RESUMO
Background: Mechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution. Methods: In this study we profiled functional expression of TRPV4 using Ca2+ imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessed in vitro using videomapping and contraction assays. Results: The TRPV4 agonist GSK1016790A evoked Ca2+ signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFRα+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+ responses by a subpopulation of glial cells, followed by sustained Ca2+ signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+ release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examined in vitro. Conclusions: We reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility.
RESUMO
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.
Assuntos
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.
Assuntos
Endossomos , Nanomedicina , Animais , Humanos , Endossomos/química , Nanomedicina/métodos , Nanopartículas/química , Polímeros/química , Concentração de Íons de Hidrogênio , Sistemas de Liberação de MedicamentosRESUMO
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.
Assuntos
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
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.
Assuntos
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
Allosteric modulators (AMs) are molecules that can fine-tune signaling by G protein-coupled receptors (GPCRs). Although they are a promising therapeutic approach for treating a range of disorders, allosteric modulation of GPCRs in the context of the enteric nervous system (ENS) and digestive dysfunction remains largely unexplored. This study examined allosteric modulation of the delta opioid receptor (DOR) in the ENS and assessed the suitability of DOR AMs for the treatment of irritable bowel syndrome (IBS) symptoms using mouse models. The effects of the positive allosteric modulator (PAM) of DOR, BMS-986187, on neurogenic contractions of the mouse colon and on DOR internalization in enteric neurons were quantified. The ability of BMS-986187 to influence colonic motility was assessed both in vitro and in vivo. BMS-986187 displayed DOR-selective PAM-agonist activity and orthosteric agonist probe dependence in the mouse colon. BMS-986187 augmented the inhibitory effects of DOR agonists on neurogenic contractions and enhanced reflex-evoked DOR internalization in myenteric neurons. BMS-986187 significantly increased DOR endocytosis in myenteric neurons in response to the weakly internalizing agonist ARM390. BMS-986187 reduced the generation of complex motor patterns in the isolated intact colon. BMS-986187 reduced fecal output and diarrhea onset in the novel environment stress and castor oil models of IBS symptoms, respectively. DOR PAMs enhance DOR-mediated signaling in the ENS and have potential benefit for the treatment of dysmotility. This study provides proof of concept to support the use of GPCR AMs for the treatment of gastrointestinal motility disorders.NEW & NOTEWORTHY 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.
Assuntos
Sistema Nervoso Entérico/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Receptores Opioides delta/efeitos dos fármacos , Xantonas/farmacologia , Analgésicos Opioides/farmacologia , Benzamidas/farmacologia , Colo/efeitos dos fármacos , Sistema Nervoso Entérico/fisiopatologia , Motilidade Gastrointestinal/fisiologia , Humanos , Receptores Opioides/efeitos dos fármacos , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Receptores Opioides mu/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The neuropeptide substance P (SP) is important in pain and inflammation. SP activates the neurokinin-1 receptor (NK1R) to signal via Gq and Gs proteins. Neurokinin A also activates NK1R, but leads to selective Gq signaling. How two stimuli yield distinct G protein signaling at the same G protein-coupled receptor remains unclear. We determined cryogenic-electron microscopy structures of active NK1R bound to SP or the Gq-biased peptide SP6-11. Peptide interactions deep within NK1R are critical for receptor activation. Conversely, interactions between SP and NK1R extracellular loops are required for potent Gs signaling but not Gq signaling. Molecular dynamics simulations showed that these superficial contacts restrict SP flexibility. SP6-11, which lacks these interactions, is dynamic while bound to NK1R. Structural dynamics of NK1R agonists therefore depend on interactions with the receptor extracellular loops and regulate G protein signaling selectivity. Similar interactions between other neuropeptides and their cognate receptors may tune intracellular signaling.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais , Animais , Inflamação/metabolismoRESUMO
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 diverse 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 example, G protein-coupled receptors (GPCRs) expressed by primary afferent neurons or other cells in inflammatory pathways utilize TRPV4 as an effector protein to amplify 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.
Assuntos
Inflamação Neurogênica/metabolismo , Nociceptividade , Canais de Cátion TRPV/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
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.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Serotonina , Canais de Cátion TRPV , Trato Gastrointestinal Superior/efeitos dos fármacos , Animais , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Pulmão/citologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Serotonina/genética , Serotonina/metabolismo , Serotonina/farmacologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Trato Gastrointestinal Superior/citologia , Trato Gastrointestinal Superior/metabolismoRESUMO
G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.
Assuntos
Analgésicos/farmacologia , Colestanol/farmacologia , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Dor/tratamento farmacológico , Substância P/análogos & derivados , Analgésicos/química , Analgésicos/uso terapêutico , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colestanol/análogos & derivados , Colestanol/uso terapêutico , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Antagonistas dos Receptores de Neurocinina-1/química , Antagonistas dos Receptores de Neurocinina-1/uso terapêutico , Dor/metabolismo , Manejo da Dor , Substância P/química , Substância P/farmacologia , Substância P/uso terapêuticoRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Macrófagos/imunologia , Macrófagos/metabolismo , Transdução de Sinais , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Animais , Proteínas de Transporte , Gerenciamento Clínico , Suscetibilidade a Doenças , Metabolismo Energético , Humanos , Ligantes , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Mecanotransdução Celular , Terapia de Alvo Molecular , Ligação ProteicaRESUMO
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.
Assuntos
Buprenorfina/química , Ácidos Graxos/química , Corantes Fluorescentes/química , Morfina/química , Ocitocina/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Buprenorfina/metabolismo , Cristalização , Avaliação Pré-Clínica de Medicamentos , Ácidos Graxos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Morfina/metabolismo , Imagem Óptica , Ocitocina/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
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 Gαi/o and ß-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.
Assuntos
Leucina Encefalina-2-Alanina/farmacologia , Inflamação/complicações , Dor/tratamento farmacológico , Dor/metabolismo , Receptores Opioides delta/agonistas , Animais , Colo/inervação , Leucina Encefalina-2-Alanina/administração & dosagem , Células HEK293 , Humanos , Camundongos , Nanopartículas/administração & dosagem , Neurônios , Nociceptores/metabolismo , Receptores Opioides delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
A key concept in nanomedicine is encapsulating therapeutic or diagnostic agents inside nanoparticles to prolong blood circulation time and to enhance interactions with targeted cells. During circulation and depending on the selected application (e.g., cancer drug delivery or immune modulators), nanoparticles are required to possess low or high interactions with cells in human blood and blood vessels to minimize side effects or maximize delivery efficiency. However, analysis of cellular interactions in blood vessels is challenging and is not yet realized due to the diverse components of human blood and hemodynamic flow in blood vessels. Here, the first comprehensive method to analyze cellular interactions of both synthetic and commercially available nanoparticles under human blood flow conditions in a microvascular network is developed. Importantly, this method allows to unravel the complex interplay of size, charge, and type of nanoparticles on their cellular associations under the dynamic flow of human blood. This method offers a unique platform to study complex interactions of any type of nanoparticles in human blood flow conditions and serves as a useful guideline for the rational design of liposomes and polymer nanoparticles for diverse applications in nanomedicine.
Assuntos
Lipossomos , Nanopartículas , Hemodinâmica , Humanos , Microvasos , PolimerizaçãoRESUMO
Endothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled receptor (GPCR) protease acctivated receptor 1 (PAR1) and the ion channel transient receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell-cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1-TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca2+ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR-TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.
Assuntos
Inflamação/genética , Receptor PAR-1/genética , Receptor PAR-2/genética , Transdução de Sinais/genética , Canais de Cátion TRPV/genética , Animais , Cálcio/metabolismo , Permeabilidade Capilar/genética , Edema/genética , Edema/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
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.
Assuntos
Analgésicos Opioides/farmacologia , Colo/metabolismo , Sistema Nervoso Entérico/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Animais , Benzamidas/farmacologia , Células CHO , Cricetulus , Sistema Nervoso Entérico/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Técnicas de Introdução de Genes , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Luminescentes/genética , Camundongos , Morfina/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Multimerização Proteica/fisiologia , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Vermelha FluorescenteRESUMO
Peripheral and central neurons in the pain pathway are well equipped to detect and respond to extracellular stimuli such as pro-inflammatory mediators and neurotransmitters through the cell surface expression of receptors that can mediate rapid intracellular signaling. Following injury or infection, activation of cell surface G protein-coupled receptors (GPCRs) initiates cell signaling processes that lead to the generation of action potentials in neurons or inflammatory responses such as cytokine secretion by immune cells. However, it is now appreciated that cell surface events alone may not be sufficient for all receptors to generate their complete signaling repertoire. Following an initial wave of signaling at the cell surface, active GPCRs can engage with endocytic proteins such as the adaptor protein ß-arrestin (ßArr) to promote clathrin-mediated internalization. Classically, ßArr-mediated internalization of GPCRs was hypothesized to terminate signaling, yet for multiple GPCRs known to contribute to pain, it has been demonstrated that endocytosis can also promote a unique "second wave" of signaling from intracellular membranes, including those of endosomes and the Golgi, that is spatiotemporally distinct from initial cell-surface events. In the context of pain, understanding the cellular and molecular mechanisms that drive spatiotemporal signaling of GPCRs is invaluable for understanding how pain occurs and persists, and how current analgesics achieve efficacy or promote side-effects. This review article discusses the importance of receptor localization for signaling outcomes of pro- and anti-nociceptive GPCRs, and new analgesic opportunities emerging through the development of "location-biased" ligands that favor binding with intracellular GPCR populations.
RESUMO
Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NK1R) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NK1R in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NK1R-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NK1R antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed non-opioid treatment option for chronic pain.
Assuntos
Aprepitanto/administração & dosagem , Dor Crônica/tratamento farmacológico , Preparações de Ação Retardada/metabolismo , Nanopartículas/metabolismo , Antagonistas dos Receptores de Neurocinina-1/administração & dosagem , Animais , Aprepitanto/farmacocinética , Aprepitanto/uso terapêutico , Linhagem Celular , Dor Crônica/metabolismo , Sistemas de Liberação de Medicamentos , Endossomos/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos Endogâmicos C57BL , Antagonistas dos Receptores de Neurocinina-1/farmacocinética , Antagonistas dos Receptores de Neurocinina-1/uso terapêutico , Ratos , Receptores da Neurocinina-1/metabolismoRESUMO
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.
Assuntos
Benzamidas/farmacologia , Clatrina/metabolismo , Colo , Endocitose , Músculo Liso , Piridinas/farmacologia , Receptores Opioides delta/metabolismo , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Triazóis/farmacologia , Analgésicos Opioides/farmacologia , Animais , Colo/metabolismo , Colo/patologia , Colo/fisiopatologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Endossomos/metabolismo , Sistema Nervoso Entérico/metabolismo , Motilidade Gastrointestinal/fisiologia , Camundongos , Músculo Liso/metabolismo , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Fosforilação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides mu/metabolismo , Transdução de Sinais , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
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.