RESUMO
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.
Assuntos
Receptores Ativados por Proteinase , Transdução de Sinais , Humanos , Transdução de Sinais/fisiologia , Receptores Acoplados a Proteínas G , Peptídeo Hidrolases/metabolismo , HomeostaseRESUMO
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
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
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.
Assuntos
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
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
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
Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR2) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR2-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR2 in NaV1.8-positive neurons (Par2Nav1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par2Nav1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice. Par2 deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR2 at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2 by biased mechanisms in HEK293 cells to induce Ca2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2 by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENT Oral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR2) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR2 Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR2 Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR2 to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, Lgmn is a biased agonist of PAR2 that evokes cancer pain.
Assuntos
Dor do Câncer/induzido quimicamente , Carcinoma de Células Escamosas/complicações , Cisteína Endopeptidases , Neoplasias Bucais/complicações , Receptor PAR-2/agonistas , Idoso , Idoso de 80 Anos ou mais , Animais , Arrestina/metabolismo , Dor do Câncer/psicologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Cisteína Endopeptidases/administração & dosagem , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteína Quinase C/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor PAR-2/genética , Microambiente Tumoral/efeitos dos fármacosRESUMO
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
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
Chronic pain is a hallmark of functional disorders, inflammatory diseases and cancer of the digestive system. The mechanisms that initiate and sustain chronic pain are incompletely understood, and available therapies are inadequate. This review highlights recent advances in the structure and function of pronociceptive and antinociceptive G protein-coupled receptors (GPCRs) that provide insights into the mechanisms and treatment of chronic pain. This knowledge, derived from studies of somatic pain, can guide research into visceral pain. Mediators from injured tissues transiently activate GPCRs at the plasma membrane of neurons, leading to sensitisation of ion channels and acute hyperexcitability and nociception. Sustained agonist release evokes GPCR redistribution to endosomes, where persistent signalling regulates activity of channels and genes that control chronic hyperexcitability and nociception. Endosomally targeted GPCR antagonists provide superior pain relief in preclinical models. Biased agonists stabilise GPCR conformations that favour signalling of beneficial actions at the expense of detrimental side effects. Biased agonists of µ-opioid receptors (MOPrs) can provide analgesia without addiction, respiratory depression and constipation. Opioids that preferentially bind to MOPrs in the acidic microenvironment of diseased tissues produce analgesia without side effects. Allosteric modulators of GPCRs fine-tune actions of endogenous ligands, offering the prospect of refined pain control. GPCR dimers might function as distinct therapeutic targets for nociception. The discovery that GPCRs that control itch also mediate irritant sensation in the colon has revealed new targets. A deeper understanding of GPCR structure and function in different microenvironments offers the potential of developing superior treatments for GI pain.
Assuntos
Dor Crônica/tratamento farmacológico , Dor Crônica/metabolismo , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Analgésicos/farmacologia , Animais , Humanos , Ligantes , Nociceptividade/efeitos dos fármacos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Fármacos do Sistema Sensorial/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vísceras/inervaçãoRESUMO
The tachykinins, exemplified by substance P, are one of the most intensively studied neuropeptide families. They comprise a series of structurally related peptides that derive from alternate processing of three Tac genes and are expressed throughout the nervous and immune systems. Tachykinins interact with three neurokinin G protein-coupled receptors. The signaling, trafficking, and regulation of neurokinin receptors have also been topics of intense study. Tachykinins participate in important physiological processes in the nervous, immune, gastrointestinal, respiratory, urogenital, and dermal systems, including inflammation, nociception, smooth muscle contractility, epithelial secretion, and proliferation. They contribute to multiple diseases processes, including acute and chronic inflammation and pain, fibrosis, affective and addictive disorders, functional disorders of the intestine and urinary bladder, infection, and cancer. Neurokinin receptor antagonists are selective, potent, and show efficacy in models of disease. In clinical trials there is a singular success: neurokinin 1 receptor antagonists to treat nausea and vomiting. New information about the involvement of tachykinins in infection, fibrosis, and pruritus justifies further trials. A deeper understanding of disease mechanisms is required for the development of more predictive experimental models, and for the design and interpretation of clinical trials. Knowledge of neurokinin receptor structure, and the development of targeting strategies to disrupt disease-relevant subcellular signaling of neurokinin receptors, may refine the next generation of neurokinin receptor antagonists.
Assuntos
Receptores de Taquicininas/metabolismo , Taquicininas/metabolismo , Animais , Humanos , Mucosa Intestinal/metabolismo , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Receptores de Taquicininas/genética , Taquicininas/genética , Bexiga Urinária/metabolismo , Bexiga Urinária/fisiopatologiaRESUMO
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
Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with ß-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.
Assuntos
Dor Crônica/etiologia , Endossomos/fisiologia , Síndrome do Intestino Irritável/fisiopatologia , Receptor PAR-2/fisiologia , Transdução de Sinais/fisiologia , Animais , Endocitose , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Nociceptividade , Nociceptores/fisiologia , Tripsina/farmacologiaRESUMO
Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR2) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR2 coupling to Gαq, Gαs, and ß-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR2 at distinct sites and activate it by biased mechanisms that induce coupling to Gαs, but not to Gαq or ß-arrestins. Because proteases activate PAR2 by irreversible cleavage, and activated PAR2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR2 from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR2-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gßγ to the Golgi, coinciding with PAR2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR2-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR2 responsiveness initially declined, consistent with PAR2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gßγ, and protein translation inhibited recovery of PAR2 responsiveness. PKD and Gßγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR2 mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteína Quinase C/metabolismo , Receptor PAR-2/metabolismo , Animais , Catepsinas/metabolismo , Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Hiperalgesia/prevenção & controle , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/antagonistas & inibidores , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Receptor PAR-2/agonistas , Transdução de Sinais/efeitos dos fármacos , Xantenos/administração & dosagem , Xantenos/farmacologiaRESUMO
Duodenogastroesophageal reflux (DGER) is associated with chronic lung disease. Bile acids (BAs) are established markers of DGER aspiration and are important risk factors for reduced post-transplant lung allograft survival by disrupting the organ-specific innate immunity, facilitating airway infection and allograft failure. However, it is unknown whether BAs also affect airway reactivity. We investigated the acute effects of 13 BAs detected in post-lung-transplant surveillance bronchial washings (BW) on airway contraction. We exposed precision-cut slices from human and mouse lungs to BAs and monitored dynamic changes in the cross-sectional luminal area of peripheral airways using video phase-contrast microscopy. We also used guinea pig tracheal rings in organ baths to study BA effects in proximal airway contraction induced by electrical field stimulation. We found that most secondary BAs at low micromolar concentrations strongly and reversibly relaxed smooth muscle and inhibited peripheral airway constriction induced by acetylcholine but not by noncholinergic bronchoconstrictors. Similarly, secondary BAs strongly inhibited cholinergic constrictions in tracheal rings. In contrast, TC-G 1005, a specific agonist of the BA receptor Takeda G protein-coupled receptor 5 (TGR5), did not cause airway relaxation, and Tgr5 deletion in knockout mice did not affect BA-induced relaxation, suggesting that this receptor is not involved. BAs inhibited acetylcholine-induced inositol phosphate synthesis in human airway smooth muscle cells overexpressing the muscarinic M3 receptor. Our results demonstrate that select BAs found in BW of patients with lung transplantation can affect airway reactivity by inhibiting the cholinergic contractile responses of the proximal and peripheral airways, possibly by acting as antagonists of M3 muscarinic receptors.
Assuntos
Acetilcolina/metabolismo , Ácidos e Sais Biliares/farmacologia , Broncoconstrição/efeitos dos fármacos , Pulmão/fisiopatologia , Animais , Broncoconstritores/farmacologia , Ácido Quenodesoxicólico/farmacologia , Estimulação Elétrica , Cobaias , Humanos , Fosfatos de Inositol/biossíntese , Pulmão/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Muscarínicos/metabolismo , Serotonina/farmacologia , Ácido Taurolitocólico/farmacologia , Traqueia/efeitos dos fármacosRESUMO
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
G-protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins. In the gastrointestinal tract, GPCRs expressed by epithelial cells sense contents of the lumen, and GPCRs expressed by epithelial cells, myocytes, neurons, and immune cells participate in communication among cells. GPCRs control digestion, mediate digestive diseases, and coordinate repair and growth. GPCRs are the target of more than one third of therapeutic drugs, including many drugs used to treat digestive diseases. Recent advances in structural, chemical, and cell biology research have shown that GPCRs are not static binary switches that operate from the plasma membrane to control a defined set of intracellular signals. Rather, GPCRs are dynamic signaling proteins that adopt distinct conformations and subcellular distributions when associated with different ligands and intracellular effectors. An understanding of the dynamic nature of GPCRs has provided insights into the mechanism of activation and signaling of GPCRs and has shown opportunities for drug discovery. We review the allosteric modulation, biased agonism, oligomerization, and compartmentalized signaling of GPCRs that control digestion and digestive diseases. We highlight the implications of these concepts for the development of selective and effective drugs to treat diseases of the gastrointestinal tract.
Assuntos
Digestão/fisiologia , Doenças do Sistema Digestório/tratamento farmacológico , Doenças do Sistema Digestório/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Regulação Alostérica , Dimerização , Descoberta de Drogas , Endossomos/metabolismo , HumanosRESUMO
BACKGROUND & AIMS: Mood disorders and constipation are often comorbid, yet their shared etiologies have rarely been explored. The neurotransmitter serotonin (5-HT) regulates central nervous system and enteric nervous system (ENS) development and long-term functions, including gastrointestinal (GI) motility and mood. Therefore, defects in neuron production of 5-HT might result in brain and intestinal dysfunction. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT biosynthesis. A variant of TPH2 that encodes the R441H substitution (TPH2-R441H) was identified in individuals with severe depression. We studied mice with an analogous mutation (TPH2-R439H), which results in a 60%-80% decrease in levels of 5-HT in the central nervous system and behaviors associated with depression in humans. Feeding chow that contains 5-HTP slow release (5-HTP SR) to TPH2-R439H mice restores levels of 5-HT in the central nervous system and reduces depressive-like behaviors. METHODS: We compared the effects of feeding chow, with or without 5-HTP SR, to mice with the TPH2-R439H mutation and without this mutation (control mice). Myenteric and submucosal plexuses were isolated from all 4 groups of mice, and immunocytochemistry was used to quantify total enteric neurons, serotonergic neurons, and 5-HT-dependent subsets of neurons. We performed calcium imaging experiments to evaluate responses of enteric neurons to tryptamine-evoked release of endogenous 5-HT. In live mice, we measured total GI transit, gastric emptying, small intestinal transit, and propulsive colorectal motility. To measure colonic migrating motor complexes (CMMCs), we isolated colons and constructed spatiotemporal maps along the proximodistal length to quantify the frequency, velocity, and length of CMMCs. We measured villus height, crypt perimeter, and relative densities of enterochromaffin and enteroendocrine cells in small intestinal tissue. RESULTS: Levels of 5-HT were significantly lower in enteric neurons from TPH2-R439H mice than from control mice. TPH2-R439H mice had abnormalities in ENS development and ENS-mediated GI functions, including reduced motility and intestinal epithelial growth. Total GI transit and propulsive colorectal motility were slower in TPH2-R439H mice than controls, and CMMCs were slower and less frequent. Villus height and crypt perimeter were significantly decreased in colon tissues from TPH2-R439H mice compared with controls. Administration of 5-HTP SR to adult TPH2-R439H mice restored 5-HT to enteric neurons and reversed these abnormalities. Adult TPH2-R439H mice given oral 5-HTP SR had normalized numbers of enteric neurons, total GI transit, and colonic motility. Intestinal tissue from these mice had normal measures of CMMCs and enteric epithelial growth CONCLUSIONS: In studies of TPH2-R439H mice, we found evidence for reduced release of 5-HT from enteric neurons that results in defects in ENS development and GI motility. Our findings indicate that neuron production of 5-HT links constipation with mood dysfunction. Administration of 5-HTP SR to mice restored 5-HT to the ENS and normalized GI motility and growth of the enteric epithelium. 5-HTP SR might be used to treat patients with intestinal dysfunction associated with low levels of 5-HT.
Assuntos
5-Hidroxitriptofano/administração & dosagem , Constipação Intestinal/tratamento farmacológico , Depressão/tratamento farmacológico , Trato Gastrointestinal/fisiopatologia , Serotonina/metabolismo , Animais , Constipação Intestinal/etiologia , Constipação Intestinal/fisiopatologia , Preparações de Ação Retardada/administração & dosagem , Depressão/complicações , Depressão/genética , Depressão/fisiopatologia , Modelos Animais de Doenças , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/fisiopatologia , Feminino , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/inervação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Resultado do Tratamento , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.
Assuntos
Proteína Semelhante a Receptor de Calcitonina/genética , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Nociceptividade/fisiologia , Dor/fisiopatologia , Transmissão Sináptica/efeitos dos fármacos , Antagonistas Adrenérgicos/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina/antagonistas & inibidores , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Capsaicina/antagonistas & inibidores , Capsaicina/farmacologia , Colestanóis/farmacologia , Clatrina/antagonistas & inibidores , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endossomos/efeitos dos fármacos , Formaldeído/antagonistas & inibidores , Formaldeído/farmacologia , Adjuvante de Freund/antagonistas & inibidores , Adjuvante de Freund/farmacologia , Regulação da Expressão Gênica , Injeções Espinhais , Masculino , Camundongos , Microtomia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nociceptividade/efeitos dos fármacos , Dor/induzido quimicamente , Dor/genética , Dor/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Ratos , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Técnicas de Cultura de TecidosRESUMO
G-CSF or CSF-3, originally defined as a regulator of granulocyte lineage development via its cell surface receptor (G-CSFR), can play a role in inflammation, and hence in many pathologies, due to its effects on mature lineage populations. Given this, and because pain is an extremely important arthritis symptom, the efficacy of an anti-G-CSFR mAb for arthritic pain and disease was compared with that of a neutrophil-depleting mAb, anti-Ly6G, in both adaptive and innate immune-mediated murine models. Pain and disease were ameliorated in Ag-induced arthritis, zymosan-induced arthritis, and methylated BSA/IL-1 arthritis by both prophylactic and therapeutic anti-G-CSFR mAb treatment, whereas only prophylactic anti-Ly6G mAb treatment was effective. Efficacy for pain and disease correlated with reduced joint neutrophil numbers and, importantly, benefits were noted without necessarily the concomitant reduction in circulating neutrophils. Anti-G-CSFR mAb also suppressed zymosan-induced inflammatory pain. A new G-CSF-driven (methylated BSA/G-CSF) arthritis model was established enabling us to demonstrate that pain was blocked by a cyclooxygenase-2 inhibitor, suggesting an indirect effect on neurons. Correspondingly, dorsal root ganglion neurons cultured in G-CSF failed to respond to G-CSF in vitro, and Csf3r gene expression could not be detected in dorsal root ganglion neurons by single-cell RT-PCR. These data suggest that G-CSFR/G-CSF targeting may be a safe therapeutic strategy for arthritis and other inflammatory conditions, particularly those in which pain is important, as well as for inflammatory pain per se.