligands including vascular endothelial growth factor A (VEGFA). Binding of this ligand to NRP-1 and the co-receptor, the tyrosine kinase receptor VEGFR2, elicits nociceptor sensitization resulting in pain through the enhancement of the activity of voltage-gated sodium and calcium channels. We previously reported that blocking the interaction between VEGFA and NRP-1 with the Spike protein of SARS-CoV-2 attenuates VEGFA-induced dorsal root ganglion (DRG) neuronal excitability and alleviates neuropathic pain, pointing to the VEGFA/NRP-1 signaling as a novel therapeutic target of pain. Here, we investigated whether peripheral sensory neurons and spinal cord hyperexcitability and pain behaviors were affected by the loss of NRP-1. Nrp-1 is expressed in both peptidergic and nonpeptidergic sensory neurons. A CRIPSR/Cas9 strategy targeting the second exon of nrp-1 gene was used to knockdown NRP-1. Neuropilin-1 editing in DRG neurons reduced VEGFA-mediated increases in CaV2.2 currents and sodium currents through NaV1.7. Neuropilin-1 editing had no impact on voltage-gated potassium channels. Following in vivo editing of NRP-1, lumbar dorsal horn slices showed a decrease in the frequency of VEGFA-mediated increases in spontaneous excitatory postsynaptic currents. Finally, intrathecal injection of a lentivirus packaged with an NRP-1 guide RNA and Cas9 enzyme prevented spinal nerve injury-induced mechanical allodynia and thermal hyperalgesia in both male and female rats. Collectively, our findings highlight a key role of NRP-1 in modulating pain pathways in the sensory nervous system.
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Neuralgia , Fator A de Crescimento do Endotélio Vascular , Animais , Feminino , Masculino , Ratos , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Células Receptoras Sensoriais/metabolismo , Sódio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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 (NK1R), Gαq/i, and ßarrestin-2. Whereas the FDA-approved NK1R 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 NK1R+ve neurons in knockin mice expressing human NK1R, 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 NK1R, 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 NK1R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK1R 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 diverse diseases.
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Endossomos , Receptores da Neurocinina-1 , Camundongos , Humanos , Animais , Receptores da Neurocinina-1/genética , Aprepitanto/farmacologia , Substância P/farmacologia , Receptores Acoplados a Proteínas G , Dor/tratamento farmacológicoRESUMO
In this perspective, we outline a new opportunity for exploiting nanoparticle delivery of antagonists to target G-protein coupled receptors localized in intracellular compartments. We discuss the specific example of antagonizing endosomal receptors involved in pain to develop long-lasting analgesics but also outline the broader application potential of this delivery approach. We discuss the materials used to target endosomal receptors and indicate the design requirements for future successful applications.
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Endossomos , Nanomedicina , Animais , Humanos , Endossomos/química , Nanomedicina/métodos , Nanopartículas/química , Polímeros/química , Concentração de Íons de Hidrogênio , Sistemas de Liberação de MedicamentosRESUMO
ABSTRACT: Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1 to 3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized nonevoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibitors of clathrin, Dnm, and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm, and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.
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Neuralgia , Nociceptores , Ratos , Animais , Humanos , Nociceptores/fisiologia , Hiperalgesia/metabolismo , Nociceptividade/fisiologia , Ratos Sprague-Dawley , Transmissão Sináptica , Neuralgia/metabolismo , Células do Corno Posterior/metabolismo , Corno Dorsal da Medula Espinal , Gânglios Espinais/fisiologiaRESUMO
Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.
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Receptores Ativados por Proteinase , Transdução de Sinais , Humanos , Transdução de Sinais/fisiologia , Receptores Acoplados a Proteínas G , Peptídeo Hidrolases/metabolismo , HomeostaseRESUMO
Granzyme K (GzmK) is a tryptic member of the granzyme family of chymotrypsin-like serine proteases produced by cells of the immune system. Previous studies have indicated that GzmK activates protease-activated receptor 1 (PAR1) enhancing activation of monocytes and wound healing in endothelial cells. Here, we show using peptides and full length proteins that GzmK and, to a lesser extent the related protease GzmA, are capable of activating PAR1 and PAR2. These cleavage events occur at the canonical arginine P1 residue and involve exosite interactions between protease and receptor. Despite cleaving PAR2 at the same point as trypsin, GzmK does not induce a classical Ca2+ flux but instead activates a distinct signalling cascade, involving recruitment of ß-arrestin and phosphorylation of ERK. In epithelial A549 cells, PAR2 activation by GzmK results in the release of inflammatory cytokines IL-6 and IL-8. These data suggest that during an immune response GzmK acts as a pro-inflammatory regulator, rather than as a cytotoxin.
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Receptor PAR-1 , Receptor PAR-2 , Endopeptidases/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Granzimas/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismoRESUMO
BACKGROUND & AIMS: More than half of Crohn's disease patients develop intestinal fibrosis-induced intestinal strictures. Elafin is a human protease inhibitor that is down-regulated in the stricturing intestine of Crohn's disease patients. We investigated the efficacy of elafin in reversing intestinal fibrosis and elucidated its mechanism of action. METHODS: We developed a new method to mimic a stricturing Crohn's disease environment and induce fibrogenesis using stricturing Crohn's disease patient-derived serum exosomes to condition fresh human intestinal tissues and primary stricturing Crohn's disease patient-derived intestinal fibroblasts. Three mouse models of intestinal fibrosis, including SAMP1/YitFc mice, Salmonella-infected mice, and trinitrobenzene sulfonic acid-treated mice, were also studied. Elafin-Eudragit FS30D formulation and elafin-overexpressing construct and lentivirus were used. RESULTS: Elafin reversed collagen synthesis in human intestinal tissues and fibroblasts pretreated with Crohn's disease patient-derived serum exosomes. Proteome arrays identified cathepsin S as a novel fibroblast-derived pro-fibrogenic protease. Elafin directly suppressed cathepsin S activity to inhibit protease-activated receptor 2 activity and Zinc finger E-box-binding homeobox 1 expression, leading to reduced collagen expression in intestinal fibroblasts. Elafin overexpression reversed ileal fibrosis in SAMP1/YitFc mice, cecal fibrosis in Salmonella-infected mice, and colonic fibrosis in trinitrobenzene sulfonic acid-treated mice. Cathepsin S, protease-activated receptor 2 agonist, and zinc finger E-box-binding homeobox 1 overexpression abolished the anti-fibrogenic effect of elafin in fibroblasts and all 3 mouse models of intestinal fibrosis. Oral elafin-Eudragit FS30D treatment abolished colonic fibrosis in trinitrobenzene sulfonic acid-treated mice. CONCLUSIONS: Elafin suppresses collagen synthesis in intestinal fibroblasts via cathepsin S-dependent protease-activated receptor 2 inhibition and decreases zinc finger E-box-binding homeobox 1 expression. The reduced collagen synthesis leads to the reversal of intestinal fibrosis. Thus, modified elafin may be a therapeutic approach for intestinal fibrosis.
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Doença de Crohn , Obstrução Intestinal , Animais , Catepsinas , Colágeno , Constrição Patológica/metabolismo , Constrição Patológica/patologia , Doença de Crohn/patologia , Elafina , Fibrose , Humanos , Obstrução Intestinal/patologia , Intestinos/patologia , Camundongos , Peptídeo Hidrolases , Ácidos Polimetacrílicos , Inibidores de Proteases , Proteoma , Receptor PAR-2 , Ácido Trinitrobenzenossulfônico/toxicidade , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The mechanisms underlying chronic bladder conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder syndrome (OAB) are incompletely understood. However, targeting specific receptors mediating neuronal sensitivity to specific stimuli is an emerging treatment strategy. Recently, irritant-sensing receptors including the bile acid receptor TGR5, have been identified within the viscera and are thought to play a key role in neuronal hypersensitivity. Here, in mice, we identify mRNA expression of TGR5 (Gpbar1) in all layers of the bladder as well as in the lumbosacral dorsal root ganglia (DRG) and in isolated bladder-innervating DRG neurons. In bladder-innervating DRG neurons Gpbar1 mRNA was 100% co-expressed with Trpv1 and 30% co-expressed with Trpa1. In vitro live-cell calcium imaging of bladder-innervating DRG neurons showed direct activation of a sub-population of bladder-innervating DRG neurons with the synthetic TGR5 agonist CCDC, which was diminished in Trpv1-/- but not Trpa1-/- DRG neurons. CCDC also activated a small percentage of non-neuronal cells. Using an ex vivo mouse bladder afferent recording preparation we show intravesical application of endogenous (5α-pregnan-3ß-ol-20-one sulphate, Pg5α) and synthetic (CCDC) TGR5 agonists enhanced afferent mechanosensitivity to bladder distension. Correspondingly, in vivo intravesical administration of CCDC increased the number of spinal dorsal horn neurons that were activated by bladder distension. The enhanced mechanosensitivity induced by CCDC ex vivo and in vivo was absent using Gpbar1-/- mice. Together, these results indicate a role for the TGR5 receptor in mediating bladder afferent hypersensitivity to distension and thus may be important to the symptoms associated with IC/BPS and OAB.
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Cistite Intersticial , Retenção Urinária , Animais , Cistite Intersticial/metabolismo , Gânglios Espinais/metabolismo , Camundongos , Neurônios Aferentes/fisiologia , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Bexiga Urinária/metabolismoRESUMO
Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NK1R) in endosomes provide efficacious yet transient relief from chronic pain. These micellar nanoparticles are unstable and rapidly release cargo, which may limit the duration of analgesia. We examined the efficacy of stable star polymer nanostars containing the NK1R antagonist aprepitant-amine for the treatment of chronic pain in mice. Nanostars continually released cargo for 24 h, trafficked through the endosomal system, and disrupted NK1R endosomal signaling. After intrathecal injection, nanostars accumulated in endosomes of spinal neurons. Nanostar-aprepitant reversed mechanical, thermal and cold allodynia and normalized nociceptive behavior more efficaciously than free aprepitant in preclinical models of neuropathic and inflammatory pain. Analgesia was maintained for >10 h. The sustained endosomal delivery of antagonists from slow-release nanostars provides effective and long-lasting reversal of chronic pain.
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Dor Crônica , Antagonistas dos Receptores de Neurocinina-1 , Animais , Aprepitanto/farmacologia , Aprepitanto/uso terapêutico , Dor Crônica/tratamento farmacológico , Endossomos , Camundongos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Antagonistas dos Receptores de Neurocinina-1/uso terapêutico , Polímeros/farmacologiaRESUMO
Opioid tolerance (OT) leads to dose escalation and serious side effects, including opioid-induced hyperalgesia (OIH). We sought to better understand the mechanisms underlying this event in the gastrointestinal tract. Chronic in vivo administration of morphine by intraperitoneal injection in male C57BL/6 mice evoked tolerance and evidence of OIH in an assay of colonic afferent nerve mechanosensitivity; this was inhibited by the δ-opioid receptor (DOPr) antagonist naltrindole when intraperitoneally injected in previous morphine administration. Patch-clamp studies of DRG neurons following overnight incubation with high concentrations of morphine, the µ-opioid receptors (MOPr) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or the DOPr agonist [D-Ala2, D-Leu5]-Enkephalin evoked hyperexcitability. The pronociceptive actions of these opioids were blocked by the DOPr antagonist SDM25N but not the MOPr antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 The hyperexcitability induced by DAMGO was reversed after a 1 h washout, but reapplication of low concentrations of DAMGO or [D-Ala2, D-Leu5]-Enkephalin restored the hyperexcitability, an effect mediated by protein kinase C. DOPr-dependent DRG neuron hyperexcitability was blocked by the endocytosis inhibitor Pitstop 2, and the weakly internalizing DOPr agonist ARM390 did not cause hyperexcitability. Bioluminescence resonance energy transfer studies in HEK cells showed no evidence of switching of G-protein signaling from Gi to a Gs pathway in response to either high concentrations or overnight incubation of opioids. Thus, chronic high-dose opioid exposure leads to opioid tolerance and features of OIH in the colon. This action is mediated by DOPr signaling and is dependent on receptor endocytosis and downstream protein kinase C signaling.SIGNIFICANCE STATEMENT Opioids are effective in the treatment of abdominal pain, but escalating doses can lead to opioid tolerance and potentially opioid-induced hyperalgesia. We found that δ-opioid receptor (DOPr) plays a central role in the development of opioid tolerance and opioid-induced hyperalgesia in colonic afferent nociceptors following prolonged exposure to high concentrations of MOPr or DOPr agonists. Furthermore, the role of DOPr was dependent on OPr internalization and activation of a protein kinase C signaling pathway. Thus, targeting DOPr or key components of the downstream signaling pathway could mitigate adverse side effects by opioids.
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Analgésicos Opioides , Morfina , Analgésicos Opioides/efeitos adversos , Animais , Tolerância a Medicamentos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/uso terapêutico , Trato Gastrointestinal , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Morfina/uso terapêutico , Antagonistas de Entorpecentes/farmacologia , Proteína Quinase C , Receptores Opioides , Receptores Opioides mu , Transdução de SinaisRESUMO
Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR2, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR2 on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR2 mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.
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Neoplasias Bucais , Microambiente Tumoral , Animais , Capsaicina/metabolismo , Capsaicina/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias Bucais/metabolismo , Neurônios Aferentes/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
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 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-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, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and ß-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 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 PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.
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Colo/metabolismo , Endocitose/fisiologia , Corantes Fluorescentes/metabolismo , Inflamação/metabolismo , Dor/metabolismo , Receptor PAR-2/metabolismo , Animais , Arrestinas/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Feminino , Gânglios Espinais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Nociceptividade/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Endossomos/metabolismo , Hiperalgesia/fisiopatologia , Células de Schwann/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Hiperalgesia/diagnóstico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/genética , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismoRESUMO
OBJECTIVE: The effectiveness of µ-opioid receptor (MOPr) agonists for treatment of visceral pain is compromised by constipation, respiratory depression, sedation and addiction. We investigated whether a fentanyl analogue, (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP), which preferentially activates MOPr in acidified diseased tissues, would inhibit pain in a preclinical model of inflammatory bowel disease (IBD) without side effects in healthy tissues. DESIGN: Antinociceptive actions of NFEPP and fentanyl were compared in control mice and mice with dextran sodium sulfate colitis by measuring visceromotor responses to colorectal distension. Patch clamp and extracellular recordings were used to assess nociceptor activation. Defecation, respiration and locomotion were assessed. Colonic migrating motor complexes were assessed by spatiotemporal mapping of isolated tissue. NFEPP-induced MOPr signalling and trafficking were studied in human embryonic kidney 293 cells. RESULTS: NFEPP inhibited visceromotor responses to colorectal distension in mice with colitis but not in control mice, consistent with acidification of the inflamed colon. Fentanyl inhibited responses in both groups. NFEPP inhibited the excitability of dorsal root ganglion neurons and suppressed mechanical sensitivity of colonic afferent fibres in acidified but not physiological conditions. Whereas fentanyl decreased defecation and caused respiratory depression and hyperactivity in mice with colitis, NFEPP was devoid of these effects. NFEPP did not affect colonic migrating motor complexes at physiological pH. NFEPP preferentially activated MOPr in acidified extracellular conditions to inhibit cAMP formation, recruit ß-arrestins and evoke MOPr endocytosis. CONCLUSION: In a preclinical IBD model, NFEPP preferentially activates MOPr in acidified microenvironments of inflamed tissues to induce antinociception without causing respiratory depression, constipation and hyperactivity.
Assuntos
Colite , Neoplasias Colorretais , Doenças Inflamatórias Intestinais , Insuficiência Respiratória , Dor Visceral , Animais , Colite/induzido quimicamente , Colo , Constipação Intestinal , Fentanila/efeitos adversos , Humanos , Doenças Inflamatórias Intestinais/complicações , Camundongos , Receptores Opioides , Microambiente TumoralRESUMO
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.
Assuntos
Ácidos e Sais Biliares/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Humanos , Canais Iônicos/agonistas , Receptores de Calcitriol/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacosRESUMO
Pain is one of the most common medical conditions and affects more Americans than diabetes, heart disease, and cancer combined. Current pain treatments mainly rely on opioid analgesics and remain unsatisfactory. The life-threatening side effects and addictive properties of opioids demand new therapeutic approaches. Nanomedicine may be able to address these challenges as it allows for sensitive and targeted treatments without some of the burdens associated with current clinical pain therapies. This review discusses the physiology of pain, the current landscape of pain treatment, novel targets for pain treatment, and recent and ongoing efforts to effectively treat pain using nanotechnology-based approaches. We highl ight advances in nanoparticle-based drug delivery to reduce side effects, gene therapy to tackle the source of pain, and nanomaterials-based scavenging to proactively mediate pain signaling.