RESUMO
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.
Assuntos
Inflamação Neurogênica/fisiopatologia , Neuropeptídeos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Modelos Animais de Doenças , Edema/fisiopatologia , Líquido Extracelular/metabolismo , Feminino , Imunofluorescência , Gânglios Espinais/metabolismo , Granulócitos/metabolismo , Soluções Hipotônicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Aferentes/metabolismo , Concentração Osmolar , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/genéticaRESUMO
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.
Assuntos
Analgésicos Opioides/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Células do Corno Posterior/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores da Calcitonina/metabolismo , Vias Aferentes/metabolismo , Vias Aferentes/ultraestrutura , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina , Ácido Glutâmico/metabolismo , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Nociceptores/metabolismo , Células do Corno Posterior/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteína 1 Modificadora da Atividade de Receptores , Proteína 2 Modificadora da Atividade de Receptores , Proteína 3 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/ultraestrutura , Transmissão Sináptica/fisiologia , Sinaptofisina/análise , Sinaptofisina/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/análise , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
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.
Assuntos
Dendritos/metabolismo , Receptores da Neurocinina-3/metabolismo , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dendritos/ultraestrutura , Dopamina/biossíntese , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Substância Negra/ultraestrutura , Transmissão Sináptica/fisiologia , Taquicininas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/ultraestruturaRESUMO
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.
Assuntos
Enteropatias Parasitárias/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/parasitologia , Neprilisina/metabolismo , Substância P/metabolismo , Trichinella spiralis , Triquinelose/metabolismo , Animais , Bradicinina/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Intestinos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neprilisina/genéticaRESUMO
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.
Assuntos
Hiperalgesia/metabolismo , Dor/metabolismo , Receptores de Trombina/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Inflamação , Masculino , Camundongos , Camundongos Knockout , Prostaglandinas/fisiologia , Ratos , Ratos Wistar , Receptor PAR-2 , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/fisiologia , Receptores de Trombina/agonistas , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Substância P/fisiologiaRESUMO
Toxin A (TxA) of Clostridium difficile induces acute inflammation of the intestine initiated by release of substance P (SP) and activation of the neurokinin-1 receptor. However, the mechanisms that terminate this response are unknown. We determined whether the SP-degrading enzyme neutral endopeptidase (NEP, EC 3.4.24.11) terminates TxA-induced enteritis. We used both genetic deletion and pharmacological inhibition of NEP to test this hypothesis. In wild-type mice, instillation of TxA (0.5-5 microg) into ileal loops for 3 h dose dependently increased ileal fluid secretion, stimulated granulocyte transmigration determined by myeloperoxidase activity, and caused histological damage characterized by depletion of enterocytes, edema, and neutrophil accumulation. Deletion of NEP reduced the threshold secretory and inflammatory dose of TxA and exacerbated the inflammatory responses by more than twofold. This exacerbated inflammation was prevented by pretreatment with recombinant NEP. Conversely, pretreatment of wild-type mice with the NEP inhibitor phosphoramidon exacerbated enteritis. Thus NEP terminates enteritis induced by C. difficile TxA, underlying the importance of SP degradation in limiting neurogenic inflammation.
Assuntos
Toxinas Bacterianas/farmacologia , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/farmacologia , Neprilisina/genética , Animais , Anti-Inflamatórios/farmacologia , Enterocolite Pseudomembranosa/imunologia , Enterocolite Pseudomembranosa/metabolismo , Glicopeptídeos/farmacologia , Granulócitos/imunologia , Mucosa Intestinal/patologia , Secreções Intestinais/metabolismo , Camundongos , Camundongos Knockout , Neprilisina/antagonistas & inibidores , Neprilisina/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND & AIMS: N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have an important role in long-term potentiation and memory processing in the central nervous system. The aims in this study were to determine whether NMDA receptors are expressed in the peripheral nervous system and identify their role in mediating behavioral pain responses to colonic distention in the normal gut. METHODS AND RESULTS: Immunohistochemical localization of the NR1 subunit showed that NMDA receptors are expressed on the cell bodies and peripheral terminals of primary afferent nerves innervating the colon. Dorsal root ganglia neurons retrogradely labeled from the colon in short-term culture responded to addition of NMDA with increased intracellular [Ca2+]. Activation of peripheral NMDA receptors in colonic tissue sections caused Ca2+-dependent release of the proinflammatory neuropeptides, calcitonin gene-related peptide and substance P. Behavioral pain responses to noxious mechanical stimulation were inhibited in a reversible, dose-dependent manner by intravenous administration of memantine, a noncompetitive antagonist of the NMDA receptor. Single fiber recordings of decentralized pelvic nerves showed that colorectal distention responsive afferent nerve activity was inhibited by memantine. CONCLUSIONS: Peripheral NMDA receptors are important in normal visceral pain transmission, and may provide a novel mechanism for development of peripheral sensitization and visceral hyperalgesia.
Assuntos
Colo/fisiopatologia , Dor/fisiopatologia , Receptores de N-Metil-D-Aspartato/fisiologia , Sequência de Aminoácidos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Gânglios Espinais/química , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/análise , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Substância P/metabolismoRESUMO
Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Cães , Dinamina I , Dinaminas , Endocitose , Imunofluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Transfecção , Células Tumorais CultivadasRESUMO
Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Dermatite Alérgica de Contato/patologia , Dermatite Alérgica de Contato/prevenção & controle , Neprilisina/fisiologia , Substância P/toxicidade , Administração Cutânea , Animais , Anti-Inflamatórios não Esteroides/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/metabolismo , Permeabilidade Capilar/genética , Permeabilidade Capilar/imunologia , Capsaicina/administração & dosagem , Óleo de Cróton/toxicidade , Dermatite Alérgica de Contato/enzimologia , Dermatite Alérgica de Contato/genética , Dermatite Irritante/enzimologia , Dermatite Irritante/genética , Dermatite Irritante/patologia , Dermatite Irritante/prevenção & controle , Inibidores Enzimáticos/administração & dosagem , Feminino , Glicopeptídeos/administração & dosagem , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neprilisina/antagonistas & inibidores , Neprilisina/deficiência , Neprilisina/metabolismo , Antagonistas dos Receptores de Neurocinina-1 , Piperidinas/administração & dosagem , Quinuclidinas/administração & dosagem , Pele/irrigação sanguínea , Pele/enzimologia , Pele/patologiaRESUMO
Substance P (SP) induces plasma extravasation and neutrophil infiltration by activating the neurokinin-1 receptor (NK1-R). We characterized the mechanisms regulating this response in the rat pancreas. Anesthetized rats were continuously infused with SP, and plasma extravasation was quantified using Evans blue (EB) dye. Continuous infusion of SP (8 nmol. kg(-1). h(-1)) resulted in a threshold increase in EB at 15 min, a peak effect at 30 min (150% increase), and a return to baseline by 60 min. The NK1-R antagonist CP-96,345 blocked SP-induced plasma extravasation. After 60 min, the NK1-R was desensitized to agonist challenge. Resensitization was first detected at 20 min and increased until full recovery was seen at 30 min. Inhibition of the cell-surface protease neutral endopeptidase (NEP) by phosphoramidon potentiated the effect of exogenous SP; therefore endogenous NEP attenuates SP-induced plasma extravasation. Thus the continuous infusion of SP stimulates plasma extravasation in the rat pancreas via activation of the NK1-R, and these effects are terminated by both desensitization of the NK1-R and the cell-surface protease NEP.
Assuntos
Permeabilidade Capilar/fisiologia , Neprilisina/metabolismo , Pâncreas/irrigação sanguínea , Receptores da Neurocinina-1/fisiologia , Substância P/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Proteínas Sanguíneas/análise , Capilares/inervação , Capilares/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Capsaicina/farmacologia , Membrana Celular/fisiologia , Azul Evans/farmacocinética , Glicopeptídeos/farmacologia , Infusões Intravenosas , Masculino , Antagonistas dos Receptores de Neurocinina-1 , Neutrófilos/fisiologia , Inibidores de Proteases/farmacologia , Ratos , Ratos Sprague-Dawley , Substância P/administração & dosagemRESUMO
BACKGROUND: The neuropeptide substance P (SP) induces plasma extravasation and neutrophil infiltration by activating the neurokinin 1-receptor (NK1-R). SP-induced neurogenic inflammation is terminated by the cell surface enzyme neutral endopeptidase (NEP), which degrades SP. We determined whether genetic deletion of the NK1-R reduces mortality and, conversely, whether genetic deletion of NEP increases mortality in a lethal model of hemorrhagic pancreatitis. METHODS: Necrotizing pancreatitis was induced by feeding mice a diet deficient in choline and supplemented with ethionine. We determined the length of survival, the severity of pancreatitis (by measuring the neutrophil enzyme myeloperoxidase [MPO] and by histologic evaluation), and the severity of pancreatitis-associated lung injury (lung MPO and histology) in NK1-R (+/+)/(-/-) and NEP (+/+)/(-/-) mice. RESULTS: Genetic deletion of the NK1-R significantly improved survival (100% vs 8% at 120 hours, P <.001) and reduced pancreatic MPO and acinar cell necrosis. Conversely, genetic deletion of NEP significantly worsened survival (0% vs 90% at 120 hours, P <.001) and exacerbated pancreatic MPO and pancreatitis-associated lung injury. CONCLUSIONS: Substance P is an important determinant of lethality in this model of necrotizing pancreatitis. Defects in NEP expression could lead to uncontrolled inflammation.
Assuntos
Deficiência de Colina/fisiopatologia , Dieta , Pulmão/fisiopatologia , Pancreatite/fisiopatologia , Receptores da Neurocinina-1/fisiologia , Substância P/fisiologia , Doença Aguda , Animais , Morte , Etionina/farmacologia , Hemorragia , Inflamação , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Neprilisina/deficiência , Neprilisina/genética , Neprilisina/metabolismo , Neutrófilos/fisiologia , Pancreatite/etiologia , Pancreatite/patologia , Peroxidase/sangue , Receptores da Neurocinina-1/deficiência , Receptores da Neurocinina-1/genéticaRESUMO
Biological responses to neuropeptides are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell-surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. We have investigated the mechanisms that terminate the proinflammatory effects of the neuropeptide substance P (SP), which are mediated by the neurokinin 1 receptor (NK1R). Neutral endopeptidase degrades SP in the extracellular fluid and is one of the first mechanisms to terminate signalling. G-protein receptor kinases and second-messenger kinases phosphorylate the NK1R to permit interaction with beta-arrestins, which uncouple the receptor from G-proteins to terminate the signal. SP-induces NK1R endocytosis by a beta-arrestin-dependent mechanism, which also involves the GTPases dynamin and Rab5a. Endocytosis contributes to desensitization by depleting receptors from the cell surface. Disruption of these mechanisms results in uncontrolled stimulation and disease. Thus the deletion of neutral endopeptidase in mice exacerbates inflammation of many tissues. There are similarities and distinct differences in the mechanisms that regulate signalling by neuropeptide receptors and other G-protein-coupled receptors, in particular those that are activated irreversibly by proteolysis.
Assuntos
Endopeptidases/metabolismo , Receptores de Neuropeptídeos/metabolismo , Transdução de Sinais , Substância P/metabolismo , Animais , Arrestinas/metabolismo , Membrana Celular/metabolismo , Dinaminas , Endocitose , GTP Fosfo-Hidrolases/metabolismo , Humanos , Mastócitos/metabolismo , Camundongos , Modelos Biológicos , Neprilisina/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Receptor PAR-2 , Receptores da Neurocinina-1/metabolismo , Receptores de Trombina/metabolismo , Serina Endopeptidases/metabolismo , Fatores de Tempo , Triptases , beta-Arrestinas , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Pancreatic oedema occurs early in the development of acute pancreatitis, and the overall extent of fluid loss correlates with disease severity. The tachykinin substance P (SP) is released from sensory nerves, binds to the neurokinin-1 receptor (NK1-R) on endothelial cells and induces plasma extravasation, oedema, and neutrophil infiltration, a process termed neurogenic inflammation. We sought to determine the importance of neurogenic mechanisms in acute pancreatitis. Pancreatic plasma extravasation was measured using the intravascular tracers Evans blue and Monastral blue after administration of specific NK1-R agonists/antagonists in rats and NK1-R(+/+)/(-/-) mice. The effects of NK1-R genetic deletion/antagonism on pancreatic plasma extravasation, amylase, myeloperoxidase (MPO), and histology in cerulein-induced pancreatitis were characterized. In rats, both SP and the NK1-R selective agonist [Sar(9) Met(O(2))(11)]SP stimulated pancreatic plasma extravasation, and this response was blocked by the NK1-R antagonist CP 96,345. Selective agonists of the NK-2 or NK-3 receptors had no effect. In rats, cerulein stimulated pancreatic plasma extravasation and serum amylase. These responses were blocked by the NK1-R antagonist CP 96,345. In wildtype mice, SP induced plasma extravasation while SP had no effect in NK1-R knockout mice. In NK1-R knockout mice, the effects of cerulein on pancreatic plasma extravasation and hyperamylasemia were reduced by 60%, and pancreatic MPO by 75%, as compared to wildtype animals. Neurogenic mechanisms of inflammation are important in the development of inflammatory oedema in acute interstitial pancreatitis.
Assuntos
Edema/fisiopatologia , Inflamação/fisiopatologia , Pancreatite/fisiopatologia , Receptores da Neurocinina-1/efeitos dos fármacos , Substância P/fisiologia , Doença Aguda , Amilases/sangue , Animais , Pressão Sanguínea/efeitos dos fármacos , Ceruletídeo , Edema/patologia , Fármacos Gastrointestinais , Inflamação/patologia , Masculino , Camundongos , Antagonistas dos Receptores de Neurocinina-1 , Pancreatite/induzido quimicamente , Pancreatite/patologia , Peroxidase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Neurogenic inflammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inflammation, the mechanisms that terminate inflammation are unknown. We examined whether neutral endopeptidase (NEP), a cell-surface enzyme that degrades SP in the extracellular fluid, terminates neurogenic inflammation in the colon. In NEP knockout mice, the SP concentration in the colon was approximately 2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was approximately 4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inflammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inflammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular fluid under basal conditions and terminates its proinflammatory effects. Because we have previously shown that intestinal inflammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inflammation.
Assuntos
Colite/etiologia , Neprilisina/fisiologia , Substância P/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Colite/prevenção & controle , Dinitrofluorbenzeno/análogos & derivados , Dinitrofluorbenzeno/toxicidade , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neprilisina/genética , Antagonistas dos Receptores de Neurocinina-1 , Peroxidase/metabolismo , Piperidinas/farmacologia , Quinuclidinas/farmacologia , Proteínas Recombinantes/uso terapêuticoRESUMO
We investigated the effects of the sensory neuropeptide substance P (SP) on amylase and fluid secretion in the isolated vascularly perfused rat pancreas. SP inhibited CCK-induced amylase release and secretin-induced juice flow via the pancreatic duct in a dose-related fashion. Threshold inhibition occurred following addition of 10(-10) M SP to the perfusate, and maximal inhibition was seen with 10(-8) M SP. The effects of SP were partially blocked by both the neurokinin-1 (NK1) and neurokinin-2 (NK2) receptor antagonists. Atropine and TTX blocked SP-induced effects on both amylase secretion (26 and 63% blockade, respectively) and pancreatic juice flow (21 and 79% blockade, respectively). Excitation of pancreatic sensory nerves using capsaicin (in the absence of SP) inhibited both amylase and pancreatic juice flow via activation of the NK1 receptor. We conclude that SP inhibits exocrine secretion via an indirect neural mechanism.
Assuntos
Pâncreas/inervação , Pâncreas/metabolismo , Substância P/farmacologia , Animais , Atropina/farmacologia , Capsaicina/farmacologia , Masculino , Bloqueio Nervoso , Fenômenos Fisiológicos do Sistema Nervoso/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Pâncreas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-1/fisiologia , Tetrodotoxina/farmacologiaRESUMO
Cells immunoreactive for the mu-opioid receptor (MOR) in laminae I-II of the spinal cord were identified as small neurons with rostro-caudal dendrites. In spinal cord slices, [D-Ala2,MePhe4-Gly-ol5]enkephalin (DAMGO) or etorphine (1 microM) caused naloxone-sensitive MOR endocytosis in 100% of these neurons, whereas the selective delta- and kappa-opioid agonists [D-Pen2,5]enkephalin (DPDPE) and spiradoline mesylate (U-62,066), respectively, produced negligible internalization at 1 microM. The EC50 for DAMGO was 30 nM, similar to its potency to inhibit cAMP accumulation and to increase [gamma-35S]GTP binding. MOR internalization followed an exponential timecourse with a half-life of 1.7 min. MOR internalization in spinal cord slices was faster and occurred at lower agonist concentrations than in MOR-transfected cells, suggesting that spinal cord neurons have a more effective coupling of MORs to intracellular components mediating endocytosis.
Assuntos
Endocitose/fisiologia , Receptores Opioides mu/metabolismo , Medula Espinal/metabolismo , Animais , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Encefalinas/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Cinética , Ratos , Receptores Opioides mu/agonistas , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia , Distribuição Tecidual/fisiologiaRESUMO
Proteases cleave proteinase-activated receptors (PARs) to expose N-terminal tethered ligands that bind and activate the cleaved receptors. The tethered ligand, once exposed, is always available to interact with its binding site. Thus, efficient mechanisms must prevent continuous activation, including receptor phosphorylation and uncoupling from G-proteins, receptor endocytosis, and lysosomal degradation. beta-Arrestins mediate uncoupling and endocytosis of certain neurotransmitter receptors, which are activated in a reversible manner. However, the role of beta-arrestins in trafficking of PARs, which are irreversibly activated, and the effects of proteases on the subcellular distribution of beta-arrestins have not been examined. We studied trafficking of PAR2 and beta-arrestin1 coupled to green fluorescent protein. Trypsin induced the following: (a) redistribution of beta-arrestin1 from the cytosol to the plasma membrane, where it co-localized with PAR2; (b) internalization of beta-arrestin1 and PAR2 into the same early endosomes; (c) redistribution of beta-arrestin1 to the cytosol concurrent with PAR2 translocation to lysosomes; and (d) mobilization of PAR2 from the Golgi apparatus to the plasma membrane. Overexpression of a C-terminal fragment of beta-arrestin-319-418, which interacts constitutively with clathrin but does not bind receptors, inhibited agonist-induced endocytosis of PAR2. Our results show that beta-arrestins mediate endocytosis of PAR2 and support a role for beta-arrestins in uncoupling of PARs.
Assuntos
Arrestinas/metabolismo , Endocitose , Receptores de Trombina/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde , Indicadores e Reagentes , Proteínas Luminescentes , Coelhos , Ratos , Receptor PAR-2 , Tripsina/metabolismo , Células Tumorais Cultivadas , beta-Arrestina 1 , beta-ArrestinasRESUMO
Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.
Assuntos
Arrestinas/metabolismo , Endocitose , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Animais , Arrestinas/genética , Transporte Biológico , Linhagem Celular , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Coelhos , Ratos , Receptores da Neurocinina-1/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , beta-ArrestinasRESUMO
The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal miro-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for micro-opioid receptors. Since the miro-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the micro-opioid receptor peptide agonist, [D-Ala2,MePhe4,Gly-ol5]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged micro-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the micro-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced micro-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins' and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle-myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA2 approximately 8.4) is consistent with antagonism at the micro-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the micro-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced micro-opioid receptor activation can be visualized by receptor endocytosis.
Assuntos
Analgésicos Opioides/farmacologia , Oligopeptídeos/farmacologia , Receptores Opioides mu/efeitos dos fármacos , Receptores Opioides mu/metabolismo , Animais , Linhagem Celular/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Encefalinas/farmacologia , Citometria de Fluxo , Cobaias , Íleo/efeitos dos fármacos , Íleo/inervação , Íleo/fisiologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Plexo Mientérico/citologia , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Ratos , Distribuição Tecidual/fisiologiaRESUMO
Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.