RESUMEN
BACKGROUND AND PURPOSE: Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists. EXPERIMENTAL APPROACH: We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. KEY RESULTS: Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca(2+) ]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. CONCLUSIONS AND IMPLICATIONS: GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.
Asunto(s)
Inflamación/metabolismo , Nociceptores/metabolismo , Oligopéptidos/farmacología , Receptor PAR-2/agonistas , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligopéptidos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptor PAR-2/deficiencia , Receptor PAR-2/metabolismo , Relación Estructura-ActividadRESUMEN
UNLABELLED: Recent studies implicate TRPV4 receptors in visceral pain signaling and intestinal inflammation. Our aim was to evaluate the role of TRPV4 in the control of gastrointestinal (GI) motility and to establish the underlying mechanisms. We used immunohistochemistry and PCR to study TRPV4 expression in the GI tract. The effect of TRPV4 activation on GI motility was characterized using in vitro and in vivo motility assays. Calcium and nitric oxide (NO) imaging were performed to study the intracellular signaling pathways. Finally, TRPV4 expression was examined in the colon of healthy human subjects. We demonstrated that TRPV4 can be found on myenteric neurons of the colon and is co-localized with NO synthase (NOS-1). In vitro, the TRPV4 agonist GSK1016790A reduced colonic contractility and increased inhibitory neurotransmission. In vivo, TRPV4 activation slowed GI motility and reduced stool production in mouse models mimicking pathophysiological conditions. We also showed that TRPV4 activation inhibited GI motility by reducing NO-dependent Ca(2+) release from enteric neurons. In conclusion, TRPV4 is involved in the regulation of GI motility in health and disease. KEY MESSAGES: ⢠Recent studies implicate TRPV4 in pain signaling and intestinal inflammation. ⢠Our aim was to characterize the role of TRPV4 in the control of GI motility. ⢠We found that TRPV4 activation reduced colonic contractility. ⢠Our studies also showed altered TRPV4 mRNA expression in IBS-C patients. ⢠TRPV4 may be a novel pharmacological target in functional GI diseases.
Asunto(s)
Colon/fisiología , Motilidad Gastrointestinal/genética , Óxido Nítrico/metabolismo , Transmisión Sináptica/genética , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Colon/efectos de los fármacos , Colon/fisiopatología , Modelos Animales de Enfermedad , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Expresión Génica , Guanilato Ciclasa/metabolismo , Humanos , Síndrome del Colon Irritable/tratamiento farmacológico , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/metabolismo , Síndrome del Colon Irritable/fisiopatología , Leucina/análogos & derivados , Leucina/farmacología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Modelos Biológicos , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Plexo Mientérico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Sulfonamidas/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidoresRESUMEN
BACKGROUND: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. METHODS: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. KEY RESULTS: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). CONCLUSIONS & INFERENCES: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.
Asunto(s)
Catepsinas/análisis , Colitis/enzimología , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Animales , Colitis/inducido químicamente , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND PURPOSE: Protease-activated receptor 2 (PAR2) is expressed on nociceptive neurons, and can sensitize transient receptor potential (TRP) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not fully understood. PAR2 causes receptor-operated activation of TRPV4 channels and TRPV4 null mice have attenuated PAR2-stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR2-induced TRPV4 channel activation and pain. EXPERIMENTAL APPROACH: Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 channels (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 channels (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (thapsigargin), Gαq (UBO-QIC), tyrosine kinases (bafetinib and dasatinib) or PI3 kinases (wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2 -induced mechanical hyperalgesia were assessed in mice. KEY RESULTS: In non-transfected HEK293 cells, PAR2 activation transiently increased intracellular calcium ([Ca(2+) ]i ). Functional expression of TRPV4 channels caused a sustained increase of [Ca(2+) ]i , inhibited by HC067047, bafetinib and wortmannin; but not by thapsigargin, UBO-QIC, dasatinib or LY294002. Bafetinib but not dasatinib inhibited PAR2-induced mechanical hyperalgesia in vivo. CONCLUSIONS AND IMPLICATIONS: This study supports a role for tyrosine kinases in PAR2-mediated receptor-operated gating of TRPV4 channels, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2-induced activation of TRPV4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy.
Asunto(s)
Hiperalgesia/metabolismo , Dolor/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Receptor PAR-2/antagonistas & inhibidores , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Células HEK293 , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Masculino , Ratones Endogámicos C57BL , Morfolinas/farmacología , Oligopéptidos/farmacología , Pirroles/farmacología , Receptor PAR-2/agonistas , Receptor PAR-2/metabolismo , Sulfonamidas/farmacología , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Tripsina/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Although serine proteases and agonists of protease-activated receptor 2 (PAR2) cause inflammation and pain, the spectrum of proteases that are activated by proinflammatory and algesic stimuli and their contribution to inflammatory pain are uncertain. EXPERIMENTAL APPROACH: Enzymic assays and selective inhibitors were used to characterize protease activity in mice after intraplantar injections of formalin, bradykinin, PAR2 activating peptide (AP) or vehicle. The capacity of these proteases and of recombinant mouse trypsin 4 to cleave fragments of PAR2 and to activate PAR2 in cell lines was determined. Protease inhibitors and par2 (-/-) mice were used to assess the contributions of proteases and PAR2 to pain and inflammation. KEY RESULTS: Intraplantar injection of formalin, bradykinin or PAR2-AP led to the activation of proteases that were susceptible to the serine protease inhibitor melagatran but resistant to soybean trypsin inhibitor (SBTI). Melagatran inhibited mouse trypsin 4, which degraded SBTI. Proteases generated in inflamed tissues cleaved PAR2-derived peptides. These proteases and trypsin 4 increased [Ca(2+) ]i in PAR2-transfected but not in untransfected cells, and melagatran suppressed this activity. Melagatran or PAR2 deletion suppressed oedema and mechanical hypersensitivity induced by intraplantar formalin, bradykinin and PAR2-AP, but had no effect on capsaicin-induced pain. CONCLUSIONS AND IMPLICATIONS: Diverse proinflammatory and algesic agents activate melagatran-sensitive serine proteases that cause inflammation and pain by a PAR2-mediated mechanism. By inducing self-activating proteases, PAR2 amplifies and sustains inflammation and pain. Serine protease inhibitors can attenuate the inflammatory and algesic effects of diverse stimuli, representing a useful therapeutic strategy.
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Inflamación/metabolismo , Dolor/metabolismo , Receptor PAR-2/metabolismo , Serina Proteasas/metabolismo , Animales , Azetidinas/farmacología , Bencilaminas/farmacología , Bradiquinina , Línea Celular , Femenino , Pie , Formaldehído , Inflamación/inducido químicamente , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligopéptidos , Dolor/inducido químicamente , Receptor PAR-2/agonistas , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Inhibidores de Serina Proteinasa/farmacología , Tripsina/metabolismoRESUMEN
Bile acids (BAs) are digestive secretions that are necessary for the emulsification and absorption of dietary fats. Given the episodic nature of BA secretion and intestinal re-absorption, the circulating and tissue levels of BAs, like those of the gut hormones, fluctuate in fasting and fed states, and BA levels and forms are markedly affected by disease. BAs exert widespread hormonal-like effects by activating receptors in the nucleus and at the plasma membrane. The nuclear steroid receptors mediate the genomic actions of BAs on BA, glucose and lipid homeostasis. GPBA (TGR5) is a G-protein coupled plasma membrane receptor for BAs that mediates many of the rapid, non-genomic actions of BAs. GPBA has been implicated in the control of glucose homeostasis, inflammation and liver functions. Recent observations have revealed an unexpected role for GPBA in the nervous system. GPBA is expressed by enteric neurons and enterochromaffin cells that control peristalsis, and GPBA mediates the prokinetic actions of BAs in the colon that have been known for millennia. GPBA is also present on primary spinal afferent and spinal neurons that are necessary for sensory transduction. BA-induced activation of GPBA in the sensory nervous system promotes scratching behaviours and analgesia, which may contribute to the pruritus and painless jaundice that are observed in some patients with chronic cholestatic disease, where circulating BA concentrations are markedly increased. Thus, GPBA has emerged as an intriguing target for diverse metabolic, inflammatory, digestive and sensory disorders, where agonists and antagonists may be of value.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Enfermedades Gastrointestinales/metabolismo , Glucosa/metabolismo , Humanos , Hepatopatías/metabolismo , Neurotransmisores/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Trastornos de la Sensación/metabolismoRESUMEN
The calcitonin-gene-related peptide (CGRP) receptor is a heterodimer of calcitonin-receptor-like receptor (CLR) and receptor-activity-modifying protein 1 (RAMP1). Despite the importance of CGRP in regulating gastrointestinal functions, nothing is known about the distribution and function of CLR/RAMP1 in the esophagus, where up to 90 % of spinal afferent neurons contain CGRP. We detected CLR/RAMP1 in the mouse esophagus using immunofluorescence and confocal laser scanning microscopy and examined their relationship with neuronal elements of the myenteric plexus. Immunoreactivity for CLR and RAMP1 colocalized with VGLUT2-positive intraganglionic laminar endings (IGLEs), which were contacted by CGRP-positive varicose axons presumably of spinal afferent origin, typically at sites of CRL/RAMP1 immunoreactivity. This provides an anatomical basis for interaction between spinal afferent fibers and IGLEs. Immunoreactive CLR and RAMP1 also colocalized in myenteric neurons. Thus, CGRP-containing spinal afferents may interact with both vagal IGLEs and myenteric neurons in the mouse esophagus, possibly modulating motility reflexes and inflammatory hypersensitivity.
Asunto(s)
Proteína Similar al Receptor de Calcitonina/metabolismo , Esófago/inervación , Esófago/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina/genética , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Neuronas Aferentes/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores/genética , Receptores de Péptido Relacionado con el Gen de Calcitonina/genética , Distribución Tisular , Proteína 2 de Transporte Vesicular de Glutamato/biosíntesisRESUMEN
BACKGROUND: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. METHODS: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. KEY RESULTS: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. CONCLUSIONS & INFERENCES: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.
Asunto(s)
Sistema Nervioso Entérico/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Animales , Western Blotting , Línea Celular , AMP Cíclico/biosíntesis , Técnica del Anticuerpo Fluorescente , Vaciamiento Gástrico , Motilidad Gastrointestinal , Tracto Gastrointestinal/anatomía & histología , Tracto Gastrointestinal/metabolismo , Inmunohistoquímica , Intestinos/inervación , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neuronas Motoras/fisiología , Plexo Mientérico/metabolismo , Óxido Nítrico/fisiología , ARN/biosíntesis , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND AND PURPOSE: Changes in extracellular fluid osmolarity, which occur after tissue damage and disease, cause inflammation and maintain chronic inflammatory states by unknown mechanisms. Here, we investigated whether the osmosensitive channel, transient receptor potential vanilloid 4 (TRPV4), mediates inflammation to hypotonic stimuli by a neurogenic mechanism. EXPERIMENTAL APPROACH: TRPV4 was localized in dorsal root ganglia (DRG) by immunofluorescence. The effects of TRPV4 agonists on release of pro-inflammatory neuropeptides from peripheral tissues and on inflammation were examined. KEY RESULTS: Immunoreactive TRPV4 was detected in DRG neurones innervating the mouse hindpaw, where it was co-expressed in some neurones with CGRP and substance P, mediators of neurogenic inflammation. Hypotonic solutions and 4alpha-phorbol 12,13-didecanoate, which activate TRPV4, stimulated neuropeptide release in urinary bladder and airways, sites of neurogenic inflammation. Intraplantar injection of hypotonic solutions and 4alpha-phorbol 12,13-didecanoate caused oedema and granulocyte recruitment. These effects were inhibited by a desensitizing dose of the neurotoxin capsaicin, antagonists of CGRP and substance P receptors, and TRPV4 gene knockdown or deletion. In contrast, antagonism of neuropeptide receptors and disruption of TRPV4 did not prevent this oedema. TRPV4 gene knockdown or deletion also markedly reduced oedema and granulocyte infiltration induced by intraplantar injection of formalin. CONCLUSIONS AND IMPLICATIONS: Activation of TRPV4 stimulates neuropeptide release from afferent nerves and induces neurogenic inflammation. This mechanism may mediate the generation and maintenance of inflammation after injury and during diseases, in which there are changes in extracellular osmolarity. Antagonism of TRPV4 may offer a therapeutic approach for inflammatory hyperalgesia and chronic inflammation.
Asunto(s)
Inflamación Neurogénica/fisiopatología , Neuropéptidos/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Modelos Animales de Enfermedad , Edema/fisiopatología , Líquido Extracelular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/metabolismo , Granulocitos/metabolismo , Soluciones Hipotónicas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Aferentes/metabolismo , Concentración Osmolar , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/genéticaRESUMEN
BACKGROUND AND PURPOSE: The metalloendopeptidase endothelin-converting enzyme 1 (ECE-1) is prominently expressed in the endothelium where it converts big endothelin to endothelin-1, a vasoconstrictor peptide. Although ECE-1 is found in endosomes in endothelial cells, the role of endosomal ECE-1 is unclear. ECE-1 degrades the pro-inflammatory neuropeptide substance P (SP) in endosomes to promote recycling and re-sensitization of its neurokinin 1 (NK(1)) receptor. We investigated whether ECE-1 regulates NK(1) receptor re-sensitization and the pro-inflammatory effects of SP in the endothelium. EXPERIMENTAL APPROACH: We examined ECE-1 expression, SP trafficking and NK(1) receptor re-sensitization in human microvascular endothelial cells (HMEC-1), and investigated re-sensitization of SP-induced plasma extravasation in rats. KEY RESULTS: HMEC-1 expressed all four ECE-1 isoforms (a-d), and fluorescent SP trafficked to early endosomes containing ECE-1b/d. The ECE-1 inhibitor SM-19712 prevented re-sensitization of SP-induced Ca2+ signals in HMEC-1 cells. Immunoreactive ECE-1 and NK(1) receptors co-localized in microvascular endothelial cells in the rat. SP-induced extravasation of Evans blue in the urinary bladder, skin and ears of the rat desensitized when the interval between two SP injections was 10 min, and re-sensitized after 480 min. SM-19712 inhibited this re-sensitization. CONCLUSIONS AND IMPLICATIONS: By degrading endocytosed SP, ECE-1 promotes the recycling and re-sensitization of NK(1) receptors in endothelial cells, and thereby induces re-sensitization of the pro-inflammatory effects of SP. Thus, ECE-1 inhibitors may ameliorate the pro-inflammatory actions of SP.
Asunto(s)
Ácido Aspártico Endopeptidasas/fisiología , Metaloendopeptidasas/fisiología , Inflamación Neurogénica/metabolismo , Receptores de Neuroquinina-1/fisiología , Animales , Ácido Aspártico Endopeptidasas/biosíntesis , Calcio/metabolismo , Señalización del Calcio , Permeabilidad Capilar , Línea Celular , Oído/irrigación sanguínea , Endocitosis , Endosomas/metabolismo , Células Endoteliales/metabolismo , Enzimas Convertidoras de Endotelina , Endotelio Vascular/citología , Humanos , Masculino , Metaloendopeptidasas/biosíntesis , Microvasos/citología , Ratas , Ratas Sprague-Dawley , Receptores de Neuroquinina-1/biosíntesis , Piel/irrigación sanguínea , Sustancia P/metabolismo , Vejiga Urinaria/irrigación sanguíneaRESUMEN
Tachykinins, acting through NK(3) receptors (NK(3)R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is dependent on protein kinase C (PKC), its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in IPANs of the guinea pig ileum, demonstrated by double staining with the IPAN marker NeuN. PKCepsilon was also colocalized with PKD1/2 in IPANs. PKCepsilon and PKD1/2 trafficking was studied in enteric neurons within whole mounts of the ileal wall. In untreated preparations, PKCepsilon and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK(3)R agonist senktide evoked a transient translocation of PKCepsilon and PKD1/2 from the cytosol to the plasma membrane and induced PKD1/2 phosphorylation at the plasma membrane. PKCepsilon translocation was maximal at 10 s and returned to the cytosol within 2 min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15 s and translocated to the cytosol by 2 min, where it remained active up to 30 min after NK(3)R stimulation. PKD1/2 activation was reduced by a PKCepsilon inhibitor and prevented by NK(3)R inhibition. NK(3)R-mediated PKCepsilon and PKD activation was confirmed in HEK293 cells transiently expressing NK(3)R and green fluorescent protein-tagged PKCepsilon, PKD1, PKD2, or PKD3. Senktide caused membrane translocation and activation of kinases within 30 s. After 15 min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus stimulation of NK(3)R activates PKCepsilon and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.
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Plexo Mientérico/fisiología , Proteína Quinasa C-epsilon/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Neuroquinina-3/fisiología , Acetatos/farmacología , Animales , Línea Celular , Diterpenos/farmacología , Femenino , Cobayas , Humanos , Íleon/inervación , Cinética , Masculino , Plexo Mientérico/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Fragmentos de Péptidos/farmacología , Forbol 12,13-Dibutirato/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteína Quinasa C-epsilon/genética , Proteína Quinasa D2 , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Quinolinas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuroquinina-3/agonistas , Receptores de Neuroquinina-3/antagonistas & inhibidores , Sustancia P/análogos & derivados , Sustancia P/farmacología , TransfecciónRESUMEN
Protease-activated receptors (PARs) belong to a family of G protein-coupled receptors activated by serine proteases via proteolytic cleavage. PARs are expressed on epithelial cells, endothelial cells, and leukocytes, indicating a role in controlling barrier function against external danger. During inflammation, microorganisms as well as host immune cells release various proteases activating PARs. Thus, PARs can be viewed as an integral component of the host antimicrobial alarm system. When stimulated, PARs regulate various functions of leukocytes in vivo and in vitro, revealing a novel pathway by which proteases affect innate immune responses. Understanding protease-immune interactions could lead to novel strategies for the treatment of infectious and immune-related diseases.
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Inmunidad Innata , Receptores Proteinasa-Activados/fisiología , Animales , Catepsina G , Catepsinas/fisiología , Células Dendríticas/fisiología , Granzimas/fisiología , Humanos , Macrófagos/fisiología , Mastocitos/fisiología , Monocitos/fisiología , Neutrófilos/fisiología , Serina Endopeptidasas/fisiología , Triptasas/fisiologíaRESUMEN
Calcitonin gene-related peptide (CGRP) is abundant in the central terminals of primary afferents. However, the function of CGRP receptors in the spinal cord remains unclear. CGRP receptors are heterodimers of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1). We studied the localization of CRLR and RAMP1 in the rat dorsal horn using well-characterized antibodies against them, which labeled numerous puncta in laminae I-II. In addition, RAMP1 was found in cell bodies, forming patches at the cell surface. The CRLR- and RAMP1-immunoreactive puncta were further characterized using double and triple labeling. Colocalization was quantified in confocal stacks using Imaris software. CRLR did not colocalize with primary afferent markers, indicating that these puncta were not primary afferent terminals. CRLR- and RAMP1-immunoreactive puncta contained synaptophysin and vesicular glutamate transporter-2 (VGLUT2), showing that they were glutamatergic presynaptic terminals. Electron microscopic immunohistochemistry confirmed that CRLR immunoreactivity was present in axonal boutons that were not in synaptic glomeruli. Using tyramide signal amplification for double labeling with the CRLR and RAMP1 antibodies, we found some clear instances of colocalization of CRLR with RAMP1 in puncta, but their overall colocalization was low. In particular, CRLR was absent from RAMP1-containing cells. Many of the puncta stained for CRLR and RAMP1 were labeled by anti-opioid and anti-enkephalin antibodies. CRLR and, to a lesser extent, RAMP1 also colocalized with adrenergic alpha(2C) receptors. Triple label studies demonstrated three-way colocalization of CRLR-VGLUT2-synaptophysin, CRLR-VGLUT2-opioids, and CRLR-opioids-alpha(2C) receptors. In conclusion, CRLR is located in glutamatergic presynaptic terminals in the dorsal horn that contain alpha(2C) adrenergic receptors and opioids. Some of these terminals contain RAMP1, which may form CGRP receptors with CRLR, but in others CRLR may form other receptors, possibly by dimerizing with RAMP2 or RAMP3. These findings suggest that CGRP or adrenomedullin receptors modulate opioid release in the dorsal horn.
Asunto(s)
Analgésicos Opioides/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Células del Asta Posterior/metabolismo , Terminales Presinápticos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Calcitonina/metabolismo , Vías Aferentes/metabolismo , Vías Aferentes/ultraestructura , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina , Ácido Glutámico/metabolismo , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Nociceptores/metabolismo , Células del Asta Posterior/ultraestructura , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteína 1 Modificadora de la Actividad de Receptores , Proteína 2 Modificadora de la Actividad de Receptores , Proteína 3 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Raíces Nerviosas Espinales/metabolismo , Raíces Nerviosas Espinales/ultraestructura , Transmisión Sináptica/fisiología , Sinaptofisina/análisis , Sinaptofisina/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/análisis , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Neurokinin-3 (NK(3)) receptors are prevalent within the substantia nigra (SN) and ventral tegmental area (VTA), where their activation can affect motor and motivational behaviors as well as cardiovascular function and stress responses. These actions are mediated, in part, by dopaminergic neurons in each region. To determine the relevant sites for activation of these receptors, we examined the electron microscopic localization of NK(3) receptors and tyrosine hydroxylase (TH), the catecholamine synthesizing enzyme in dopaminergic neurons in the SN and VTA of rat brain. In each region, immunogold-silver labeling for NK(3) receptors was detected in many somatodendritic profiles, some of which contained TH-immunoreactivity. NK(3)-immunogold particles were largely associated with endomembranes resembling smooth endoplasmic reticulum, and only occasionally located on the plasma membrane in TH-labeled dendrites. In comparison with these dendrites, non-TH immunoreactive dendrites contained significantly more total (VTA) and more plasmalemmal (VTA and SN) NK(3)-immunogold particles. In each region, NK(3) gold particles also were seen in axonal as well as glial profiles, some of which contacted TH-immunoreactive dendrites. The NK(3)-labeled axon terminals formed either symmetric or asymmetric, excitatory-type synapses, the latter of which were significantly more prevalent in the VTA, compared with SN. These results provide the first ultrastructural evidence indicating that NK(3) receptors are available in cytoplasmic reserve in dopaminergic neurons, but more immediately accessible at the plasmalemmal surface of non-dopaminergic dendrites in both the SN and VTA. The activation of these receptors, together with the NK(3) receptors in either the presynaptic axon terminals or glia may contribute to the diverse physiological effects of tachykinins in each region, and most prominently involving excitatory inputs to the VTA.
Asunto(s)
Dendritas/metabolismo , Receptores de Neuroquinina-3/metabolismo , Sustancia Negra/metabolismo , Área Tegmental Ventral/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Dendritas/ultraestructura , Dopamina/biosíntesis , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Neuroglía/metabolismo , Neuroglía/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Sustancia Negra/ultraestructura , Transmisión Sináptica/fisiología , Taquicininas/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/ultraestructuraRESUMEN
BACKGROUND AND AIMS: Substance P (SP) release from sensory nerves induces neurogenic inflammation. Neutral endopeptidase (NEP) degrades SP, thereby limiting its proinflammatory effects. Intestinal inflammation following Trichinella spiralis infection markedly downregulates NEP, resulting in diminished SP degradation, with unknown functional consequences. We hypothesised that diminished expression of NEP would exacerbate T spiralis induced enteritis. METHODS: NEP knockout (NEP-/-) and wild-type (NEP+/+) mice were infected with T spiralis and studied at 6, 12, 24, and 48 hours post infection (PI). Tissue inflammation was quantified by computerised cell counting and myeloperoxidase activity (MPO). The leucocyte adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), and SP were assessed by immunohistochemistry. RESULTS: Before infection, the lack of NEP was not associated with changes in mucosal cellularity or MPO activity. Twelve hours PI, NEP-/- mice showed a 2.5-fold increase in MPO activity at a time when values in NEP+/+ mice were still within normal limits. MPO activity and cellularity peaked at 24 hours PI. This was accompanied by increased staining for both ICAM-1 and SP in NEP-/- mice. Infusion of rhNEP to NEP-/- mice significantly reduced MPO activity 24 hours PI. CONCLUSIONS: These findings demonstrate that NEP downregulates the early onset of nematode intestinal inflammation and that increased bioavailability of SP and overexpression of ICAM-1 in NEP-/- mice likely play a role in the earlier onset of intestinal inflammation.
Asunto(s)
Parasitosis Intestinales/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/parasitología , Neprilisina/metabolismo , Sustancia P/metabolismo , Trichinella spiralis , Triquinelosis/metabolismo , Animales , Bradiquinina/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Intestinos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neprilisina/genéticaRESUMEN
BACKGROUND: Protease activated receptor-2 (PAR-2) is a transmembrane G protein coupled receptor preferentially activated by trypsin and tryptase. The protease activated receptors play an important role in most components of injury responses including cell proliferation, migration, matrix remodelling, and inflammation. Cigarette smoking causes an inflammatory process in the central airways, peripheral airways, lung parenchyma, and adventitia of pulmonary arteries. METHODS: To quantify the expression of PAR-2 in the central airways of smokers and non-smokers, surgical specimens obtained from 30 subjects undergoing lung resection for localised pulmonary lesions (24 with a history of cigarette smoking and six non-smoking control subjects) were examined. Central airways were immunostained with an antiserum specific for PAR-2 and PAR-2 expression was quantified using light microscopy and image analysis. RESULTS: PAR-2 expression was found in bronchial smooth muscle, epithelium, glands, and in the endothelium and smooth muscle of bronchial vessels. PAR-2 expression was similar in the central airways of smokers and non-smokers. When smokers were divided according to the presence of symptoms of chronic bronchitis and chronic airflow limitation, PAR-2 expression was increased in smooth muscle (median 3.8 (interquartile range 2.9-5.8) and 1.4 (1.07-3.4) respectively); glands (33.3 (18.2-43.8) and 16.2 (11.5-22.2), respectively); and bronchial vessels (54.2 (48.7-56.8) and 40.0 (36-40.4), respectively) of smokers with symptoms of chronic bronchitis with normal lung function compared with smokers with chronic airflow limitation (COPD), but the increase was statistically significant (p<0.005) only for bronchial vessels. CONCLUSIONS: PAR-2 is present in bronchial smooth muscle, glands, and bronchial vessels of both smokers and non-smokers. An increased expression of PAR-2 was found in bronchial vessels of patients with bronchitis compared with those with COPD.
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Bronquios/metabolismo , Receptores de Trombina/metabolismo , Fumar/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Volumen Espiratorio Forzado/fisiología , Humanos , Masculino , Músculo Liso/metabolismo , Receptor PAR-2 , Músculos Respiratorios/metabolismo , Fumar/patología , Fumar/fisiopatología , Capacidad Vital/fisiologíaRESUMEN
The effects of proteinase-activated receptor 2 (PAR2) agonists on the electrical properties of intact guinea pig ileal myenteric neurons were measured with intracellular microelectrodes. Approximately 52% of AH neurons and 41% of S neurons responded to pressure ejection of SLIGRL-NH(2) or trypsin with a prolonged depolarization that was often accompanied by increased excitability. When added to the bathing solution, trypsin caused a concentration-dependent depolarization of responding neurons with an estimated EC(50) value of 87 nM. Collectively, these novel observations indicate that PAR2 excites a proportion of myenteric neurons, which may contribute to dysmotility during intestinal inflammation.
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Íleon/efectos de los fármacos , Receptores de Trombina/agonistas , Animales , Motilidad Gastrointestinal , Cobayas , Íleon/inervación , Técnicas In Vitro , Potenciales de la Membrana , Microelectrodos , Plexo Mientérico/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Oligopéptidos/farmacología , Receptor PAR-2 , Tripsina/farmacologíaRESUMEN
Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction.
Asunto(s)
Bronquios/metabolismo , Músculo Liso/metabolismo , Receptores de Trombina/biosíntesis , Receptores de Trombina/fisiología , Bronquios/química , Bronquios/efectos de los fármacos , Células Cultivadas , Humanos , Macrófagos/fisiología , Contracción Muscular/efectos de los fármacos , Músculo Liso/química , Músculo Liso/efectos de los fármacos , ARN Mensajero/análisis , Receptor PAR-2 , Receptores de Trombina/agonistas , Receptores de Trombina/genéticaRESUMEN
Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.
Asunto(s)
Permeabilidad Capilar/fisiología , Neuronas/fisiología , Receptores de Trombina/metabolismo , Animales , Calcio/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Edema/inducido químicamente , Edema/metabolismo , Esófago/irrigación sanguínea , Esófago/efectos de los fármacos , Esófago/inervación , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Yeyuno/irrigación sanguínea , Yeyuno/efectos de los fármacos , Yeyuno/inervación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas del Receptor de Neuroquinina-1 , Neuronas/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Oligopéptidos/farmacología , Páncreas/irrigación sanguínea , Páncreas/efectos de los fármacos , Páncreas/inervación , Piperidinas/farmacología , Quinuclidinas/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptor PAR-1 , Receptores de Trombina/agonistas , Receptores de Trombina/química , Transducción de Señal/efectos de los fármacos , Estómago/irrigación sanguínea , Estómago/efectos de los fármacos , Estómago/inervación , Trombina/farmacología , Vejiga Urinaria/irrigación sanguínea , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/inervaciónRESUMEN
Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.