RESUMO
Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK(Y397F) reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Melanoma/metabolismo , Melanoma/patologia , Metaloporfirinas/farmacologia , Camundongos , Proteínas de Ligação a Fosfato , Fosfoproteínas/metabolismo , Fosforilação , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Quinases da Família src/genéticaRESUMO
The instant blood-mediated inflammatory reaction (IBMIR) leads to massive destruction of transplanted islets. Islet isolation and time of culture may elicit the release of potent activators of Toll-like receptors (TLRs) signaling pathways during IBMIR. This work sought to evaluate the role of TLR signaling pathways to mediate inflammatory reactions. Isolated rat pancreatic islets were cultured for 12, 24, or 48 hours. Their viability was assessed by fluorescein diacetate/propidium iodide and their functionality, by glucose stimulation tests. Endotoxin levels were quantified using the Limulus Amebocyte Lysate assays. After RNA extraction and reverse transcription, we performed polymerase chain reaction (PCR) arrays. Samples obtained immediately after isolation were defined as controls. Eighty-four genes belonging to the TLR signaling pathways, were compared with control samples. After culture, islets were viable and functional with low endotoxin levels (< 0.1 endotoxin units/mL) showed TLR activation not due to exogenous contamination. Analysis of PCR arrays highlighted significant up-regulation of TLR-2. After 24 hours of culture, TLR-2 was up-regulated to 6.8 ± 0.6-fold (P < .001) compared with controls but decreased to 4.3 ± 1.4-fold after 48 hours. In the same way, expression of myeloid differentiation primary response gene 88 (Myd88) was significantly up-regulated (3.2 ± 0.4-fold [P < .001]) compared with controls. After 12 hours of culture, interleukin-10 gene expression was significantly up-regulated at 11.6 ± 3.7- fold (P < .05), reaching 17.5 ± 8.3 after 24 hours. Finally, the cyclo-oxygenase-2 gene expression was up-regulated to 509 ± 67.1-fold (P < .05) after 12 hours of culture. These data confirmed the implication of TLR signaling pathways in early inflammatory events.
Assuntos
Inflamação/patologia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Receptores Toll-Like/metabolismo , Animais , Sobrevivência Celular , Regulação da Expressão Gênica , Insulina/metabolismo , Masculino , Modelos Biológicos , Ratos , Ratos Wistar , Transdução de Sinais , Receptor 2 Toll-Like/biossínteseRESUMO
BACKGROUND AND PURPOSE: Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells. EXPERIMENTAL APPROACHES: Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured. KEY RESULTS: Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G(i)-proteins and intracellular Ca(2+) increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls. CONCLUSIONS AND IMPLICATIONS: Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.
Assuntos
Produtos Finais de Glicação Avançada/farmacologia , Mastócitos/efeitos dos fármacos , Receptores Imunológicos/fisiologia , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/imunologia , Relação Dose-Resposta a Droga , Exocitose/efeitos dos fármacos , Produtos Finais de Glicação Avançada/metabolismo , Liberação de Histamina/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Microscopia de Fluorescência , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Mast cells are found in the brain, where they contribute to immune responses. They have been implicated in multiple sclerosis, but their potential role in Alzheimers disease (AD), another inflammatory disease of the central nervous system, remains elusive. In the present study, we examined mast cell responses to amyloid beta (Abeta) peptides 1-40 and 1-42, the major components of the Alzheimer amyloid plaques. Rat peritoneal mast cells were used as experimental model for human brain serosal mast cells. Fibrillar Abeta1-40 and Ami1-42 peptides induced concentration-dependent exocytosis, as assessed by measurement of histamine secretion; exocytosis was reduced by pre-treatment with pertussis toxin and with antibodies against the CD47 receptor and the beta1-integrin subunit. Fibrillar Abeta1-40 and Abeta1- 42 peptides coated on heat-inactivated yeast particles and soluble fibrillar Abeta1-40 and Abeta1-42 peptides were also recognized and phagocyted by mast cells. Uptake of the peptides was decreased in the presence of 4N1, a peptide agonist of the CD47 receptor, but remained unchanged in the presence of 4NGG, a peptide derived from 4N1 which does not bind to CD47. Non-fibrillar forms of Abeta1-40 and 1-42 peptides were unable to elicit mast cell responses. These results show that fibrillar Abeta peptides can trigger mast cells and elicit exocytosis and phagocytosis. The Abeta-induced activation of mast cells operates through a CD47/beta1-integrin membrane complex coupled with Gi-protein. The present data support the hypothesis that mast cells, similarly to microglial cells, could play a major role in AD pathogenesis.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Antígeno CD47/metabolismo , Exocitose , Liberação de Histamina , Mastócitos/imunologia , Fragmentos de Peptídeos/metabolismo , Fagocitose , Animais , Anticorpos , Antígeno CD47/imunologia , Células Cultivadas , Exocitose/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Liberação de Histamina/efeitos dos fármacos , Humanos , Integrina beta1/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Microscopia de Fluorescência , Toxina Pertussis/farmacologia , Fagocitose/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcepsilonRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the betagamma dimer of a G(i) protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G(i) protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G(i) protein complex.
Assuntos
Antígeno CD47/fisiologia , Liberação de Histamina/fisiologia , Histamina/biossíntese , Mastócitos/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígeno CD47/imunologia , Membrana Celular/fisiologia , Colesterol/fisiologia , Exocitose , Subunidades beta da Proteína de Ligação ao GTP/imunologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/imunologia , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Técnicas In Vitro , Cadeias beta de Integrinas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Mastócitos/efeitos dos fármacos , Microdomínios da Membrana/fisiologia , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Toxina Pertussis/farmacologia , Multimerização Proteica , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Espermina/farmacologia , Fosfolipases Tipo C/metabolismo , Venenos de Vespas/farmacologiaRESUMO
Amyloid peptides 1-40 and 1-42 (Abeta 1-40 and Abeta 1-42) are major components of diffuse and neuritic senile plaques present in the brain of patients with Alzheimers disease. Their interaction with microglial cells was studied using a system partly mimicking these plaques, which consisted in heat-killed yeast particles coated with either Abeta 1-40 or Abeta 1-42. Using these particles, it has been shown in our laboratory that LRP is involved mainly in the elimination of Abeta 1-42-coated heat-killed yeast particles and partly in that of Abeta 1-40-coated heat-killed yeast particles by microglial cells in culture. We show here that in the presence of calcium and magnesium ions extracellular chelators, namely EDTA (for both ions) and EGTA (for calcium ions), the internalization of coated heat-killed particles was impaired. In the presence of BAPTA-AM, an intracellular chelator of calcium ions and thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump, no effect was observed on the phagocytosis of Abeta 1-40-coated heat-killed yeast particles, whereas that of Abeta 1-42-coated heat-killed yeast particles was affected. These results suggest that different signaling mechanisms are involved after the internalization of Abeta 1-40 and Abeta 1-42.
Assuntos
Peptídeos beta-Amiloides/farmacocinética , Cálcio/fisiologia , Magnésio/fisiologia , Microglia/metabolismo , Fragmentos de Peptídeos/farmacocinética , Animais , Linhagem Celular , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Transdução de Sinais , Leveduras/imunologiaRESUMO
Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.
Assuntos
Apoptose/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Mitose , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Flavonoides/farmacologia , Cinetina/farmacologia , Camundongos , Fatores de Crescimento Neural/deficiência , Piperidinas/farmacologia , Purinas/farmacologia , RoscovitinaRESUMO
Heptahelical receptors are coupled to heterotrimeric GTP-binding proteins (G-proteins) which transduce most signals through their alpha and betagamma subunits to effectors, enzymes and ion channels. Of the 367 heptahelical receptors for endogenous ligands, about 330 are potential targets for drug discovery with agonist, antagonist or inverse agonist properties. The term G-protein-coupled receptors (GPCRs) is a broader functional definition rather than a structural one referring to heptahelical receptors specifically. Non-heptahelical putative GPCRs include some transmembrane receptors with tyrosine-kinase activity on their cytosolic endings (EGF, insulin and IGF-1 receptors), other transmembrane receptors (mannose-6-phosphate/IGF-2 receptor and integrin-associated protein IAP or CD47), and some receptors belonging to the class of glycosylphosphatidylinositol (GPI)-anchored proteins and located on the outer face of the plasma membrane. Also, activators of G-protein signaling (AGS) proteins that regulate vesicular trafficking activate heterotrimeric G-proteins in the Golgi independently of receptor activation. Main effectors activated through their direct interactions with alpha subunits or betagamma dimers of heterotrimeric G-proteins include adenylylcyclases, cGMP-phosphodiesterase, phospholipases Cbeta, phosphoinositide 3-kinase gamma, Ca(V2) calcium channels, GIRK/Kir3 potassium channels, and guanine nucleotide exchange factors RasGEF and RhoGEF leading to small G-proteins and MAP-kinases activation. Current signaling cascades leading to final cell responses are depicted.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Membrana Celular/metabolismo , GMP Cíclico/metabolismo , Desenho de Fármacos , Ativação Enzimática , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipases/efeitos dos fármacos , Fosfolipases/metabolismo , Diester Fosfórico Hidrolases/efeitos dos fármacos , Diester Fosfórico Hidrolases/metabolismo , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Toxinas Biológicas/farmacologiaRESUMO
Neurotrophic growth factors are involved in cell survival. However, natural growth factors have a very limited therapeutic use because of their short half-life. In the present study, we investigated the mechanism of action of a non-peptidic neurotrophic drug, Xaliproden, a potential molecule for the treatment of motoneuron diseases, since the transduction pathways of this synthetic 5-HT1A agonist are very poorly understood. Xaliproden does not activate the Trk receptor but causes a rapid increase in the activities of the ERK1 and ERK2 isoforms of MAP kinase, which then rapidly decrease to the basal level. We demonstrate that isoforms of the SHC adapter protein are phosphorylated independently of each other and are probably not the source of the Xaliproden-induced MAP kinases activation. The inhibitor of Ras farnesylation, FPT-1, and the protein kinase C inhibitors, GF 109203X and chelerythrine, inhibited the Xaliproden-induced MAP kinase activation, suggesting p21Ras and PKC involvement. Moreover, the observations that the 5-HT1A antagonist, pindobind, and pertussis toxin abolished the Xaliproden-induced ERK stimulation suggested that Xaliproden activates the MAP kinase pathways by stimulating the G protein-coupled receptor, 5-HT1A. These results demonstrate clearly that the non-peptidic compound, Xaliproden, exerts its neurotrophic effects through a mechanism of action differing from that of neurotrophins. These findings suggest that this compound does not involve MAPK activation by TrkA receptor stimulation but acts by MAP kinase pathway by a pertussis toxin-sensitive mechanism involving 5-HT1A receptors, p21 Ras and MEK-1 and by PKC and Akt pathways.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Naftalenos/farmacologia , Piridinas/farmacologia , Receptor 5-HT1A de Serotonina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Immunoblotting , Células PC12 , Fosforilação , Ratos , Receptor trkA/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina , Antagonistas da Serotonina/farmacologia , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
Motoneurons require neurotrophic factors for their survival and their differentiation. Xaliproden (SR57746A) is a synthetic compound that exhibits in vivo and in vitro neurotrophic effects in several experimental studies. Here we demonstrate that neuroprotective effects of Xaliproden on motoneuron cultures are mediated by the activation of the mitogen activated protein kinase pathway. It is inhibited by PD98059, a selective and irreversible inhibitor of MEK1. The activation of this pathway seems to involve two different proteins, the protein kinase C and the Ras. Indeed, we show that Xaliproden is able to activate the MAP kinases ERK1/2 and PKC in motoneurons. In addition, the use of a 5-hydroxytryptamine 1A receptor antagonist, Pindobind and pertussis toxin, inhibits the effect of Xaliproden on motoneuron survival, suggesting the involvement of this G-protein coupled receptor. Morever, 8-OH-DPAT, an agonist of 5-hydroxytryptamine 1A receptor, increases the survival of mouse motoneurons but not by the same extent as BDNF or xaliproden. Since 8-OH-DPAT does not act synergistically with Xaliproden, it is likely that their neuroprotective properties involve a similar pathway. Taken together, these results indicate that neuroprotective effects of Xaliproden on mouse motoneurons are dependent on the mitogen-activated protein kinase activation via 5-hydroxytryptamine 1A receptor.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Naftalenos/farmacologia , Fármacos Neuroprotetores/farmacologia , Piridinas/farmacologia , Receptor 5-HT1A de Serotonina/fisiologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Immunoblotting , Imuno-Histoquímica , MAP Quinase Quinase 1/fisiologia , Camundongos , Microscopia Confocal , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Neurônios Motores/efeitos dos fármacos , Fenótipo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor 5-HT1A de Serotonina/efeitos dos fármacosRESUMO
Compounds possessing neurotrophic properties may represent a possible treatment for neurodegenerative disorders such as amyotrophic lateral sclerosis. Xaliproden (SR57746A), an orally-active non-peptide compound, which has been found to exhibit neurotrophic effects in vitro and in vivo, increased the lifespan and delayed the progression of the motor neuron degeneration in PMN mice. We have used a quantitative reverse transcription/polymerase chain reaction amplification technique to study the regulation of neurotrophin mRNA and trk mRNA expression in PMN mice. NGF and NT-3 mRNA are downregulated in PMN mice. These deficiencies can be overcome by a treatment with xaliproden. Such an effect could contribute to neurotrophic effects of xaliproden in vivo and in vitro.
Assuntos
Naftalenos/farmacologia , Fatores de Crescimento Neural/biossíntese , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Piridinas/farmacologia , RNA Mensageiro/biossíntese , Animais , Química Encefálica/genética , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Primers do DNA , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Masculino , Camundongos , Fator de Crescimento Neural/biossíntese , Fator de Crescimento Neural/efeitos dos fármacos , Fatores de Crescimento Neural/genética , Neurotrofina 3/biossíntese , Neurotrofina 3/genética , RNA Mensageiro/genética , Receptor trkA/biossíntese , Receptor trkA/genética , Receptor trkC/biossíntese , Receptor trkC/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismoRESUMO
Heptahelical receptors are coupled to heterotrimeric GTP-binding proteins (G proteins) which transduce most signals through their alpha and betagamma subunits to effectors including adenylylcyclases, ion channels, phospholipases Cbeta, and phosphoinositide 3-kinases. The diversity of G proteins, their effectors and regulators (RGS proteins), supports the interest of these protein families as potential drug targets.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Transdução de Sinais/fisiologia , Animais , Desenho de Fármacos , Reguladores de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/efeitos dos fármacos , Humanos , Subunidades Proteicas , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
We hypothesized that inflammatory mediators such as interleukin-1beta (IL-1beta) might be responsible for the hyperreactivity to bradykinin observed in asthmatic patients. We reported previously that IL-1beta induced a prostanoid-dependent increase in the density of bradykinin B2 receptors in cultured human bronchial smooth muscle cells. Our experiments demonstrate that the rapid prostaglandin E2 (PGE2) synthesis induced by IL-1beta is abolished by cycloheximide, suggesting the involvement of protein synthesis. The formation of PGE2 is preceded by the phosphorylation of cPLA2 and the expression of cyclooxygenase-2 (Cox-2). The inhibition of p38 MAP kinase inhibited PGE2 synthesis, cPLA2 phosphorylation and abolished Cox-2 expression. The inhibition of Cox-2 expression correlated with a decrease of bradykinin B2 receptor expression. These data demonstrate that the activation of p38 MAP kinase elicited by IL-1beta leads to the phosphorylation of cPLA2 and Cox-2 overexpression, allowing rapid synthesis of PGE2 as a prerequisite for bradykinin B2 gene expression in human bronchial smooth muscle cells which could explain the hyperresponsiveness of asthmatic patients to bradykinin.
Assuntos
Brônquios/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Interleucina-1/farmacologia , Isoenzimas/biossíntese , Proteínas Quinases Ativadas por Mitógeno , Fosfolipases A/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Receptores da Bradicinina/efeitos dos fármacos , Brônquios/enzimologia , Brônquios/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Células Cultivadas , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana , Fosfolipases A2 , Fosforilação/efeitos dos fármacos , RNA Mensageiro/isolamento & purificação , Receptor B2 da Bradicinina , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Experimental probing of the model of the muscarinic M2 receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8-15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [3H] N-methylscopolamine and [3H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [3H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [3H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (< 10-fold) to the binding site of the M2 receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M2 receptor binding site but W155 is important for proper ligand binding on the muscarinic M2 receptor, as predicted by the proposed model.
Assuntos
Receptores Muscarínicos/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Mutagênese Sítio-Dirigida , Mutação , N-Metilescopolamina/metabolismo , Oligopeptídeos , Peptídeos/genética , Quinuclidinil Benzilato/metabolismo , Ensaio Radioligante , Receptor Muscarínico M2 , Receptores Muscarínicos/química , Proteínas Recombinantes de Fusão/genética , TrítioRESUMO
1. The effects of spermine and methoctramine, a selective M2 muscarinic receptor antagonist, were studied on the high-affinity GTPase activity of G proteins, and on ligand binding to M2 muscarinic receptors in pig heart sarcolemma. 2. The spontaneous GTP hydrolysis by pig heart sarcolemma and its stimulation by mastoparan or carbachol were prevented by pertussis toxin and inhibited by methoctramine (IC50s: 21, 13 and 0.005 microM, respectively), and spermine (IC50s: 967, 278 and 11 microM). Spermine and methoctramine also inhibited spontaneous GTP hydrolysis by rat peritoneal mast cell membranes which do not respond to carbachol. 3. The neutral muscarinic antagonists, AF-DX 116 and atropine, did not modify the inhibitory effect of high concentrations of methoctramine, indicating that this effect was not related to the antagonist binding site of muscarinic receptors. We suggest that methoctramine behaves as a receptor antagonist at nanomolar concentrations and interacts with G proteins at micromolar concentrations. 4. Spermine did not modify the binding of the tritiated muscarinic antagonist [3H]-NMS, but decreased the binding of the agonist [3H]-Oxo-M. Spermine elicited a rightward shift of the carbachol/[3H]-NMS binding isotherm with a decrease in the proportion of sites with high-affinity for carbachol, suggesting that polyamines uncouple Gi proteins from receptors. 5. The inhibition of GTPase activity by polyamines, preventing the re-association of alpha and betagamma subunits of Gi proteins, might sustain the regulatory effect of Gi subunits on downstream effectors. The level of intracellular polyamines might be important for the control of the transduction of extracellular signals through Gi protein-coupled receptors.
Assuntos
Diaminas/farmacologia , GTP Fosfo-Hidrolases/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Agonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacologia , Receptores Muscarínicos/metabolismo , Espermina/farmacologia , Animais , N-Metilescopolamina/metabolismo , Especificidade de Órgãos , Oxotremorina/análogos & derivados , Oxotremorina/metabolismo , Ratos , Receptor Muscarínico M2 , SuínosRESUMO
1. Motor responses to des-Arg9-bradykinin and bradykinin were studied in the isolated mouse trachea (precontracted with carbachol, 10 microM) and the urinary bladder of either Swiss, C57B1/6J or bradykinin B2 receptor knockout (Bk2r(-/-)) mice after 1-6 h in vitro. The expression of mRNA for the mouse B1 receptor in tracheal and urinary bladder tissues was also studied by using Northern blot analysis. 2. In isolated tracheae, des-Arg9-bradykinin produced a relaxant response that increased over time: no response was observed after 1 h of incubation, whereas after 6 h the maximum response (1 microM) was 68-84% of the relaxation produced by isoproterenol (1 microM) in the three mouse strains. The relaxant response to bradykinin (1 microM) observed at 1 h (38-51% of isoproterenol) was increased (62-65% of isoproterenol) after 6 h in Swiss and C57B1/6J mice, but was absent in Bk2r(-/-) mice. In the presence of cycloheximide, des-Arg9-bradykinin did not cause any response at 6 h. 3. Similar findings were obtained in the urinary bladder: at 1 h des-Arg9-bradykinin (1 microM) did not cause any motor effect, whereas at 6 h it caused a contraction that was 28-59% of that produced by carbachol (1 microM) in the three mouse strains. Cycloheximide blocked the response to des-Arg9-bradykinin. Bradykinin (1 microM) contracted urinary bladders at 1 h (34-35% of carbachol), as well as at 6 h (66-77% of carbachol) in Swiss and C57B1/6J strains, but was without effect in Bk2r(-/-) mice. 4. Northern blot hybridization with a specific cDNA probe against mouse B1 receptor mRNA using total RNA extracted from tracheae and urinary bladders freshly removed from Swiss and Bk2r(-/-) mice revealed minimal expression. However, marked hybridization was detected 150 min after in vitro exposure in both tissues. 5. Evidence is provided that in vitro exposure of mouse trachea and urinary bladder causes a time-dependent induction of B1 receptors that cause relaxation and contraction, respectively.
Assuntos
Receptores da Bradicinina/genética , Traqueia/metabolismo , Bexiga Urinária/metabolismo , Animais , Northern Blotting , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor B1 da Bradicinina , Fatores de Tempo , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiologiaRESUMO
1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*<
Assuntos
Antagonistas Muscarínicos/farmacologia , Pirenzepina/farmacologia , Receptores Muscarínicos/metabolismo , Sarcolema/efeitos dos fármacos , Animais , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos , Receptor Muscarínico M2 , Receptores Muscarínicos/efeitos dos fármacos , Sarcolema/enzimologia , Sarcolema/metabolismo , Suínos , Venenos de Vespas/farmacologiaRESUMO
We investigated the effect of the glucocorticoid methylprednisolone on the modulation of the expression of the bradykinin B2 receptors in cultured, guinea-pig, tracheal, smooth muscle cells. These receptors are implicated in the pathogenesis of human asthma. Untreated cells expressed a single population of binding sites for [3H]bradykinin with a dissociation constant, Kd, of 87.7+/-12.0 pM and a maximum binding site density, Bmax, of 245.4+/-71 fmol/mg protein. Treatment of the cultured guinea-pig tracheal smooth muscle cells with methylprednisolone 10(-5) M for 6 h increased the number of bradykinin receptors; this response reached a maximum of 78% and returned to the basal value after 12 h. Bradykinin (10(-12) M) elicited a six-fold higher calcium level in treated cells than in control cells. To investigate bradykinin B2 receptor mRNA expression in guinea-pig cells, we used the reverse transcription polymerase chain reaction (RT-PCR) technique to synthesize a specific bradykinin B2 cDNA probe of 296 bp corresponding to nucleotides 456-751 of the human sequence. This guinea-pig cDNA had 88%, 86% and 83% homology with the corresponding human, mouse and rat sequences, respectively, but no homology with any other known sequences. Following methylprednisolone treatment, Northern blot hybridization indicated that mRNA increased fourfold after 3 h compared with control cells, and returned to basal level within 7 h. The rate of gene transcription, assessed by nuclear run-on assays, increased fourfold after 3 h treatment with 10(-5) M methylprednisolone. These results indicate that glucocorticoids induce early up-regulation of bradykinin B2 receptors in cultured guinea-pig tracheal smooth muscle cells by increasing the rate of transcription of the bradykinin B2 receptor gene.
Assuntos
Regulação da Expressão Gênica , Glucocorticoides/farmacologia , Metilprednisolona/farmacologia , Músculo Liso/metabolismo , Receptores da Bradicinina/efeitos dos fármacos , Traqueia/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Northern Blotting , Cálcio/metabolismo , Técnicas de Cultura de Células , Cobaias , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Receptores da Bradicinina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Fatores de TempoRESUMO
We studied the effect of the glucocorticoids, dexamethasone and budesonide, on the interleukin-1beta-induced increase of bradykinin B2 receptors in cultured human bronchial smooth muscle cells, a cellular model of bronchial hyperreactivity. Both compounds prevented the increase of the bradykinin B2 mRNA and the bradykinin-induced inositol phosphate accumulation. These results demonstrate a direct effect of glucocorticoids on airway smooth muscle hyperresponsiveness mediated through inhibition of the over-expression of receptors for contractile mediators induced by inflammatory mediators.
Assuntos
Anti-Inflamatórios/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/ultraestrutura , Broncodilatadores/farmacologia , Budesonida/farmacologia , Dexametasona/farmacologia , Interleucina-1/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/ultraestrutura , Receptores da Bradicinina/biossíntese , Brônquios/metabolismo , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/metabolismo , Células Cultivadas , Interações Medicamentosas , Humanos , Fosfatos de Inositol/biossíntese , Interleucina-1/antagonistas & inibidores , Músculo Liso/metabolismo , RNA Mensageiro/metabolismo , Receptor B2 da BradicininaRESUMO
We investigated the hypothesis that inflammatory mediators such as interleukin-1beta (IL-1beta) might be responsible for the hyperreactivity of asthmatic patients to bradykinin. In cultured human bronchial smooth muscle cells, IL-1beta elicited a rapid and transient increase in the density of bradykinin B2 receptors without affecting their affinity for ligands. The increase in B2 receptors was correlated to an enhancement of inositol phosphate formation elicited by bradykinin, indicating its relevance to the contractile response of smooth muscle cells to bradykinin. The increase in receptor density was related to an increase in B2 receptor mRNA level corresponding to a 5-fold enhancement of the transcriptional rate and to a lengthened half-life of mRNA. These effects of IL-1beta were largely inhibited by indomethacin, suggesting the involvement of a prostanoid pathway in IL-1beta transduction process. An increase in prostaglandin E2 levels preceded the mRNA increase, confirming this involvement. Moreover, IL-1beta and prostaglandin E2 led to cAMP formation. We propose this predominant transduction pathway of IL-1beta to stimulate the transcription of the bradykinin B2 gene in human bronchial smooth muscle cells as a major mechanism involved in the hyperresponsiveness of asthmatic patients to bradykinin.