RESUMEN
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.
Asunto(s)
Dolor Abdominal/etiología , Páncreas/enzimología , Pancreatitis/complicaciones , Transducción de Señal , Tripsina/metabolismo , Dolor Abdominal/enzimología , Dolor Abdominal/patología , Dolor Abdominal/prevención & control , Enfermedad Aguda , Amilasas/sangre , Analgésicos/uso terapéutico , Animales , Azetidinas/farmacología , Bencilaminas/farmacología , Ceruletida , Modelos Animales de Enfermedad , Enteropeptidasa/metabolismo , Activación Enzimática , Humanos , Cinética , Masculino , Dimensión del Dolor , Páncreas/efectos de los fármacos , Páncreas/patología , Pancreatitis/inducido químicamente , Pancreatitis/tratamiento farmacológico , Pancreatitis/enzimología , Pancreatitis/patología , Peroxidasa/sangre , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor PAR-2/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Soja/farmacología , Médula Espinal/enzimología , Inhibidores de Tripsina/farmacologíaRESUMEN
Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased [Ca(2+)](i) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca(2+)](i) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.
Asunto(s)
Señalización del Calcio , Hiperalgesia/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/fisiología , Tripsina/metabolismo , Animales , Aprotinina/química , Señalización del Calcio/efectos de los fármacos , Capsaicina/farmacología , Edema/inducido químicamente , Edema/genética , Edema/metabolismo , Edema/patología , Enteropeptidasa/química , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Granulocitos/metabolismo , Granulocitos/patología , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/genética , Hiperalgesia/patología , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Nociceptores/metabolismo , Nociceptores/patología , Dimensión del Dolor , Ratas , Ratas Sprague-Dawley , Receptor PAR-1/deficiencia , Receptor PAR-2/deficiencia , Receptores Proteinasa-Activados/metabolismo , Receptores de Trombina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Tripsina/química , Tripsina/genética , Tripsina/farmacología , Inhibidores de Tripsina/químicaRESUMEN
BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.
Asunto(s)
Toxinas Bacterianas , Catepsina C/metabolismo , Enteritis/inducido químicamente , Enterotoxinas , Péptido Hidrolasas/metabolismo , Receptor PAR-2/metabolismo , Animales , Toxinas Bacterianas/farmacología , Catepsina C/deficiencia , Células Cultivadas , Colon/citología , Colon/metabolismo , Enteritis/etiología , Enteritis/metabolismo , Enteritis/patología , Enterotoxinas/farmacología , Granulocitos/patología , Ileítis/etiología , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/patología , Ratones , Ratones Noqueados , Sistema Nervioso/metabolismo , Peroxidasa/metabolismo , Receptor PAR-2/deficiencia , Receptores de Neuroquinina-1/metabolismo , Tripsina/metabolismo , Inhibidores de Tripsina/farmacología , Triptasas/farmacología , Regulación hacia ArribaRESUMEN
Intestinal epithelial cells can be induced to secrete the chemokine interleukin (IL)-8 during inflammation. The PAR-2 receptor is believed to play a proinflammatory role and is expressed in gut epithelial cells. The aim was to investigate PAR-2 signaling in Caco-2 intestinal epithelial cells, with respect to chemokine secretion. Activation of PAR-2 by high concentrations of the synthetic activating peptide (SLIGKV) did not induce secretion of IL-8, in contrast to stimulation with IL-1beta. However, upon simultaneous treatment with activating peptide and IL-1beta, a potentiating effect of PAR-2 stimulation was seen, resulting in a fivefold increase of IL-8. Available data suggest that NF-kappaB activation is required for IL-8 gene expression. Unlike IL-1beta, PAR-2 stimulation did not activate NF-kappaB, which may explain the lack of IL-8 expression. However, PAR-2 stimulation led to rapid phosphorylation of two MAP kinases, p38 MAPK and ERK1/2. ERK1/2 is known to activate the transcription factor AP-1, also involved in upregulation of IL-8 gene transcription. Inhibition of p38 MAPK led to decreased IL-8 following stimulation with IL-1beta and/or activating peptide. These results suggest that maximal IL-8 expression requires coordination of several signaling pathways. Thus, identifying antagonists to the PAR-2 receptor may be beneficial by inhibiting potentiation of a proinflammatory response, through inhibition of p38 and ERK MAP kinases.
Asunto(s)
Células Epiteliales/citología , Interleucina-1/biosíntesis , Intestinos/citología , Sistema de Señalización de MAP Quinasas , Receptor PAR-2/metabolismo , Animales , Western Blotting , Células CACO-2 , Línea Celular Tumoral , Quimiocinas/metabolismo , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Células HeLa , Humanos , Inflamación , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Ligandos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Péptidos/química , Fosforilación , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Factor de Transcripción AP-1/biosíntesis , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The glucocorticosteroid budesonide is one of the mainstay treatments in inflammatory bowel disease. The aim of this study was to investigate its effects on Caco-2 cells upon coculture with LPS stimulated macrophages, or with conditioned medium (CM) from the same. The mRNA expression of the chemokines IL-8 and ENA-78 were upregulated in Caco-2 cells, which was always more pronounced in the coculture, as compared to the CM situation, with the ENA-78 induction being higher than of IL-8. Addition of budesonide to the apical side of Caco-2 cells, decreased both IL-8 and ENA-78 mRNA levels in a dose dependent manner. A reduction in trans epithelial electrical resistance (TEER) and an increase in the apical-basolateral transport of fluoresceinated sulfonic acid (FS) were observed in the Caco-2 cells both in cocultures and CM experiments. The reduction in TEER was counteracted by budesonide, whereas it had no effect on the FS transport. We conclude that budesonide exert an anti-inflammatory effect directly on intestinal epithelial cells, which may contribute to the overall action of budesonide in the treatment of inflammatory bowel diseases.