RESUMO
The mostly widely used bronchodilators in asthma therapy are ß2-adrenoreceptor (ß2AR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that ß2AR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that ß2AR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models. Our data show that inhibition of ß2AR signaling with an aerosolized antagonist attenuates airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus-production responses to IL-13, whereas treatment with an aerosolized agonist worsens these phenotypes, suggesting that ß2AR signaling on resident lung cells modulates the asthma phenotype. Labeling with a fluorescent ß2AR ligand shows the receptors are highly expressed in airway epithelium. In ß2AR-/- mice, transgenic expression of ß2ARs only in airway epithelium is sufficient to rescue IL-13-induced AHR, inflammation, and mucus production, and transgenic overexpression in WT mice exacerbates these phenotypes. Knockout of ß-arrestin-2 (ßarr-2-/-) attenuates the asthma phenotype as in ß2AR-/- mice. In contrast to eosinophilic inflammation, neutrophilic inflammation was not promoted by ß2AR signaling. Together, these results suggest ß2ARs on airway epithelial cells promote the asthma phenotype and that the proinflammatory pathway downstream of the ß2AR involves ßarr-2. These results identify ß2AR signaling in the airway epithelium as capable of controlling integrated responses to IL-13 and affecting the function of other cell types such as airway smooth muscle cells.
Assuntos
Asma/etiologia , Eosinófilos/patologia , Células Epiteliais/metabolismo , Pulmão/patologia , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Asma/patologia , Brônquios/citologia , Modelos Animais de Doenças , Epinefrina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-13/toxicidade , Pulmão/citologia , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Receptores Adrenérgicos beta 2/genética , Transdução de SinaisRESUMO
BACKGROUND: Our previous studies suggested certain ß-adrenoceptor blockers (ß-blockers) attenuate the asthma phenotype in ovalbumin driven murine models of asthma. However, the ovalbumin model has been criticized for lack of clinical relevance. METHODS: We tested the non-selective ß-blockers, carvedilol and nadolol, in house dust mite (HDM) driven murine asthma models where drugs were administered both pre- and post-development of the asthma phenotype. We measured inflammation, mucous metaplasia, and airway hyper-responsiveness (AHR). We also measured the effects of the ß-blockers on extracellular-signal regulated kinase (ERK 1/2) phosphorylation in lung homogenates. RESULTS: We show that nadolol, but not carvedilol, attenuated inflammation and mucous metaplasia, and had a moderate effect attenuating AHR. Following HDM exposure, ERK1/2 phosphorylation was elevated, but the level of phosphorylation was unaffected by ß-blockers, suggesting ERK1/2 phosphorylation becomes dissociated from the asthma phenotype. CONCLUSION: Our findings in HDM models administering drugs both pre- and post-development of the asthma phenotype are consistent with previous results using ovalbumin models and show differential effects for nadolol and carvedilol on the asthma phenotype. Lastly, our data suggest that ERK1/2 phosphorylation may be involved in development of the asthma phenotype, but may have a limited role in maintaining the phenotype.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Asma/tratamento farmacológico , Carbazóis/farmacologia , Nadolol/farmacologia , Propanolaminas/farmacologia , Animais , Asma/imunologia , Asma/patologia , Carvedilol , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ovalbumina/imunologia , Fenótipo , Fosforilação , Pyroglyphidae/imunologia , Hipersensibilidade Respiratória/tratamento farmacológicoRESUMO
Mice lacking the endogenous ß2-adrenoceptor (ß2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of ß2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various ß2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous ß2AR agonists on allergic lung inflammation can be explained by qualitative ß2AR signaling. The ß2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a ß-arrestin-dependent pathway. Previous studies suggest that ß-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the ß2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing ß2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of ß2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by ß-agonists.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/efeitos adversos , Asma/tratamento farmacológico , Feniletanolamina N-Metiltransferase/deficiência , Inibidores da Fosfodiesterase 4/uso terapêutico , Aminopiridinas/administração & dosagem , Aminopiridinas/farmacologia , Animais , Asma/complicações , Asma/patologia , Asma/fisiopatologia , Benzamidas/administração & dosagem , Benzamidas/farmacologia , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/fisiopatologia , Ciclopropanos/administração & dosagem , Ciclopropanos/farmacologia , Quimioterapia Combinada , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Fumarato de Formoterol/administração & dosagem , Fumarato de Formoterol/farmacologia , Inflamação/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Camundongos Knockout , Muco/metabolismo , Fenótipo , Feniletanolamina N-Metiltransferase/metabolismo , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/farmacologiaRESUMO
The effect of stains in asthma is mediated through targeting several signaling molecules that are involved in the development of asthma phenotype. In vitro and in vivo studies revealed that statins reduce airway smooth muscle cells proliferation and inflammatory mediators' release. Statins reduce chemokine release and mucus production from airway epithelial cells besides attenuating subepithelial fibrosis and eosinophils recruitment. In acute and chronic allergen driven animal models of asthma, statins reduce airway hyper-responsiveness, inflammation and remodeling. However, the effectiveness of statins in clinical trials results in contradictory conclusions based on study design and treatment protocol. Therefore, more clinical trials are needed to evaluate their role in asthma patients.
Assuntos
Asma/tratamento farmacológico , Asma/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Remodelação das Vias Aéreas/efeitos dos fármacos , Remodelação das Vias Aéreas/fisiologia , Animais , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/metabolismo , Ensaios Clínicos como Assunto/métodos , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismoRESUMO
ß(2)-Adrenoceptor (ß2AR) agonists are the most effective class of bronchodilators and a mainstay of asthma management. The first potent ß2AR agonist discovered and widely used in reversing the airway constriction associated with asthma exacerbation was the endogenous activator of the ß2AR, epinephrine. In this study, we demonstrate that activation of the ß2AR by epinephrine is paradoxically required for development of the asthma phenotype. In an antigen-driven model, mice sensitized and challenged with ovalbumin showed marked elevations in three cardinal features of the asthma phenotype: inflammatory cells in their bronchoalveolar lavage fluid, mucin over production, and airway hyperresponsiveness. However, genetic depletion of epinephrine using mice lacking the enzyme to synthesize epinephrine, phenylethanolamine N-methyltransferase, or mice that had undergone pharmacological sympathectomy with reserpine to deplete epinephrine, had complete attenuation of these three cardinal features of the asthma phenotype. Furthermore, administration of the long-acting ß2AR agonist, formoterol, a drug currently used in asthma treatment, to phenylethanolamine N-methyltransferase-null mice restored the asthma phenotype. We conclude that ß2AR agonist-induced activation is needed for pathogenesis of the asthma phenotype. These findings also rule out constitutive signaling by the ß2AR as sufficient to drive the asthma phenotype, and may help explain why chronic administration of ß2AR agonists, such as formoterol, have been associated with adverse outcomes in asthma. These data further support the hypothesis that chronic asthma management may be better served by treatment with certain "ß-blockers."
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Asma/induzido quimicamente , Modelos Animais de Doenças , Etanolaminas/farmacologia , Animais , Asma/fisiopatologia , Brônquios/fisiopatologia , Líquido da Lavagem Broncoalveolar , Cromatografia Líquida de Alta Pressão , Epinefrina/metabolismo , Fumarato de Formoterol , Camundongos , Camundongos Knockout , Mucinas/metabolismo , FenótipoRESUMO
Chronic regular use of beta(2)-adrenoceptor (beta(2)-AR) agonists in asthma is associated with a loss of disease control and increased risk of death. Conversely, we have found that administration of beta(2)-AR inverse agonists results in attenuation of the asthma phenotype in an allergen-driven murine model. Besides antagonizing agonist-induced signaling and reducing signaling by empty receptors, beta-AR inverse agonists can also activate signaling by novel pathways. To determine the mechanism of the beta-AR inverse agonists, we compared the asthma phenotype in beta(2)-AR-null and wild-type mice. Antigen challenge of beta(2)-AR-null mice produced results similar to what was observed with chronic beta(2)-AR inverse agonist treatment, namely, reductions in mucous metaplasia, airway hyperresponsiveness (AHR), and inflammatory cells in the lungs. These results indicate that the effects of beta(2)-AR inverse agonists are caused by inhibition of beta(2)-AR signaling rather than by the induction of novel signaling pathways. Chronic administration of alprenolol, a beta-blocker without inverse agonist properties, did not attenuate the asthma phenotype, suggesting that it is signaling by empty receptors, rather than agonist-induced beta(2)-AR signaling, that supports the asthma phenotype. In conclusion, our results demonstrate that, in a murine model of asthma, beta(2)-AR signaling is required for the full development of three cardinal features of asthma: mucous metaplasia, AHR, and the presence of inflammatory cells in the lungs.
Assuntos
Asma/genética , Asma/patologia , Receptores Adrenérgicos beta 2/metabolismo , Alprenolol/farmacologia , Animais , Broncoconstritores/farmacologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Pulmão/patologia , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Transdução de SinaisRESUMO
For the beta(2)-adrenergic receptor (beta(2)AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of beta(2)AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Citocalasina D/farmacologia , Endocitose/efeitos dos fármacos , Humanos , Cinética , Tiazolidinas/farmacologiaRESUMO
Single-dose administration of beta-adrenoceptor agonists produces bronchodilation and inhibits airway hyperresponsiveness (AHR), and is the standard treatment for the acute relief of asthma. However, chronic repetitive administration of beta-adrenoceptor agonists may increase AHR, airway inflammation, and risk of death. Based upon the paradigm shift that occurred with the use of beta-blockers in congestive heart failure, we previously determined that chronic administration of beta-blockers decreased AHR in a murine model of asthma. To elucidate the mechanisms for the beneficial effects of beta-blockers, we examined the effects of chronic administration of several beta-adrenoceptor ligands in a murine model of allergic asthma. Administration of beta-blockers resulted in a reduction in total cell counts, eosinophils, and the cytokines IL-13, IL-10, IL-5, and TGF-beta1 in bronchoalveolar lavage, and attenuated epithelial mucin content and morphologic changes. The differences in mucin content also occurred if the beta-blockers were administered only during the ovalbumin challenge phase, but administration of beta-blockers for 7 days was not as effective as administration for 28 days. These results indicate that in a murine model of asthma, chronic administration of beta-blockers reduces inflammation and mucous metaplasia, cardinal features of asthma that may contribute to airflow obstruction and AHR. Similar to heart failure, our results provide a second disease model in which beta-blockers producing an acutely detrimental effect may provide a therapeutically beneficial effect with chronic administration.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Asma/tratamento farmacológico , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Mucinas/metabolismo , Administração Oral , Antagonistas Adrenérgicos beta/administração & dosagem , Animais , Asma/fisiopatologia , Feminino , Bombas de Infusão , Injeções Intraperitoneais , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nadolol/administração & dosagem , Nadolol/farmacologia , Ovalbumina , Propanolaminas/administração & dosagem , Propanolaminas/farmacologia , Organismos Livres de Patógenos EspecíficosRESUMO
Activated beta2-adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. The phosphorylation of the GRK site Ser355,356 during a 10 microM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. In contrast, phosphorylation of the PKA site Ser262 during a 10 microM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. The results indicate that the kinetics of beta2-adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.
Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dinamina I/análise , Endocitose/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G , Quinase 3 de Receptor Acoplado a Proteína G , Humanos , Fosforilação , Receptores Adrenérgicos beta 2/química , Serina/metabolismo , Quinases de Receptores Adrenérgicos beta/fisiologiaRESUMO
Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes ß2-adrenoceptor (ß2AR) agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether ß2AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential ß2AR antagonist, but not by CGP-20712A, a preferential ß1AR antagonist. Constitutive ß2AR activity was not sufficient for IL-13 induced mucin production and ß-agonist-induced signaling is required. A clinically important long-acting ß-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that ß2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that ß2AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of ß2ARs on epithelial cells.
Assuntos
Brônquios/citologia , Epinefrina/farmacologia , Células Epiteliais/metabolismo , Interleucina-13/farmacologia , Mucina-5AC/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Bovinos , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacosRESUMO
Hormone-stimulated receptor internalization and desensitization occur widely in the G protein-coupled receptor (GPCR) family. A critical first step in both these processes is thought to be receptor phosphorylation, a reaction which has been extensively characterized in cell culture. However, little is known about GPCR phosphorylation in vivo. The somatostatin (SS) receptor subtype (sst)2A is widely distributed in human neuroendocrine tumors, and SS analogs are commonly used to target this receptor for both therapy and diagnosis. In cultured pituitary cells sst2A is rapidly phosphorylated and internalized after hormone binding. The aim of the present study was to go one crucial step further and characterize the phosphorylation state of this receptor in human neuroendocrine tumors using a newly developed gel-shift assay. The receptor from a somatostatinoma was completely phosphorylated. In contrast, only unphosphorylated sst2A was present in human tumors that were not exposed to autocrine stimulation. Both in vivo and in cultured cells, the phosphorylation state of the sst2A receptor was correlated with its subcellular localization: phosphorylated receptor was mostly intracellular, whereas unphosphorylated receptor was localized at the cell surface. These results are the first to demonstrate ligand-stimulated GPCR phosphorylation in human tissue in situ, providing a crucial step toward a better understanding of receptor regulation in vivo. Analysis of sst2A phosphorylation promises to provide a sensitive indicator of the effectiveness of SS analogs in diagnostic and therapeutic situations in tumor patients.
Assuntos
Tumores Neuroendócrinos/metabolismo , Receptores de Somatostatina/metabolismo , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Transporte Biológico , Células CHO , Tumor Carcinoide/metabolismo , Cricetinae , Humanos , Neoplasias do Íleo/metabolismo , Neoplasias Pancreáticas/metabolismo , Feocromocitoma/metabolismo , Fosforilação , Receptores de Somatostatina/agonistas , Somatostatinoma/metabolismo , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
1. We have investigated the role of G protein-coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human beta(2)-adrenoceptors. 2. Expressing GRK5 at a high level significantly increased the extent of internalization of wild-type beta(2)-adrenoceptors and of an internalization-defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. 3. Overexpressing GRK5 did not alter beta(2)-adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. 4. These data indicate that GRK5 does not regulate the sorting of beta(2)-adrenoceptors in the endocytic pathway.
Assuntos
Endossomos/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Linhagem Celular , Regulação para Baixo/fisiologia , Endossomos/genética , Quinase 5 de Receptor Acoplado a Proteína G , Humanos , Mutação , Fosforilação , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Receptores Adrenérgicos beta 2/genéticaRESUMO
Nadolol (NAD) is a ß-adrenergic receptor blocker with inverse agonist activity at ßARs. Previous studies in our laboratory showed that chronic treatment with NAD decreased airway resistance response (R (aw)) to the muscarinic agonist methacholine in a murine model of asthma while acute treatment with NAD increased R (aw) (Callaerts-Vegh et al., Proc Natl Acad Sci U S A 101:4948-4953, 2004). Chronic treatment with NAD also caused decreased airway inflammation and mucin content in a murine asthma model (Nguyen et al., Am J Respir Cell Mol Biol 38:256-262, 2008). In this study, we examined the effects of nadolol on ß(2)AR levels and signaling components downstream of the ß(2)AR using a line of HEK293 cells expressing human ß(2)ARs. Chronic treatment with NAD increased ß(2)AR protein levels and decreased receptor degradation, consistent with receptor stabilization by the inverse agonist. Basal cAMP levels decreased after 5 min of treatment with NAD but increased after a 24-h treatment. A 5-min treatment with NAD decreased forskolin-stimulated phosphorylation at the ß(2)AR PKA site Ser 262 while a 24-h treatment with NAD increased it. In contrast, chronic treatment with NAD had no effect on phosphorylation of the ß(2)AR GRK site at Ser 355, 356. Chronic treatment with NAD upregulated cellular levels of G(α)s but had no effect on G(α)i. Chronic NAD treatment therefore increases cellular cAMP levels by mechanisms that include the upregulation of ß(2)AR and G(α)s. This effect may explain in part the beneficial effects of chronic nadolol treatment on airway contractility.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Nadolol/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antagonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Asma/tratamento farmacológico , Asma/metabolismo , Técnicas de Cultura de Células , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Immunoblotting , Ligantes , Nadolol/administração & dosagem , Receptores Adrenérgicos beta 2/genética , TransfecçãoRESUMO
The internalization of beta(2)-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on beta(2)-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the beta(2)-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. beta(2)-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A beta(2)-adrenoceptors. The mutant beta(2)-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type beta(2)-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A beta(2)-adrenoceptors was reduced to approximately 25% that of wild type beta(2)-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A beta(2)-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type beta(2)-adrenoceptors. Therefore, although the role of L339,L340 in beta(2)-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient beta(2)-adrenoceptors endocytosis.
Assuntos
Endocitose , Quinases de Receptores Acoplados a Proteína G/metabolismo , Leucina/genética , Leucina/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Receptores Adrenérgicos beta 2/metabolismo , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Regulação para Baixo , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Fosforilação/genética , Transporte Proteico , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Serina/metabolismoRESUMO
BACKGROUND: We have previously reported that chronic treatment with certain 'beta-blockers' reduces airway hyperresponsiveness (AHR) to methacholine in a murine model of asthma. METHODS: Airway resistance was measured using the forced oscillation technique in ovalbulmin-sensitized and ovalbulmin-challenged mice treated with several beta-adrenoceptor (beta-AR) ligands. We used the selective beta 2-AR ligand ICI 118,551 and the preferential beta 1-AR ligand metoprolol to investigate the receptor subtype mediating the beneficial effect. Expression of beta-ARs was evaluated using immunofluorescence. We evaluated several signaling proteins by western blot using lung homogenates, and measured the relaxation of the isolated trachea produced by EP2 and IP receptor agonists. RESULTS: Four findings were associated with the decreased AHR after chronic beta-blocker treatment: (1) the highly selective beta 2-AR antagonist/inverse agonist, ICI 118,551 produced the bronchoprotective effect; (2) beta 2-AR up-regulation resulted from chronic 'beta-blocker' treatment; (3) reduced expression of certain proteins involved in regulating bronchial tone, namely, Gi, phosphodiesterase 4D and phospholipase C-beta 1; and (4) an enhanced bronchodilatory response to prostanoid agonists for the IP and EP2 receptors. CONCLUSIONS: These data suggest that in the murine model of asthma, several compensatory changes associated with either increased bronchodilator signaling or decreased bronchoconstrictive signaling, result from the chronic administration of certain 'beta-blockers'.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Asma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metoprolol/farmacologia , Propanolaminas/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas Adrenérgicos beta/administração & dosagem , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Asma/imunologia , Asma/metabolismo , Western Blotting , Testes de Provocação Brônquica , Esquema de Medicação , Imunofluorescência , Masculino , Cloreto de Metacolina , Metoprolol/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Nadolol/farmacologia , Ovalbumina/imunologia , Propanolaminas/administração & dosagemRESUMO
Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the beta2-adrenergic receptor (beta2AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t(1/2) of 9 min (k = 0.08 +/- 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 microM isoproterenol for 5 min exhibited a lag phase of approximately 5 min, after which dephosphorylation proceeded slowly with a t(1/2) of 18 min (k = 0.039 +/- 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated beta2AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated beta2AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5- and 10-fold faster (k = 0.43 +/- 0.009/min; t(1/2) = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.
Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Adenilil Ciclases/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Humanos , Isoproterenol/farmacologia , Rim , Cinética , Fosfosserina/metabolismo , Receptores Adrenérgicos beta 2/efeitos dos fármacosRESUMO
Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Cromonas/farmacologia , Endocitose/efeitos dos fármacos , Morfolinas/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Transporte Proteico/efeitos dos fármacos , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Salmeterol is a long-acting beta(2)-adrenergic receptor (beta(2)AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other beta-agonists in that it has a very low intrinisic efficacy, especially when compared with the other available long-acting beta-agonist, formoterol. Receptor desensitization and down-regulation has been described with the chronic use of beta-agonists. This effect may not be the same with all beta-agonists and may be related to their stabilization of altered receptor states. The extreme hydrophobicity and high-affinity quasi-irreversible binding of salmeterol have rendered studies examining the mechanisms by which it mediates receptor desensitization, down-regulation, and internalization difficult. We determined the capacity of salmeterol to induce beta(2)AR endocytosis, G protein-coupled receptor kinase (GRK)-site phosphorylation, degradation, and beta-arrestin2 translocation in HEK293 cells as compared with other agonists of varying intrinsic efficacies. Despite stimulating GRK-mediated phosphorylation of Ser355,356 after 30 min and 18 h to an extent similar to that observed with agonists of high intrinsic efficacy, such as epinephrine and formoterol, salmeterol did not induce significant beta(2)AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-beta-arrestin2 chimera (EGFP-beta-arrestin2) to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP-beta-arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit beta-arrestin or undergo significant endocytosis or degradation despite stimulating considerable GRK-site phosphorylation. Defects in these components of salmeterol-induced receptor desensitization may be important determinants of its sustained bronchodilation with chronic use.
Assuntos
Albuterol/análogos & derivados , Endocitose/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Arrestinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Epinefrina/farmacologia , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Adrenérgicos beta 2/análise , Xinafoato de Salmeterol , Quinases de Receptores Adrenérgicos beta/metabolismoRESUMO
There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.