RESUMO
Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins ß-arrestin (ßARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and ßARR1/2 terminate plasma membrane Ca(2+) signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. ßARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from ßARRs, to recycle. Thus, both ECE-1 and ßARRs mediate the resensitization of NK1R Ca(2+) signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or ßARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of ßARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation.
Assuntos
Arrestinas/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Metaloendopeptidases/metabolismo , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Arrestinas/antagonistas & inibidores , Arrestinas/genética , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Membrana Celular/metabolismo , Endossomos/metabolismo , Enzimas Conversoras de Endotelina , Transferência Ressonante de Energia de Fluorescência , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloendopeptidases/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores da Neurocinina-1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , beta-ArrestinasRESUMO
BACKGROUND & AIMS: Abnormal delivery of bile acids (BAs) to the colon as a result of disease or therapy causes constipation or diarrhea by unknown mechanisms. The G protein-coupled BA receptor TGR5 (or GPBAR1) is expressed by enteric neurons and endocrine cells, which regulate motility and secretion. METHODS: We analyzed gastrointestinal and colon transit, as well as defecation frequency and water content, in wild-type, knockout, and transgenic mice (trg5-wt, tgr5-ko, and tgr5-tg, respectively). We analyzed colon tissues for contractility, peristalsis, and transmitter release. RESULTS: Deoxycholic acid inhibited contractility of colonic longitudinal muscle from tgr5-wt but not tgr5-ko mice. Application of deoxycholic acid, lithocholic acid, or oleanolic acid (a selective agonist of TGR5) to the mucosa of tgr5-wt mice caused oral contraction and caudal relaxation, indicating peristalsis. BAs stimulated release of the peristaltic transmitters 5-hydroxytryptamine and calcitonin gene-related peptide; antagonists of these transmitters suppressed BA-induced peristalsis, consistent with localization of TGR5 to enterochromaffin cells and intrinsic primary afferent neurons. tgr5-ko mice did not undergo peristalsis or transmitter release in response to BAs. Mechanically induced peristalsis and transmitter release were not affected by deletion of tgr5. Whole-gut transit was 1.4-fold slower in tgr5-ko than tgr5-wt or tgr5-tg mice, whereas colonic transit was 2.2-fold faster in tgr5-tg mice. Defecation frequency was reduced 2.6-fold in tgr5-ko and increased 1.4-fold in tgr5-tg mice compared with tgr5-wt mice. Water content in stool was lower (37%) in tgr5-ko than tgr5-tg (58%) or tgr5-wt mice (62%). CONCLUSIONS: The receptor TGR5 mediates the effects of BAs on colonic motility, and deficiency of TGR5 causes constipation in mice. These findings might mediate the long-known laxative properties of BAs, and TGR5 might be a therapeutic target for digestive diseases.
Assuntos
Colo/efeitos dos fármacos , Colo/fisiologia , Defecação/efeitos dos fármacos , Trânsito Gastrointestinal/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colo/metabolismo , Defecação/genética , Ácido Desoxicólico/farmacologia , Células Enterocromafins/efeitos dos fármacos , Células Enterocromafins/metabolismo , Fezes/química , Trânsito Gastrointestinal/genética , Mucosa Intestinal/efeitos dos fármacos , Ácido Litocólico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Ácido Oleanólico/farmacologia , Peristaltismo , Receptores Acoplados a Proteínas G/genética , Serotonina/metabolismo , Água/análiseRESUMO
Chronic pancreatitis (CP) is a devastating disease characterized by persistent and uncontrolled abdominal pain. Our lack of understanding is partially due to the lack of experimental models that mimic the human disease and also to the lack of validated behavioral measures of visceral pain. The ligand-gated cation channel transient receptor potential ankyrin 1 (TRPA1) mediates inflammation and pain in early experimental pancreatitis. It is unknown if TRPA1 causes fibrosis and sustained pancreatic pain. We induced CP by injecting the chemical agent trinitrobenzene sulfonic acid (TNBS), which causes severe acute pancreatitis, into the pancreatic duct of C57BL/6 trpa1(+/+) and trpa1(-/-) mice. Chronic inflammatory changes and pain behaviors were assessed after 2-3 wk. TNBS injection caused marked pancreatic fibrosis with increased collagen-staining intensity, atrophy, fatty replacement, monocyte infiltration, and pancreatic stellate cell activation, and these changes were reflected by increased histological damage scores. TNBS-injected animals showed mechanical hypersensitivity during von Frey filament probing of the abdomen, decreased daily voluntary wheel-running activity, and increased immobility scores during open-field testing. Pancreatic TNBS also reduced the threshold to hindpaw withdrawal to von Frey filament probing, suggesting central sensitization. Inflammatory changes and pain indexes were significantly reduced in trpa1(-/-) mice. In conclusion, we have characterized in mice a model of CP that resembles the human condition, with marked histological changes and behavioral measures of pain. We have demonstrated, using novel and objective pain measurements, that TRPA1 mediates inflammation and visceral hypersensitivity in CP and could be a therapeutic target for the treatment of sustained inflammatory abdominal pain.
Assuntos
Pancreatite Crônica/genética , Canais de Potencial de Receptor Transitório/genética , Animais , Sensibilização do Sistema Nervoso Central/genética , Modelos Animais de Doenças , Fibrose/genética , Inflamação/genética , Escala de Gravidade do Ferimento , Locomoção/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/patologia , Pancreatite Crônica/fisiopatologia , Canal de Cátion TRPA1 , Ácido Trinitrobenzenossulfônico/farmacologia , Dor Visceral/genéticaRESUMO
GPCR (G-protein-coupled receptor) signalling at the plasma membrane is under tight control. In the case of neuropeptides such as SP (substance P), plasma membrane signalling is regulated by cell-surface endopeptidases (e.g. neprilysin) that degrade extracellular neuropeptides, and receptor interaction with ß-arrestins, which uncouple receptors from heterotrimeric G-proteins and mediate receptor endocytosis. By recruiting GPCRs, kinases and phosphatases to endocytosed GPCRs, ß-arrestins assemble signalosomes that can mediate a second wave of signalling by internalized receptors. Endosomal peptidases, such as ECE-1 (endothelin-converting enzyme-1), can degrade SP in acidified endosomes, which destabilizes signalosomes and allows receptors, freed from ß-arrestins, to recycle and resensitize. By disassembling signalosomes, ECE-1 terminates ß-arrestin-mediated endosomal signalling. These mechanisms have been studied in model cell systems, and the relative importance of plasma membrane and endosomal signalling to complex pathophysiological processes, such as inflammation, pain and proliferation, is unclear. However, deletion or inhibition of metalloendopeptidases that control neuropeptide signalling at the plasma membrane and in endosomes has marked effects on inflammation. Neprilysin deletion exacerbates inflammation because of diminished degradation of pro-inflammatory SP. Conversely, inhibition of ECE-1 attenuates inflammation by preventing receptor recycling/resensitization, which is required for sustained pro-inflammatory signals from the plasma membrane. ß-Arrestin deletion also affects inflammation because of the involvement of ß-arrestins in pro-inflammatory signalling and migration of inflammatory cells. Knowledge of GPCR signalling in specific subcellular locations provides insights into pathophysiological processes, and can provide new opportunities for therapy. Selective targeting of ß-arrestin-mediated endosomal signalling or of mechanisms of receptor recycling/resensitization may offer more effective and selective treatments than global targeting of cell-surface signalling.
Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Inflamação/fisiopatologia , Neuropeptídeos/fisiologia , Transdução de Sinais , HumanosRESUMO
To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking providing up to 4-log-fold protection. In vivo, HER2-XPAT protein induces protease-dependent antitumor activity and is proteolytically stable in healthy tissues. In non-human primates, HER2-XPAT protein demonstrates a strong safety margin (>400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT protein cleavage is low and similar in plasma samples from healthy and diseased humans and non-human primates, supporting translatability of stability to patients. EGFR-XPAT protein confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.
Assuntos
Neoplasias , Linfócitos T , Animais , Humanos , Antígenos de Neoplasias/metabolismo , Receptores ErbB , Imunoterapia/efeitos adversos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Complexo CD3/metabolismoRESUMO
Cathepsins regulate premature trypsinogen activation within acinar cells, a key initial step in pancreatitis. The identity, origin, and causative roles of activated cathepsins in pancreatic inflammation and pain are not defined. By using a near infrared-labeled activity-based probe (GB123) that covalently modifies active cathepsins, we localized and identified activated cathepsins in mice with cerulein-induced pancreatitis and in pancreatic juice from patients with chronic pancreatitis. We used inhibitors of activated cathepsins to define their causative role in pancreatic inflammation and pain. After GB123 administration to mice with pancreatitis, reflectance and confocal imaging showed significant accumulation of the probe in inflamed pancreas compared with controls, particularly in acinar cells and macrophages, and in spinal cord microglia and neurons. Biochemical analysis of pancreatic extracts identified them as cathepsins B, L, and S (Cat-B, Cat-L, and Cat-S, respectively). These active cathepsins were also identified in pancreatic juice from patients with chronic pancreatitis undergoing an endoscopic procedure for the treatment of pain, indicating cathepsin secretion. The cathepsin inhibitor K11777 suppressed cerulein-induced activation of Cat-B, Cat-L, and Cat-S in the pancreas and ameliorated pancreatic inflammation, nocifensive behavior, and activation of spinal nociceptive neurons. Thus pancreatitis is associated with an increase in the active forms of the proteases Cat-B, Cat-L, and Cat-S in pancreatic acinar cells and macrophages, and in spinal neurons and microglial cells. Inhibition of cathepsin activation ameliorated pancreatic inflammation and pain. Activity-based probes permit identification of proteases that are predictive biomarkers of disease progression and response to therapy and may be useful noninvasive tools for the detection of pancreatic inflammation.
Assuntos
Catepsina B/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Pâncreas/metabolismo , Pancreatite/metabolismo , Células Acinares/metabolismo , Amilases/metabolismo , Animais , Feminino , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Neurônios/metabolismo , Dor/metabolismoRESUMO
BACKGROUND & AIMS: Transient receptor potential ankyrin (TRPA) 1, an excitatory ion channel expressed by sensory neurons, mediates somatic and visceral pain in response to direct activation or noxious mechanical stimulation. Although the intestine is routinely exposed to irritant alimentary compounds and inflammatory mediators that activate TRPA1, there is no direct evidence for functional TRPA1 receptors on enteric neurons, and the effects of TRPA1 activation on intestinal function have not been determined. We characterized expression of TRPA1 by enteric neurons and determined its involvement in the control of intestinal contractility and transit. METHODS: TRPA1 expression was characterized by reverse-transcription polymerase chain reaction and immunofluorescence analyses. TRPA1 function was examined by Ca(2+) imaging and by assays of contractile activity and transit. RESULTS: We detected TRPA1 messenger RNA in the mouse intestine and TRPA1 immunoreactivity in enteric neurons. The cecum and colon had immunoreactivity for neuronal TRPA1, but the duodenum did not. TRPA1 immunoreactivity was also detected in inhibitory motoneurons and descending interneurons, cholinergic neurons, and intrinsic primary afferent neurons. TRPA1 activators, including cinnamaldehyde, allyl isothiocyanate (AITC), and 4-hydroxynonenal, increased [Ca(2+)](i) in myenteric neurons. These were reduced by a TRPA1 antagonist (HC-030031) or deletion of Trpa1. TRPA1 activation inhibited contractility of the segments of colon but not stomach or small intestine of Trpa1(+/+) but not Trpa1(-/-) mice; this effect was reduced by tetrodotoxin or N(G)-nitro-l-arginine methyl ester. Administration of AITC by gavage did not alter gastric emptying or small intestinal transit, but luminal AITC inhibited colonic transit via TRPA1. CONCLUSIONS: Functional TRPA1 is expressed by enteric neurons, and activation of neuronal TRPA1 inhibits spontaneous neurogenic contractions and transit of the colon.
Assuntos
Esvaziamento Gástrico/fisiologia , Motilidade Gastrointestinal/fisiologia , Interneurônios/metabolismo , Neurônios Motores/metabolismo , Neurônios Aferentes/metabolismo , RNA Mensageiro/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Aldeídos/farmacologia , Animais , Carbacol/farmacologia , Ceco/efeitos dos fármacos , Ceco/inervação , Ceco/metabolismo , Ceco/fisiologia , Colo/efeitos dos fármacos , Colo/inervação , Colo/metabolismo , Colo/fisiologia , Duodeno/efeitos dos fármacos , Duodeno/inervação , Duodeno/metabolismo , Duodeno/fisiologia , Células Epiteliais/metabolismo , Feminino , Imunofluorescência , Gânglios/metabolismo , Mucosa Gástrica/metabolismo , Motilidade Gastrointestinal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Íleo/inervação , Íleo/metabolismo , Íleo/fisiologia , Interneurônios/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Isotiocianatos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estômago/efeitos dos fármacos , Estômago/inervação , Estômago/fisiologia , Substância P/farmacologia , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/agonistasRESUMO
BACKGROUND & AIMS: Although proteases control inflammation and pain, the identity, cellular origin, mechanism of action, and causative role of proteases that are activated during disease are not defined. We investigated the activation and function of cysteine cathepsins (Cat) in colitis. METHODS: Because protease activity, rather than expression, is regulated, we treated mice with fluorescent activity-based probes that covalently modify activated cathepsins. Activated proteases were localized by tomographic imaging of intact mice and confocal imaging of tissues, and were identified by electrophoresis and immunoprecipitation. We examined the effects of activated cathepsins on excitability of colonic nociceptors and on colonic pain, and determined their role in colonic inflammatory pain by gene deletion. RESULTS: Tomography and magnetic resonance imaging localized activated cathepsins to the inflamed colon of piroxicam-treated il10(-/-) mice. Confocal imaging detected activated cathepsins in colonic macrophages and spinal neurons and microglial cells of mice with colitis. Gel electrophoresis and immunoprecipitation identified activated Cat-B, Cat-L, and Cat-S in colon and spinal cord, and Cat-S was preferentially secreted into the colonic lumen. Intraluminal Cat-S amplified visceromotor responses to colorectal distension and induced hyperexcitability of colonic nociceptors, which required expression of protease-activated receptor-2. Cat-S deletion attenuated colonic inflammatory pain induced with trinitrobenzene sulfonic acid. CONCLUSIONS: Activity-based probes enable noninvasive detection, cellular localization, and proteomic identification of proteases activated during colitis and are potential diagnostic tools for detection of predictive disease biomarkers. Macrophage cathepsins are activated during colitis, and Cat-S activates nociceptors to induce visceral pain via protease-activated receptor-2. Cat-S mediates colitis pain and is a potential therapeutic target.
Assuntos
Catepsinas/metabolismo , Colite/complicações , Colite/metabolismo , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Receptor PAR-2/metabolismo , Dor Visceral/metabolismo , Animais , Catepsina B/metabolismo , Catepsina L/metabolismo , Colite/induzido quimicamente , Colo/metabolismo , Colo/patologia , Doença de Crohn , Modelos Animais de Doenças , Deleção de Genes , Interleucina-10/genética , Interleucina-10/metabolismo , Macrófagos/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nociceptores/metabolismo , Piroxicam/efeitos adversos , Receptor PAR-2/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND & AIMS: To investigate the peripheral sensory effects of repeated stress in patients with postinfectious irritable bowel syndrome (IBS), we tested whether stress following self-limiting bacterial colitis increases colonic dorsal root ganglia (DRG) nociceptive signaling. METHODS: C57BL/6 mice were infected with Citrobacter rodentium. Stress was induced using a 9-day water avoidance paradigm (days 21-30 after infection). Colonic DRG neuronal excitability was measured using perforated patch clamp techniques, in vitro multi-unit afferent recordings, and measurements of visceromotor reflexes. RESULTS: Combined stress and prior infection increased corticosterone and epinephrine levels, compared with infected animals, but did not alter the resolution of colonic inflammation. These changes were associated with increased neuronal excitability and parallel changes in multi-unit afferent recordings and visceromotor reflex thresholds. Protease activity was increased at day 30 following infection with C rodentium. Protease inhibitors markedly reduced the effects of colonic supernatants on neuronal excitability from C rodentium but not stressed animals. Colonic DRG neurons expressed messenger RNAs for the ß(2) adrenergic and glucocorticoid receptors; incubation with stress mediators recapitulated the effects on neuronal excitability observed with chronic stress alone. PAR2 activation with concentrations of the activating peptide SLIGRL that had no effect on neuronal excitability in controls caused marked increases in excitability when applied to neurons from chronically stressed animals. CONCLUSIONS: Stress, combined with prior acute colitis, results in exaggerated peripheral nociceptive signaling. Proteases and stress mediators can signal directly to colonic DRG neurons; further analysis of these pathways could provide new targets for treatment of patients with postinfectious IBS.
Assuntos
Citrobacter rodentium , Colite/complicações , Infecções por Enterobacteriaceae/fisiopatologia , Síndrome do Intestino Irritável/fisiopatologia , Nociceptores/fisiologia , Transdução de Sinais/fisiologia , Estresse Psicológico/fisiopatologia , Potenciais de Ação , Animais , Colo/enzimologia , Corticosterona/sangue , Infecções por Enterobacteriaceae/enzimologia , Infecções por Enterobacteriaceae/microbiologia , Ensaio de Imunoadsorção Enzimática , Epinefrina/sangue , Gânglios Espinais/fisiopatologia , Síndrome do Intestino Irritável/enzimologia , Síndrome do Intestino Irritável/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Peptídeo Hidrolases/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Psicológico/sangueRESUMO
Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR(2)), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR(2) and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Injections of a subinflammatory dose of exogenous trypsin increased c-Fos immunoreactivity, indicative of spinal nociceptive activation, but did not cause inflammation, as assessed by measuring serum amylase and myeloperoxidase activity and by histology. The same dose of trypsin IV and P23 increased some inflammatory end points and caused a more robust effect on nociception, which was blocked by melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant trypsin isoforms. To determine the contribution of endogenous activation of trypsin and its minor isoforms, recombinant enterokinase (ENK), which activates trypsins in the duodenum, was administered into the pancreas. Intraductal ENK caused nociception and inflammation that were diminished by polypeptide inhibitors, including soybean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by melagatran. Finally, the secretagogue cerulein induced pancreatic nociceptive activation and nocifensive behavior that were reversed by melagatran. Thus trypsin and its minor isoforms mediate pancreatic pain and inflammation. In particular, the inhibitor-resistant isoforms trypsin IV and P23 may be important in mediating prolonged pancreatic inflammatory pain in pancreatitis. Our results suggest that inhibitors of these isoforms could be novel therapies for pancreatitis pain.
Assuntos
Dor Abdominal/etiologia , Pâncreas/enzimologia , Pancreatite/complicações , Transdução de Sinais , Tripsina/metabolismo , Dor Abdominal/enzimologia , Dor Abdominal/patologia , Dor Abdominal/prevenção & controle , Doença Aguda , Amilases/sangue , Analgésicos/uso terapêutico , Animais , Azetidinas/farmacologia , Benzilaminas/farmacologia , Ceruletídeo , Modelos Animais de Doenças , Enteropeptidase/metabolismo , Ativação Enzimática , Humanos , Cinética , Masculino , Medição da Dor , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/enzimologia , Pancreatite/patologia , Peroxidase/sangue , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor PAR-2/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Soja/farmacologia , Medula Espinal/enzimologia , Inibidores da Tripsina/farmacologiaRESUMO
The excitatory ion channel transient receptor potential ankyrin-1 (TRPA1) is prominently expressed by primary afferent neurons and is a mediator of inflammatory pain. Inflammatory agents can directly activate [e.g., hydroxynonenal (HNE), prostaglandin metabolites] or indirectly sensitize [e.g., agonists of protease-activated receptor (PAR(2))] TRPA1 to induce somatic pain and hyperalgesia. However, the contribution of TRPA1 to visceral pain is unknown. We investigated the role of TRPA1 in visceral hyperalgesia by measuring abdominal visceromotor responses (VMR) to colorectal distention (CRD) after intracolonic administration of TRPA1 agonists [mustard oil (MO), HNE], sensitizing agents [PAR(2) activating peptide (PAR(2)-AP)], and the inflammatory agent trinitrobenzene sulfonic acid (TNBS) in trpa1(+/+) and trpa1(-/-) mice. Sensory neurons innervating the colon, identified by retrograde tracing, coexpressed immunoreactive TRPA1, calcitonin gene-related peptide, and substance P, expressed TRPA1 mRNA and responded to MO with depolarizing currents. Intracolonic MO and HNE increased VMR to CRD and induced immunoreactive c-fos in spinal neurons in trpa1+/+ but not in trpa1(-/-) mice. Intracolonic PAR(2)-AP induced mechanical hyperalgesia in trpa1+/+ but not in trpa1(-/-) mice. TNBS-induced colitis increased in VMR to CRD and induced c-fos in spinal neurons in trpa1(+/+) but not in trpa1(-/-) mice. Thus TRPA1 is expressed by colonic primary afferent neurons. Direct activation of TRPA1 causes visceral hyperalgesia, and TRPA1 mediates PAR(2)-induced hyperalgesia. TRPA1 deletion markedly reduces colitis-induced mechanical hyperalgesia in the colon. Our results suggest that TRPA1 has a major role in visceral nociception and may be a therapeutic target for colonic inflammatory pain.
Assuntos
Colite/fisiopatologia , Hiperalgesia/fisiopatologia , Dor/fisiopatologia , Canais de Potencial de Receptor Transitório/metabolismo , Fibras Aferentes Viscerais/fisiologia , Aldeídos/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colite/induzido quimicamente , Colo/inervação , Colo/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Vias Eferentes/fisiologia , Feminino , Hiperalgesia/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mostardeira , Nociceptores/efeitos dos fármacos , Nociceptores/fisiologia , Dor/induzido quimicamente , Óleos de Plantas/farmacologia , Gravidez , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/metabolismo , Medula Espinal/fisiologia , Substância P/metabolismo , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/genética , Fibras Aferentes Viscerais/efeitos dos fármacosRESUMO
The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.
Assuntos
Dor/metabolismo , Pancreatite/complicações , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Aldeídos/toxicidade , Animais , Inibidores de Cisteína Proteinase/toxicidade , Feminino , Gânglios Espinais/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Irritantes/toxicidade , Masculino , Camundongos , Camundongos Knockout , Mostardeira/toxicidade , Nociceptores/fisiologia , Dor/etiologia , Pâncreas/efeitos dos fármacos , Pâncreas/inervação , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/metabolismo , Óleos de Plantas/toxicidade , Medula Espinal/metabolismo , Canal de Cátion TRPA1 , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/genética , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/genéticaRESUMO
BACKGROUND: Volatile anesthetics such as isoflurane and halothane have been in clinical use for many years and represent the group of drugs most commonly used to maintain general anesthesia. However, despite their widespread use, the molecular mechanisms by which these drugs exert their effects are not completely understood. Recently, a seemingly paradoxical effect of general anesthetics has been identified: the activation of peripheral nociceptors by irritant anesthetics. This mechanism may explain the hyperalgesic actions of inhaled anesthetics and their adverse effects in the airways. METHODS: To test the hypothesis that irritant inhaled anesthetics activate the excitatory ion-channel transient receptor potential (TRP)-A1 and thereby contribute to hyperalgesia and irritant airway effects, we used the measurement of intracellular calcium concentration in isolated cells in culture. For our functional experiments, we used models of isolated guinea pig bronchi to measure bronchoconstriction and withdrawal threshold to mechanical stimulation with von Frey filaments in mice. RESULTS: Irritant inhaled anesthetics activate TRPA1 expressed in human embryonic kidney cells and in nociceptive neurons. Isoflurane induces mechanical hyperalgesia in mice by a TRPA1-dependent mechanism. Isoflurane also induces TRPA1-dependent constriction of isolated bronchi. Nonirritant anesthetics do not activate TRPA1 and fail to produce hyperalgesia and bronchial constriction. CONCLUSIONS: General anesthetics induce a reversible loss of consciousness and render the patient unresponsive to painful stimuli. However, they also produce excitatory effects such as airway irritation and they contribute to postoperative pain. Activation of TRPA1 may contribute to these adverse effects, a hypothesis that remains to be tested in the clinical setting.
Assuntos
Anestésicos Gerais/farmacologia , Broncoconstrição/fisiologia , Hiperalgesia/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Anestésicos Gerais/toxicidade , Animais , Broncoconstrição/efeitos dos fármacos , Linhagem Celular , Cobaias , Humanos , Hiperalgesia/induzido quimicamente , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, ß-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Neoplasias Colorretais , Mutação , Proteínas de Neoplasias , Paclitaxel/farmacologia , Taxoides/farmacologia , Trombospondinas , Microambiente Tumoral/efeitos dos fármacos , Via de Sinalização Wnt , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trombospondinas/antagonistas & inibidores , Trombospondinas/genética , Trombospondinas/metabolismo , Microambiente Tumoral/genética , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/ß-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.
Assuntos
Antineoplásicos/farmacologia , Mitose/efeitos dos fármacos , Taxoides/farmacologia , Proteínas Wnt/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Paclitaxel/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidoresRESUMO
Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements.
Assuntos
Tomografia por Emissão de Pósitrons/métodos , Embolia Pulmonar/diagnóstico por imagem , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Radiografia , Trombina/farmacologiaRESUMO
Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucine-enkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide- and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.
Assuntos
Ácidos e Sais Biliares/metabolismo , Percepção da Dor/efeitos dos fármacos , Dor/metabolismo , Prurido/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Potenciais de Ação , Animais , Ácidos e Sais Biliares/farmacologia , Ácidos e Sais Biliares/fisiologia , Capsaicina/farmacologia , Células Cultivadas , Colestase/complicações , Colestase/metabolismo , Derme/patologia , Encefalina Leucina/metabolismo , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Peptídeo Liberador de Gastrina/metabolismo , Expressão Gênica , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peptídeos Opioides/metabolismo , Peptídeos Opioides/fisiologia , Especificidade de Órgãos , Dor/etiologia , Técnicas de Patch-Clamp , Prurido/etiologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única , Medula Espinal/metabolismoRESUMO
In this study, the involvement of 5-HT2A receptors on mesenteric ischemia-reperfusion injury was examined in mice. Intestinal ischemia produced by 45 min occlusion of superior mesenteric artery was followed by 24h reperfusion (I/R). The 5-HT2A selective antagonist, ketanserin (0.5 mgkg(-1)) or the 5-HT2A agonist DOI (0.25 mgkg(-1)) was intravenously administered before ischemia and 8h after the beginning of reperfusion. The effects were compared with those obtained in sham operated animals (S). Ketanserin prevented the upper gastrointestinal transit delay induced by I/R (P<0.01), protected intestine from leukocyte recruitment as indicated by jejunal myeloperoxidase activity (P<0.05) and reverted Evans Blue extravasation elicited by I/R in lung, colon and jejunum (P<0.05). On the other hand, 5-HT2A activation by DOI mimicked the effects of I/R in S mice prolonging small intestine transit (P<0.05) and enhancing neutrophil accumulation in jejunal tissues (P<0.05). Furthermore, the reduction of ADP-induced platelet aggregation in plasma of I/R mice was prevented by ketanserin treatment. All together, these findings support the critical involvement of 5-HT2A receptor subtype in mediating the damage induced by mesenteric I/R in mice.
Assuntos
Receptor 5-HT2A de Serotonina/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Anfetaminas/farmacologia , Animais , Permeabilidade Capilar , Feminino , Trânsito Gastrointestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Intestinos/fisiologia , Ketanserina/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Peroxidação de Lipídeos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Malondialdeído/metabolismo , Artéria Mesentérica Superior , Camundongos , Peroxidase/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina , Antagonistas do Receptor 5-HT2 de Serotonina , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Fator de Necrose Tumoral alfa/sangueRESUMO
Nitric oxide (NO) involvement in intestinal ischemia-reperfusion (I/R) injury has been widely suggested but its protective or detrimental role remains still question of debate. Here, we examine the impact of supplementation or inhibition of NO availability on intestinal dysmotility and inflammation caused by mesenteric I/R in mice. Ischemia 45min and reperfusion 24h were performed by superior mesenteric artery occlusion in female Swiss mice. Saline-treated sham-operated (S) or normal mice without surgery (N) served as controls. Drugs were subcutaneously injected 0, 4, 8, and 18 h after ischemia. Upper gastrointestinal transit (GIT, estimated through black marker gavage), intestinal myeloperoxidase activity (MPO), intestinal malondialdehyde levels (MDA), Evans blue extravasation (EB), intestinal histological damage, and mean arterial pressure (MAP) were considered. In I/R mice, GIT was significantly delayed compared to S and N groups; MPO activity and EB extravasation enhanced, whereas MDA levels did not change. Compared to N and S groups, in I/R mice selective iNOS inhibitor P-BIT significantly prevented motor, MPO and EB changes; putative iNOS inhibitor aminoguanidine significantly counteracted GIT delay but not neutrophil recruitment and the increase in vascular permeability; NOS inhibitor l-NAME and NO precursor l-arginine were scarcely or no effective. Furthermore, in S mice aminoguanidine caused a significant increase of MPO activity reverted by H(1) histamine receptor antagonist pre-treatment. Unlike P-BIT, aminoguanidine and l-NAME injection increased MAP. These findings confirm a detrimental role for iNOS-derived NO overproduction during reperfusion. Aminoguanidine-associated neutrophil recruitment suggests that this drug could act through mechanisms additional to iNOS inhibition involving both eNOS blockade, as indicated by its hemodynamic effects, and indirect activation of H(1) histamine receptors.
Assuntos
Intestino Delgado/irrigação sanguínea , Intestino Delgado/enzimologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Animais , Pressão Sanguínea/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Trânsito Gastrointestinal/efeitos dos fármacos , Guanidinas/farmacologia , Intestino Delgado/patologia , Malondialdeído/análise , Camundongos , Camundongos Endogâmicos , NG-Nitroarginina Metil Éster/farmacologia , Peroxidase/análise , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
We hypothesized that proteinase-activated receptor-2 (PAR(2)) modulates intestinal injuries induced by ischemia/reperfusion. Ischemia (1 hour) plus reperfusion (6 hours) significantly delayed gastrointestinal transit (GIT) compared with sham operation. Intraduodenal injection of PAR(2)-activating peptide SLIGRL-NH(2) significantly accelerated transit in ischemia/reperfusion but not in sham-operated rats. GIT was significantly delayed in ischemia/reperfusion and sham-operated PAR(2)(-/-) mice compared with PAR(2)(+/+). SLIGRL-NH(2) significantly accelerated transit in ischemia/reperfusion in PAR(2)(+/+) but not in PAR(2)(-/-) mice. Prevention of mast cell degranulation with cromolyn, ablation of visceral afferents with capsaicin, and antagonism of calcitonin gene-related peptide (CGRP) and neurokinin-1 receptors with CGRP(8-37) and RP67580, respectively, abolished the SLIGRL-NH(2)-induced stimulatory effect on transit in ischemia/reperfusion. Tissue damage was significantly reduced by SLIGRL-NH(2); this effect was not observed in cromolyn-, capsaicin-, or RP67580-treated rats but was detected following CGRP(8-37). Intestinal PAR(2) mRNA levels were not affected by SLIGRL-NH(2) in ischemia/reperfusion. We propose that PAR(2) modulates GIT and tissue damage in intestinal ischemia/reperfusion by a mechanism dependent on mast cells and visceral afferents. PAR(2) effect on transit might be mediated by CGRP and substance P, whereas the effect on tissue damage appears to involve substance P but not CGRP. PAR(2) might be a signaling system in the neuroimmune communication in intestinal ischemia/reperfusion.