RESUMO
Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g., ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultrafast benchtop method for radical generation for HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond time scale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g., ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that effective hydroxyl radical yields in FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be compensated on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.
Assuntos
Adenina/química , Fibrinopeptídeo B/química , Radical Hidroxila/química , Mioglobina/química , Pegadas de Proteínas/métodos , Adenina/análise , Radical Hidroxila/efeitos da radiação , Oxirredução , Espectrofotometria Ultravioleta , Raios UltravioletaRESUMO
Hydroxyl radical protein footprinting (HRPF) is a powerful and flexible technique for probing changes in protein topography. With the development of the fast photochemical oxidation of proteins (FPOP), it became possible for researchers to perform HRPF in their laboratory on a very short time scale. While FPOP has grown significantly in popularity since its inception, adoption remains limited due to technical and safety issues involved in the operation of a hazardous Class IV UV laser and irreproducibility often caused by improper laser operation and/or differential radical scavenging by various sample components. Here, we present a new integrated FOX (Flash OXidation) Protein Footprinting System. This platform delivers sample via flow injection to a facile and safe-to-use high-pressure flash lamp with a flash duration of 10 µs fwhm. Integrated optics collect the radiant light and focus it into the lumen of a capillary flow cell. An inline radical dosimeter measures the hydroxyl radical dose delivered and allows for real-time compensation for differential radical scavenging. A programmable fraction collector collects and quenches only the sample that received the desired effective hydroxyl radical dose, diverting the carrier liquid and improperly oxidized sample to waste. We demonstrate the utility of the FOX Protein Footprinting System by determining the epitope of TNFα recognized by adalimumab. We successfully identify the surface of the protein that serves as the epitope for adalimumab, identifying four of the five regions previously noted by X-ray crystallography while seeing no changes in peptides not involved in the epitope interface. The FOX Protein Footprinting System allows for FPOP-like experiments with real-time dosimetry in a safe, compact, and integrated benchtop platform.
Assuntos
Pegadas de Proteínas/instrumentação , Pegadas de Proteínas/métodos , Cromatografia Líquida , Epitopos/química , Desenho de Equipamento , Células HEK293 , Humanos , Oxirredução , Conformação Proteica , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
The Brown-Norway rat is often used to study the allergic pulmonary response. However, relatively little is known about the delayed phase reactions after allergen challenge in this species. To evaluate the temporal changes in lung function and elucidate the mechanisms involved in the delayed phase response, Brown-Norway rats were sensitized and challenged to aerosolized ovalbumin and lung functions were measured by forced expiratory maneuvers and forced oscillation for up to 10 days after a single antigen challenge. Statistically significant (P < 0.05) reductions in inspiratory capacity, forced vital capacity, functional residual capacity, peak expiratory flow and maximum mid-expiratory flow and increases in respiratory system resistance and elastance were seen by 1 to 3 days after ovalbumin challenge that returned to baseline by 10 days. The reductions in lung function after ovalbumin challenge were blocked by the corticosteroid, betamethasone (1 mg/kg, p.o.). Histological evaluation of lung tissue of sensitized rats demonstrated evidence of interstitial pulmonary edema, an increase in tissue eosinophils and an increase in Periodic Acid Schiff-positive cells in the airway epithelium. Bronchoalveolar lavage fluid samples showed large numbers of eosinophils and increased mucin content up to 6 days after antigen challenge. There was also an increase in wet-to-dry lung weight ratio in the lungs of sensitized rats after antigen. These results demonstrate that prolonged reductions in lung function occur after a single antigen challenge in Brown-Norway rats that is probably due to inflammatory processes producing interstitial pulmonary edema, mucus secretion and cellular influx into the lungs.
Assuntos
Pulmão/fisiopatologia , Hipersensibilidade Respiratória/fisiopatologia , Capacidade Vital/fisiologia , Alérgenos/imunologia , Animais , Antiasmáticos/farmacologia , Betametasona/farmacologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Mucinas/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ovalbumina/imunologia , Ratos , Ratos Endogâmicos BN , Testes de Função Respiratória , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Fatores de Tempo , Capacidade Vital/efeitos dos fármacosRESUMO
Among the three isoenzymes of neuraminidase (Neu) or sialidase, Neu-1 has been suggested to be induced by cell activation and to be involved in IL (interleukin)-4 biosynthesis in murine T-cells. In the present study, we found that antigen-induced airway eosinophilia, a typical response dependent on Th2 (T-helper cell type 2) cytokines, as well as mRNA expression of Th2 cytokines, including IL-4, are suppressed in Neu-1-deficient mice, thereby demonstrating the in vivo role of murine Neu-1 in regulation of Th2 cytokines. To elucidate the roles of various sialidases in human T-cell activation, we investigated their tissue distribution, gene induction and function. Neu-1 is the predominant isoenzyme at the mRNA level in most tissues and cells in both mice and humans, including T-cells. T-cells also have significant levels of Neu-3 mRNAs, albeit much lower than those of Neu-1, whereas the levels of Neu-2 mRNAs are minimal. In human T-cells, both Neu-1 and Neu-3 mRNAs are significantly induced by T-cell-receptor stimulation, as is sialidase activity against 4-methylumbelliferyl- N -acetylneuramic acid (a substrate for both Neu-1 and Neu-3) and the ganglioside G(D1a) [NeuAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-cer] (a substrate for Neu-3, but not for Neu-1). The expression of the two sialidase genes may be under differential regulation. Western blot analysis and enzymic comparison with recombinant sialidases have revealed that Neu-3 is induced as a major isoform in activated cells. The induction of Neu-1 and Neu-3 in T-cells is unique. In human monocytes and neutrophils stimulated with various agents, the only observation of sialidase induction has been by IL-1 in neutrophils. Functionally, a major difference has been observed in Jurkat T-cell lines over-expressing Neu-1- and Neu-3. Upon T-cell receptor stimulation, IL-2, interferon-gamma, IL-4 and IL-13 are induced in the Neu-1 line, whereas in the Neu-3 line the same cytokines are induced, with the exception of IL-4. Taken together, these results suggest an important immunoregulatory role for both Neu-1 and Neu-3 in humans.
Assuntos
Membrana Celular/enzimologia , Lisossomos/enzimologia , Neuraminidase/biossíntese , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/enzimologia , Animais , Antígenos CD28/farmacologia , Complexo CD3/farmacologia , Linfócitos T CD4-Positivos/enzimologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Eosinofilia/etiologia , Eosinofilia/patologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Insetos/citologia , Isoenzimas/biossíntese , Isoenzimas/fisiologia , Células Jurkat/enzimologia , Contagem de Leucócitos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Neuraminidase/deficiência , Neuraminidase/fisiologia , Especificidade de Órgãos/fisiologia , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/enzimologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Ativação TranscricionalRESUMO
Previously, four splice variants of human cGMP-specific phosphodiesterase (PDE) 9A (PDEs 9A1, 9A2, 9A3 and 9A4) have been identified. In this study, we have cloned a cDNA representing a new human PDE9A variant (PDE9A5). PDE9A5 encodes a protein of 492 amino acids, smaller than PDEs 9A1 and 9A2 but larger than PDEs 9A3 and 9A4. The exon structure of PDE9A5 is different from those of PDEs 9A1, 9A2, 9A3 and 9A4 in that, of the 20 exons of PDE9A gene, it lacks exons 2 and 5. PDE9A5 has been characterized in comparison with PDE9A1, the longest PDE9A variant. PDEs 9A5 and 9A1 have similar enzymatic properties. They both have a high affinity for cGMP with similar Km values (0.39 and 0.25 microM, respectively), although they have slightly different Vmax values (2.55 and 0.96 micromol/min/mg, respectively). They exhibit very similar divalent metal ion dependency and inhibitor sensitivity. Real-time quantitative PCR analysis shows that PDEs 9A5 and 9A1 exhibit differential tissue distribution. They are highly expressed in immune tissues (spleen, lymph node and thymus) and are more abundant in T cells than in B cells, neutrophils and monocytes. When transiently expressed in HEK293 cells, PDEs 9A5 and 9A1 proteins exhibit differential subcellular localization. PDE9A5 localizes exclusively in the cytoplasm, whereas PDE9A1 localizes in the nucleus only. The nuclear localization of PDE9A1 is dependent on a unique pat7 motif. By Western blot analysis, native PDE9A1 is detectable in the nucleus but not in the cytoplasm of T cells. Thus, to our knowledge, PDE9A1 is the only PDE isoform found to localize exclusively in the nucleus. We speculate that the physiological role of the PDE9A diversity may be imparting cGMP-metabolizing ability to specific cellular compartments in appropriate tissues.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/genética , Processamento Alternativo , 1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Linhagem Celular , Clonagem Molecular , GMP Cíclico/metabolismo , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Imidazóis/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Dados de Sequência Molecular , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Purinas , Purinonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Citrato de Sildenafila , SulfonasRESUMO
1. Endogenous neuronal lipid mediator anandamide, which can be synthesized in the lung, is a ligand of both cannabinoid (CB) and vanilloid receptors (VR). The tussigenic effect of anandamide has not been studied. The current study was designed to test the direct tussigenic effect of anandamide in conscious guinea-pigs, and its effect on VR1 receptor function in isolated primary guinea-pig nodose ganglia neurons. 2. Anandamide (0.3-3 mg.ml(-1)), when given by aerosol, induced cough in conscious guinea-pigs in a concentration dependent manner. When guinea-pigs were pretreated with capsazepine, a VR1 antagonist, the anandamide-induced cough was significantly inhibited. Pretreatment with CB1 (SR 141716A) and CB2 (SR 144528) antagonists had no effect on anandamide-induced cough. These results indicate that anandamide-induced cough is mediated through the activation of VR1 receptors. 3. Anandamide (10-100 micro M) increased intracellular Ca(2+) concentration estimated by Fluo-4 fluorescence change in isolated guinea-pig nodose ganglia cells. The anandamide-induced Ca(2+) response was inhibited by two different VR1 antagonists: capsazepine (1 micro M) and iodo-resiniferatoxin (I-RTX, 0.1 micro M), indicating that anandamide-induced Ca(2+) response was through VR1 channel activation. In contrast, the CB1 (SR 141716A, 1 micro M) and CB2 (SR 144528, 0.1 micro M) receptor antagonists had no effect on Ca(2+) response to anandamide. 4. In conclusion, these results provide evidence that anandamide activates native vanilloid receptors in isolated guinea-pig nodose ganglia cells and induces cough through activation of VR1 receptors.
Assuntos
Ácidos Araquidônicos/farmacologia , Capsaicina/análogos & derivados , Tosse/induzido quimicamente , Dronabinol/análogos & derivados , Receptores de Droga/fisiologia , Aerossóis , Animais , Ácidos Araquidônicos/administração & dosagem , Cálcio/metabolismo , Canfanos/farmacologia , Capsaicina/administração & dosagem , Capsaicina/farmacologia , Células Cultivadas , Estado de Consciência , Dronabinol/farmacologia , Endocanabinoides , Antagonistas de Aminoácidos Excitatórios/farmacologia , Cobaias , Masculino , Gânglio Nodoso/citologia , Gânglio Nodoso/efeitos dos fármacos , Gânglio Nodoso/metabolismo , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas , Pirazóis/farmacologia , Receptores de Canabinoides , Receptores de Droga/agonistas , Receptores de Droga/antagonistas & inibidores , RimonabantoRESUMO
Human photoreceptor cGMP-phosphodiesterases (PDE6s) are important reagents in PDE inhibitor discovery. However, recombinant human PDE6s have not been expressed, and isolation of native human PDE6s is highly difficult. In this study, the catalytic subunit(s) of human rod and cone PDE6s (PDE6alphabeta and PDE6alpha', respectively) were co-expressed or expressed separately as catalytically active enzymes. Sildenafil inhibited both the recombinant PDE6s in a dose-dependent manner with Ki values of 94 and 98 nM, respectively. These Ki values were four-fold higher than that (25 nM) of a human native PDE6 preparation. Similarly, 3-isobutyl-1-methylxanthine (IBMX)'s Ki values for the recombinant PDE6s were five- to eight-fold higher than that of the native enzyme. However, E4021 and zaprinast exhibited much (30-80-fold) lower potencies for the recombinant PDE6s than for the native enzyme. Additional PDE5 inhibitors representing other structural classes and possessing different selectivity against native PDE6 also showed different potencies against the recombinant and native PDE6s. In particular, one class of xanthine analogues exhibited significantly (5-15-fold) higher potencies for the recombinant PDE6s than for the native enzyme. Our data demonstrates that the recombinant and native PDE6s exhibit differential sensitivity to inhibitors, and cautions the use of recombinant catalytic subunits of PDE6 in drug discovery or in structural/functional studies.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Inibidores de Fosfodiesterase/farmacologia , Células Fotorreceptoras/metabolismo , Piperazinas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , 3',5'-GMP Cíclico Fosfodiesterases/genética , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Humanos , Insetos/citologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Purinas , Citrato de Sildenafila , Sulfonas , TransfecçãoRESUMO
Receptor activator of NF-kappaB ligand (RANKL), produced by osteoblastic lineage cells and activated T cells, is an essential factor for osteoclast differentiation, activation, and survival. Therefore, RANKL is a focal point of therapies targeting bone diseases where there is an imbalance of bone metabolism in favor of bone resorption. The present study assesses the effects of exogenous RANKL on growing bone. RANKL (100 microg x kg-1x day-1 for 7 days) administered to Sprague-Dawley weanling rats caused major deficits in growth, appearance, and bone mineral densities (BMD). Urinary deoxypyridinoline crosslinks, a measure of bone turnover, were higher in the RANKL-treated rats (P = 0.031), and the bone mineral content was lower (P < 0.001). The final BMD in the RANKL-treated rats was lower (P = 0.039) than in the control rats (19 +/- 7 vs. 38 +/- 5 mg/cm3). Moreover, calculated cortical bone density in each bone slice (total BMD - trabecular BMD) indicated there was only 5% cortical bone remaining in RANKL-treated rats. We conclude that therapies targeting RANKL are likely to have effects on cortical as well as trabecular bone density.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Proteínas de Transporte/farmacologia , Glicoproteínas de Membrana/farmacologia , Aminoácidos/química , Aminoácidos/urina , Animais , Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Proteínas de Transporte/administração & dosagem , Relação Dose-Resposta a Droga , Glicoproteínas de Membrana/administração & dosagem , Camundongos , Ligante RANK , Ratos , Ratos Sprague-Dawley , Receptor Ativador de Fator Nuclear kappa-B , Tomografia Computadorizada por Raios XRESUMO
This study investigated the effects of antigen challenge on the cough reflex in dogs that were neonatally sensitized to ragweed. Tidal volume (V(T)), respiratory rate (f), pulmonary resistance (R(L)), dynamic lung compliance (C(Dyn)) and the number and amplitude (increase in mean peak expiratory pressure) of coughs induced by mechanical stimulation of the intrathoracic trachea were measured in propofol-anesthetized dogs. Aerosolized ragweed challenge had no effect to induce spontaneous cough but increased f and R(L) and reduced V(T) and C(Dyn). Mechanical stimulation of the intrathoracic trachea at this time produced 19+/-5 coughs with an average increase in cough amplitude of 11+/-1 cm H(2)O which differed significantly from the number (9+/-2 coughs) and amplitude (30+/-5.5 cm H(2)O) of mechanically induced coughs after treatment with aerosolized saline. Both the number and amplitude of mechanically induced coughs returned to baseline values by 24-48 h after the ragweed challenge. Similar results were obtained after challenge with aerosolized histamine (0.3-1% histamine) that did not induce spontaneous coughs but increased f, reduced V(T) and decreased C(Dyn) and increased the number but reduced the amplitude of the mechanically induced coughs. In conclusion, both antigen and histamine bronchoprovocation changed the characteristics of the mechanically induced cough in dogs to a response of increased cough number but reduced mean expiratory cough amplitude.
Assuntos
Tosse/fisiopatologia , Hipersensibilidade/fisiopatologia , Reflexo , Aerossóis , Animais , Testes de Provocação Brônquica , Tosse/imunologia , Modelos Animais de Doenças , Cães , Histamina/imunologia , Hipersensibilidade/imunologia , Masculino , Estimulação FísicaRESUMO
Experiments were performed to characterize the pharmacology of SCH 206272 [(R,R)-1'[5-[(3,5-dichlorobenzoyl)methylamino]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl]-N-methyl-2-oxo-[1,4'bipiperidine]-3-acetamide] as a potent and selective antagonist of tachykinin (NK) NK(1), NK(2), and NK(3) receptors. SCH 206272 inhibited binding at human tachykinin NK(1), NK(2), and NK(3) receptors (K(i) = 1.3, 0.4, and 0.3 nM, respectively) and antagonized [Ca(2+)](i) mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human tachykinin NK(1), NK(2), or NK(3) receptors. SCH 206272 inhibited relaxation of the human pulmonary artery (pK(b) = 7.7 +/- 0.3) induced by the tachykinin NK(1) receptor agonist, [Met-O-Me] substance P and contraction of the human bronchus (pK(b = 8.2 +/- 0.3) induced by the tachykinin NK(2) receptor agonist, neurokinin A. In isolated guinea pig tissues, SCH 206272 inhibited substance P-induced enhancement of electrical field stimulated contractions of the vas deferens, (pK(b = 7.6 +/- 0.2), NKA-induced contraction of the bronchus (pK(b) = 7.7 +/- 0.2), and senktide-induced contraction of the ileum. In vivo, oral SCH 206272 (0.1-10 mg/kg, p.o.) inhibited substance P-induced airway microvascular leakage and neurokinin A-induced bronchospasm in the guinea pig. In a canine in vivo model, SCH 206272 (0.1-3 mg/kg, p.o.) inhibited NK(1) and NK(2) activities induced by exogenous substance P and neurokinin A. Furthermore, in guinea pig models involving endogenously released tachykinins, SCH 206272 inhibited hyperventilation-induced bronchospasm, capsaicin-induced cough, and airway microvascular leakage induced by nebulized hypertonic saline. These data demonstrate that SCH 206272 is a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist. This compound may have beneficial effects in diseases thought to be mediated by tachykinins, such as cough, asthma, and chronic obstructive pulmonary disease.
Assuntos
Acetamidas/farmacologia , Antagonistas dos Receptores de Neurocinina-1 , Piperidinas/farmacologia , Receptores da Neurocinina-2/antagonistas & inibidores , Receptores da Neurocinina-3/antagonistas & inibidores , Administração Oral , Animais , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/fisiologia , Células CHO , Permeabilidade Capilar , Capsaicina/farmacologia , Tosse/induzido quimicamente , Tosse/tratamento farmacológico , Cricetinae , Cães , Relação Dose-Resposta a Droga , Cobaias , Humanos , Íleo/efeitos dos fármacos , Íleo/fisiologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Ensaio Radioligante , Receptores da Neurocinina-1/metabolismo , Receptores da Neurocinina-2/metabolismo , Receptores da Neurocinina-3/metabolismo , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiologiaRESUMO
Mast cells are the central mediating cells of allergic reactions. Binding of allergen specific IgE to high affinity IgE receptor (Fcepsilon RI) and subsequent binding of allergen by the IgE causes receptor cross-linking and activation. In a study examining the differential gene expression in human cord blood-derived mast cells (CBMCs) mediated by activation of Fcepsilon RI both with IgE and IgE followed by cross-linking with alpha-IgE, the chemokine I-309 was found to be upregulated. I-309 is the ligand for the CCR8 receptor and is responsible for chemoattraction of TH2 type T-cells. Interestingly, I-309 RNA and protein levels were elevated not only in response to IgE/alpha-IgE activation but also by IgE alone. In addition, the I-309 levels were augmented by growth of the CBMCs in the presence of the proinflammatory cytokine IL-4. GM-CSF and MIP-1alpha secretion was also induced by IgE. These results suggest that IgE, through the production and release of cytokines such as I-309, GM-CSF and MIP-1alpha could promote an inflammatory reaction in the absence of antigen stimulation of mast cells.
Assuntos
Quimiocinas CC/metabolismo , Sangue Fetal/citologia , Imunoglobulina E/farmacologia , Mastócitos/fisiologia , Quimiocina CCL1 , Quimiocinas/metabolismo , Reagentes de Ligações Cruzadas , Citocinas/metabolismo , Primers do DNA , Expressão Gênica , Histamina/metabolismo , Liberação de Histamina/efeitos dos fármacos , Humanos , Interleucina-4/biossíntese , Interleucina-4/genética , Análise de Sequência com Séries de Oligonucleotídeos , Receptores CCR8 , Receptores de Quimiocinas/metabolismo , Receptores de IgE/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neurokinin A (NKA) potentiates airway cholinergic neurotransmission in several species. In this study, the role of cholinergic reflexes on the bronchoconstrictor response to NKA was evaluated in non-sensitized dogs and in allergic dogs neonatally sensitized to ragweed in which heightened bronchoconstrictor reactivity to NKA has previously been observed. Cardiopulmonary functions, including pulmonary resistance (R(L)) were measured in anesthetized, spontaneously breathing dogs before and after increasing concentrations of aerosolized NKA. The provocative concentrations of NKA increasing R(L) by 25% above the baseline (PC(25)) was measured before and after ( approximately 10 min) aerosolized saline or ipratropium bromide (0.01%). This concentration of ipratropium produced a 250-fold shift in the methacholine dose-response curve. In sensitized dogs, NKA bronchoconstrictor reactivity (PC(25)=0.050+/-0.011%) was 2.5 times more potent than that of non-sensitized controls (PC(25)=0.177+/-0.031%). Ipratropium bromide inhibited the bronchoconstrictor response to NKA in both sensitized and non-sensitized dogs and after ipratropium, NKA reactivity was 5.2-fold less in allergic dogs (PC(25)=0.246+/-0.048%) as compared to 3.5 fold less in non-sensitized controls (PC(25)=0.622+/-0.106%). In conclusion, cholinergic reflexes are important components of the bronchoconstrictor response to NKA in dogs particularly in those sensitized neonatally to ragweed. It is speculated that heightened activity of cholinergic reflexes contributes to the bronchial hyperresponsiveness seen in allergic dogs.
Assuntos
Alérgenos/efeitos adversos , Antagonistas Colinérgicos/farmacologia , Imunização , Ipratrópio/farmacologia , Neurocinina A/farmacologia , Reflexo/efeitos dos fármacos , Aerossóis , Alérgenos/efeitos dos fármacos , Animais , Testes de Provocação Brônquica , CãesRESUMO
Anesthetized Sprague-Dawley weanling rats were scanned for bone mineral density (BMD) values after 7 days of treatment to determine whether resorption/growth at the proximal tibia can be quantified by peripheral quantitative computed tomography scanning techniques. Because the weanling rat is in a rapid growth stage, all groups showed significant increases in change from baseline values of BMD. Bisphosphonate treatment produced significant dose-related changes in BMD with average increases of 195 and 241% (10 and 20 microg/kg) vs. 86% in control rats. We further characterized this model to determine effects of steroids on growing bone. Graded doses of glucocorticoid (3.5, 7.0, 10.5, 14.0, 28.0, and 42.0 mg x kg(-1) x wk(-1)) caused no significant differences in trabecular BMD in 7 days between control and treated rats. Significant decreases in growth (weights) and increases in cortical bone area were observed, indicating that this model may be useful in comparing effects of nonsteroid, anti-inflammatory alternatives on juvenile bone. Although the relevance of this model to adult disease remains to be elucidated, it also provides a tool for mechanistic evaluation of therapeutic modalities or efficacy assessment for dose selection for longerterm models.
Assuntos
Envelhecimento , Desenvolvimento Ósseo/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Esteroides/farmacologia , Tomografia Computadorizada por Raios X/métodos , Alendronato/administração & dosagem , Animais , Densidade Óssea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fêmur/anatomia & histologia , Glucocorticoides/farmacologia , Masculino , Metilprednisolona/farmacologia , Tamanho do Órgão , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The role of IL-5-induced eosinophilia in airway hyperresponsiveness has been questioned. In addition, eosinophil-independent IL-5-induced airway hyperresponsiveness has been demonstrated in animals. OBJECTIVE: In this study, IL-5 was investigated for direct effects on human bronchial responsiveness. METHODS: Human muscle preparations were isolated from organ donor and surgical tissue. Bronchus, jejunum, and saphenous vein were incubated for 24 hours in vitro with recombinant human (rh) IL-5. Contractility to acetylcholine (bronchus, jejunum) and phenylephrine (saphenous vein) was then investigated. RT-PCR was used to evaluate IL-5 receptor alpha (IL-5R(alpha)) expression in various tissues and to assess bronchus and saphenous vein eosinophils through use of CCR3 expression. RESULTS: rhIL-5 primed bronchus for an exaggerated contraction to acetylcholine. The acetylcholine concentration that produced 50% of the control maximum response was reduced 17- to 20-fold in bronchus treated with 1 and 10 nmol/L rhIL-5. The lower concentration of 0.1 nmol/L rhIL-5 had no effect. The rhIL-5 effect on bronchial contractility was attenuated by antibodies to IL-5 (TRFK-5; 100 nmol/L) and human IL-5R(alpha) (100 nmol/L). rhIL-5 (10 nmol/L) did not enhance contractility of saphenous vein or jejunum. When RT-PCR was used, IL-5R(alpha) expression was strong in bronchus muscle, weak in trachealis, saphenous vein, and atrial muscle, and undetectable in jejunum, urinary bladder, and pulmonary and renal artery muscle. Comparable weak expression of CCR3 was identified in bronchus and saphenous vein. CONCLUSION: The findings are consistent with an airway tissue-selective expression of the IL-5 receptor that mediates IL-5-induced airway hyperresponsiveness independent of eosinophils. In asthma, in which IL-5 expression is elevated, IL-5 might directly induce bronchial hyperresponsiveness.
Assuntos
Brônquios/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição , Interleucina-5/farmacologia , Receptores de Interleucina/metabolismo , Acetilcolina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Brônquios/fisiologia , Feminino , Humanos , Interleucina-5/genética , Jejuno/efeitos dos fármacos , Jejuno/fisiologia , Masculino , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Fenilefrina/farmacologia , Receptores CCR3 , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-5 , Proteínas Recombinantes/farmacologia , Veia Safena/efeitos dos fármacos , Veia Safena/fisiologiaRESUMO
Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.
Assuntos
Brônquios/citologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Miócitos de Músculo Liso/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Expressão Gênica , Cobaias , Humanos , Soluções Hipotônicas/farmacologia , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Canais de Cátion TRPV , Traqueia/citologia , Traqueia/fisiologia , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
Cough is an important defensive pulmonary reflex that removes irritants, fluids or foreign materials from the airways. However, often cough is non-productive and requires suppression. Opioid mu receptor agonists, such as codeine are commonly used as antitussive agents and are among the most widely administered drugs in the world. Codeine suppresses the responsiveness of one or more components of the central reflex pathway for cough and is an efficacious antitussive drug for cough due to diverse aetiologies. However, opioids produce side effects that include sedation, addiction potential and constipation. Therefore, novel cough suppressant therapies should maintain or improve upon the antitussive efficacy profile of opioids. Moreover, these novel therapies should have a safety profile significantly better than current antitussive therapies. Presently, we discuss preclinical findings showing that activation of the 'opioid-like' receptor (NOP(1)) inhibits cough in the guinea pig and cat.
Assuntos
Antitussígenos/uso terapêutico , Tosse/tratamento farmacológico , Peptídeos Opioides/uso terapêutico , Receptores Opioides/agonistas , Vasodilatadores/uso terapêutico , Animais , Gatos , Cobaias , NociceptinaRESUMO
ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.
Assuntos
Metaloendopeptidases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas ADAM , Animais , Domínio Catalítico , Linhagem Celular , Glicosilação , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/genética , Camundongos , Mutação , Polimorfismo Genético , Inibidores de Proteases/metabolismo , Distribuição Tecidual , Extratos de Tecidos/metabolismo , Transfecção , alfa-Macroglobulinas/metabolismoRESUMO
We present the pharmacological and pharmacokinetic profiles of a novel histamine H3 receptor antagonist, N-(3,5-dichlorophenyl)-N'-[[4-(1H-imidazol-4-ylmethyl)phenyl]-methyl]-urea (SCH 79687). The H3-receptor binding Ki values for SCH 79687 were 1.9 and 13 nM in the rat and guinea pig (GP), respectively. The Ki values for SCH 79687 at histamine H1 and H2 receptors were greater than 1 microM. SCH 79687 showed a 41- and 82-fold binding selectivity for the H3 receptor over alpha 2A-adrenoceptors and imidazoline I2, and >500-fold H3 selectivity compared with over 60 additional receptors. The pA2 value for SCH 79687 in the GP ileum electrical field-stimulated (EFS) contraction was 9.6 +/- 0.3. Similar H3 antagonist activity was observed in the EFS cryopreserved and fresh tissue isolated human saphenous vein (HSV) assays (pKb = 9.4 +/- 0.3 and 10.1 +/- 0.4). SCH 79687 (30 nM) did not block clonidine-induced inhibition of EFS-induced contractions in HSV. SCH 79687 (ED50 = 0.3 mg/kg i.v.) attenuated (R)-alpha-methylhistamine inhibition of sympathetic hypertensive responses in the GP. At the time of activity evaluation, the GP plasma SCH 79687 concentration was 25 ng/ml at the dose of 0.3 mg/kg i.v. In feline nasal studies, combined administration of SCH 79687 (3 mg/kg i.v.) and the H1-antagonist loratadine (3 mg/kg i.v.), at individual doses that do not produce decongestion, inhibited the compound 48/80-induced congestion by 47%. The alpha-adrenergic agonist phenylpropanolamine (PPA; 1 mg/kg i.v.) also attenuated compound 48/80 nasal responses by 42%. Unlike the H3/H1 combination that did not affect blood pressure (BP), PPA (1 mg/kg i.v.) significantly increased BP compared with control animals by a maximum of 31 mm Hg. Orally, SCH 79687 (10 mg/kg) plus loratadine (10 mg/kg) also produced decongestion without effects on BP. In pharmacokinetic studies, oral dosing with SCH 79687 in the rat (10 mg/kg) and monkey (3 mg/kg) achieved plasma Cmax and area under the curve values greater than 1.5 and 12.1 microg. h/ml, respectively. SCH 79687 is an orally active H3 antagonist with a good pharmacokinetic profile that, in combination with an H1 antagonist, demonstrates decongestant efficacy comparable with oral sympathomimetic decongestants but without hypertensive liabilities.
Assuntos
Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Imidazóis/farmacologia , Compostos de Fenilureia/farmacologia , Receptores Histamínicos H3/metabolismo , Animais , Gatos , Interações Medicamentosas , Feminino , Cobaias , Haplorrinos , Agonistas dos Receptores Histamínicos/farmacocinética , Antagonistas dos Receptores Histamínicos/farmacocinética , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Loratadina/farmacologia , Masculino , Metilistaminas/farmacocinética , Metilistaminas/farmacologia , Descongestionantes Nasais/farmacologia , Ratos , Receptores Histamínicos H3/efeitos dos fármacos , Veia Safena/efeitos dos fármacos , Veia Safena/metabolismoRESUMO
N-(3,5-Dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinoline carboxamide (SCH 351591) has been identified as a potent (IC(50) = 58 nM) and highly selective type 4 phosphodiesterase (PDE4) inhibitor with oral bioactivity in several animal models of lung inflammation. N-(3,5-Dichloro-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinoline carboxamide (SCH 365351), the only significant in vivo metabolite, is also a potent and highly selective PDE4 inhibitor (IC(50) = 20 nM). Both SCH 351591 and SCH 365351 inhibited cytokine production in human blood mononuclear cell preparations. Oral SCH 351591 significantly attenuated allergen-induced eosinophilia and airway hyperreactivity in allergic guinea pigs at doses as low as 1 mg/kg. In this model, oral SCH 365351 showed similar potency. When SCH 351591 was administered orally to allergic cynomolgus monkeys at 3 mg/kg, Ascaris suum-induced lung eosinophilia was blocked. Hyperventilation-induced bronchospasm in nonallergic guinea pigs, a model for exercise-induced asthma, was also suppressed significantly by oral SCH 351591 at 0.3 mg/kg. Cilomilast (SB 207499; Ariflo), a PDE4 inhibitor currently being developed for asthma and chronic obstructive pulmonary disease (COPD), was 10- to 30-fold less potent than SCH 351591 at inhibiting guinea pig lung eosinophilia and hyperventilation-induced bronchospasm. In a ferret model of emesis, maximum nonemetic oral doses of SCH 351591 and cilomilast were 5 and 1 mg/kg, respectively. Comparison of plasma levels at these nonemetic doses in ferrets to those at doses inhibiting hyperventilation-induced bronchospasm in guinea pigs gave a therapeutic ratio of 16 for SCH 351591 and 4 for cilomilast. Thus, SCH 351591 exhibits a promising preclinical profile as a treatment for asthma and COPD.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Óxidos N-Cíclicos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Quinolinas/farmacologia , Adolescente , Adulto , Idoso , Animais , Antiasmáticos/farmacologia , Ligação Competitiva/efeitos dos fármacos , Hiper-Reatividade Brônquica/prevenção & controle , Espasmo Brônquico/prevenção & controle , Broncodilatadores/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Eméticos/farmacologia , Feminino , Furões , Cobaias , Humanos , Hiperventilação/fisiopatologia , Interleucina-12/biossíntese , Interleucina-5/biossíntese , Macaca fascicularis , Masculino , Camundongos , Pessoa de Meia-Idade , Ratos , Rolipram/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Asthma is a common respiratory disorder characterized by recurrent episodes of coughing, wheezing and breathlessness. Although environmental factors such as allergen exposure are risk factors in the development of asthma, both twin and family studies point to a strong genetic component. To date, linkage studies have identified more than a dozen genomic regions linked to asthma. In this study, we performed a genome-wide scan on 460 Caucasian families and identified a locus on chromosome 20p13 that was linked to asthma (log(10) of the likelihood ratio (LOD), 2.94) and bronchial hyperresponsiveness (LOD, 3.93). A survey of 135 polymorphisms in 23 genes identified the ADAM33 gene as being significantly associated with asthma using case-control, transmission disequilibrium and haplotype analyses (P = 0.04 0.000003). ADAM proteins are membrane-anchored metalloproteases with diverse functions, which include the shedding of cell-surface proteins such as cytokines and cytokine receptors. The identification and characterization of ADAM33, a putative asthma susceptibility gene identified by positional cloning in an outbred population, should provide insights into the pathogenesis and natural history of this common disease.