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1.
Leukemia ; 32(3): 820-827, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28819285

RESUMO

Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Fusão bcr-abl/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína SOS1/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Camundongos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tirosina/metabolismo , Proteínas rac de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP/metabolismo
2.
Eur Biophys J ; 39(6): 947-57, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19455321

RESUMO

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.


Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/métodos , Fenômenos Biomecânicos , Células , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Movimento/efeitos da radiação , Mutação
3.
Cell Mol Life Sci ; 62(9): 955-70, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15868099

RESUMO

Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.


Assuntos
Citoesqueleto de Actina/fisiologia , Movimento Celular/fisiologia , Transdução de Sinais/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Proteínas do Citoesqueleto/fisiologia , Humanos , Proteínas dos Microfilamentos/fisiologia , Modelos Biológicos , Pseudópodes/metabolismo , Proteínas rac de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia
4.
J Cell Biol ; 154(5): 1031-44, 2001 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-11524436

RESUMO

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas/metabolismo , Proteína SOS1/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Proteínas de Transporte/genética , Fracionamento Celular , Extensões da Superfície Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Citocalasina D/farmacologia , Proteínas do Citoesqueleto , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genes Reporter , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microscopia de Fluorescência , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SOS1/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas ras/genética
5.
EMBO J ; 19(11): 2393-8, 2000 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10835338

RESUMO

Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase-->activator--> GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.


Assuntos
GTP Fosfo-Hidrolases/fisiologia , Transdução de Sinais/fisiologia , Animais , GTP Fosfo-Hidrolases/classificação , Guanosina Trifosfato/fisiologia , Mamíferos/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Proteínas ras/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia
6.
J Allergy Clin Immunol ; 100(5): 669-78, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9389298

RESUMO

BACKGROUND: In allergic subjects with asthma, the migration of CD4+ T cells to the lungs in the hours after allergen exposure may contribute to allergic inflammation in the target organ. OBJECTIVE: We studied allergen-specific T cells from the peripheral blood and lungs of allergic subjects with asthma at baseline and after allergen challenge. METHODS: In each patient, blood samples were taken 10 minutes before and 24 hours after the inhalation of a major sensitizing allergen. In vitro proliferation of peripheral blood CD4+ T cells specific for the same allergen used in the in vivo challenge was assessed. In one patient two Dermatophagoides pteronyssinus-specific T-cell clones (TCCs) were derived from peripheral blood, and their T-cell receptors were sequenced to determine their clonotypic determinants on the beta chains. The T-cell receptor determinants of the allergen-specific TCCs were sought in blood and bronchoalveolar lavage samples taken from this patient. RESULTS: We found that allergen inhalation is followed by a decrement in the specific proliferation of peripheral CD4+ T cells to the same allergen used for bronchial provocation. In one patient the clonotypic determinants of two allergen-specific TCCs diminished in the peripheral blood, whereas they were simultaneously expanded in the lower respiratory tract. CONCLUSION: Our data suggest that allergen-specific T cells are recruited from the peripheral blood to the bronchial lumen after allergen challenge.


Assuntos
Alérgenos/farmacologia , Asma/imunologia , Pulmão/citologia , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/citologia , Células Clonais , Feminino , Humanos , Depleção Linfocítica , Masculino , Ácaros , Receptores de Antígenos de Linfócitos T alfa-beta/química , Linfócitos T/citologia
7.
J Allergy Clin Immunol ; 99(6 Pt 1): 788-97, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9215247

RESUMO

BACKGROUND: In allergic asthma, CD4+ T lymphocytes are a fundamental component of local chronic inflammation. Their cytokine profile is oriented toward a TH2 phenotype, characterized by production of IL-4, IL-5, IL-10, and IL-13. Egress of T cells from blood to airways after allergen challenge has been described. OBJECTIVE: We have studied a cohort of six patients with asthma who had multiple allergies to investigate how exposure to allergen affects the proliferation of peripheral CD4+ T lymphocytes with different allergen specificities and lymphokine profiles. METHODS: For each patient, CD4+ T-cell lines were generated by in vitro stimulation with sensitizing and with nonsensitizing allergens, and IL-4 and interferon-gamma production by these lines was assessed. Proliferation of peripheral blood CD4+ T lymphocytes in response to the same allergens was measured before and 24 hours after inhalation challenge with a sensitizing allergen. RESULTS: We found that each single sensitizing allergen can deplete peripheral blood of TH2-type CD4+ T lymphocytes specific for all sensitizing allergens, but not of TH1-type CD4+ T lymphocytes. CONCLUSIONS: Our results suggest the existence of mechanisms capable of sorting disease-associated antigen specificities together with defined lymphokine patterns into T lymphocytes that can migrate to target organs, in allergic asthma.


Assuntos
Alérgenos/sangue , Alérgenos/imunologia , Asma/imunologia , Depleção Linfocítica , Células Th2/imunologia , Administração por Inalação , Adulto , Alérgenos/administração & dosagem , Animais , Citocinas/biossíntese , Epitopos/imunologia , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Ácaros/imunologia , Poaceae/imunologia , Células Th2/metabolismo
8.
Pharmacol Res ; 35(5): 439-42, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9299208

RESUMO

The effect of polymyxin B and KN-62 on the [3H]5-HT release induced by depolarization and by 0.5 microM D-fenfluramine (dF) from superfused rat hippocampal synaptosomes was examined. Polymyxin B and KN-62 were initially characterized as inhibitors, respectively, of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (Ca/CaM-KII), although both compounds were subsequently described as inhibitors of the depolarization-induced Ca2+ influx through voltage-operated Ca2+ channels, at concentrations similar to those interacting with the CaM systems. Three micromolar KN-62 significantly inhibited the dF- and the depolarization-induced [3H]5-HT release, by 25% and 33%. Polymyxin B, tested at concentrations from 30 to 1000 IU ml-1, dose-dependently inhibited both the dF- and the depolarization-induced [3H]5-HT release with similar potency, with complete inhibition at the highest concentration tested. These compounds did not significantly alter 5-HT transporter function. Moreover dF had no direct effect on Ca/CaM-KII activity. These results further support the suggestion that the [3H]5-HT release induced by low concentrations of dF (0.5 microM), previously found to be Ca(2+)-dependent, actually involves a dF-induced Ca2+ influx into the nerve terminal and the subsequent exocytosis.


Assuntos
Cálcio/fisiologia , Exocitose/efeitos dos fármacos , Fenfluramina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Antibacterianos/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Polimixina B/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
9.
Neurochem Int ; 29(2): 129-34, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8837041

RESUMO

It has been suggested that the Ca(2+)-dependent enzyme transglutaminase (TGase) may suppress vesicular neurotransmitter release and mediate the inhibitory effect of tetanus toxin (TetTx) on exocytosis. The aim of the present study was to test this in a model of [3H]5-HT release from superfused rat cortical synaptosomes. Monodansylcadaverine, a synthetic amine that acts as an alternative substrate for TGase, showed dose-dependent releasing activity which, however, was Ca(2+)-independent, being maintained in a Ca(2+)-free buffer (containing EGTA) or using synaptosomes preloaded with the intracellular Ca2+ chelator BAPTA. Preincubation of synaptosomes with RS-48373, an irreversible TGase inactivator, resulted in marked (64%) and persistent inhibition of endogenous TGase but did not alter basal and K(+)-induced [3H]5-HT release. Preincubation of synaptosomes with 10 nM TetTx resulted in 52% inhibition of K(+)-induced [3H]5-HT release, and this effect was not antagonized in RS-48373-treated synaptosomes. The inhibitory effect of TetTx was significantly antagonized by 20 mM captopril, a metalloendoprotease inhibitor, confirming in rat brain synaptosomes that TetTx inhibits exocytosis by acting as a metalloendoprotease. These results suggest that TGase is not involved in controlling [3H]5-HT release from resting and depolarized synaptosomes, or in the inhibitory effect of TetTx.


Assuntos
Serotonina/metabolismo , Sinaptossomos/metabolismo , Toxina Tetânica/farmacologia , Transglutaminases/metabolismo , Animais , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Cálcio/farmacologia , Córtex Cerebral/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos , Isoxazóis/farmacologia , Masculino , Potássio/farmacologia , Ratos , Transglutaminases/antagonistas & inibidores , Trítio
10.
Naunyn Schmiedebergs Arch Pharmacol ; 353(6): 641-7, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8738297

RESUMO

The effects of repeated subcutaneous (s.c) injections of dexfenfluramine (d-F; 10 mg/kg, twice daily, for 4 days) on the contents of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brain were assessed in primates (cynomolgus and rhesus monkeys) and compared with the regional brain concentrations of unchanged drug and its active metabolite, dexnorfenfluramine (d-NF). This four-day, high-dose, regimen caused a large depletion of 5-HT (more than 95%) and of 5-HIAA (80-90%) in all brain areas studied (cortex, hippocampus, putamen, caudate nucleus and hypothalamus) 2 h after the last injection of d-F. Analysis of the plasma and brain contents of d-F and d-NF confirmed that both compounds were concentrated as in other species, in regions of the primate brain. However, d-NF was concentrated to a greater extent than d-F, and there were differences between the two primate species. Unlike in the rat brain, concentrations of d-NF greatly exceeded those of d-F in the primate brain suggesting that in these primates the d-NF may play a major role in the overall neurochemical response. The effects of d-F and d-NF on different in vitro parameters of serotoninergic neuronal function did not show appreciable differences between cynomolgus or rhesus monkeys when compared to rats, the ability of the two compounds to inhibit 5-HT reuptake, to enhance its release, and to affect the binding of [3H] -d-F or of [3H] -mesulergine (a ligand for 5-HT2C receptors) being similar. Kinetic differences in the disposition of d-F appear to have more relevance than biochemical effects in providing an explanation for the more marked brain depletion induced by d-F in primates than in rodents.


Assuntos
Depressores do Apetite/farmacologia , Química Encefálica/efeitos dos fármacos , Fenfluramina/farmacologia , Norfenfluramina/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/análise , Serotonina/análise , Animais , Depressores do Apetite/metabolismo , Encéfalo/metabolismo , Fenfluramina/sangue , Fenfluramina/metabolismo , Técnicas In Vitro , Macaca fascicularis , Macaca mulatta , Masculino , Norfenfluramina/análogos & derivados , Norfenfluramina/sangue , Ratos , Receptores de Serotonina/metabolismo , Serotonina/análogos & derivados , Serotonina/metabolismo , Serotonina/farmacocinética , Especificidade da Espécie
11.
Eur Respir J ; 9(2): 319-27, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8777971

RESUMO

We reasoned that persistent exposure to a limited set of airborne antigens could drive the preferential expansion of single T-cell clones in the lower respiratory tract of normal individuals. To explore this issue, the normal human alpha/beta T-cell receptor repertoire was studied in lung lymphocytes obtained by bronchoalveolar lavage (BAL) from the humen of the lower respiratory tract. BAL T-cells obtained from five healthy volunteers were first analysed using polymerase chain reaction to amplify all known V alpha and V beta genes of the T-cell receptor. T-cells from peripheral blood were used as an internal control. Heteroduplex analysis of the amplified products was then performed, to assess the clonal composition of the repertoire of lung- versus blood-derived T-lymphocytes within each amplified variable gene family. In all subjects, the T-cell repertoire in the lung was largely as heterogeneous as peripheral blood in terms of clonal composition. This indicated lack of preferential expansion of single T-cell clones. A few T-cell clones were simultaneously expanded in blood and lung in all individuals within a limited number of V beta (mean 2.4; range 2-4) and V alpha (mean 1.6; range 1-3) genes. We also found that lung T-lymphocytes expressed all of the V gene families of the T-cell receptor that were expressed by peripheral blood T-cells. Our results indicate that T-cell clones in the lower respiratory tract of normal individuals are distributed according to a largely polyclonal pattern, which corresponds to that found in peripheral blood.


Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Lavagem Broncoalveolar , Ácidos Nucleicos Heteroduplexes/análise , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Adulto , Sequência de Bases , DNA/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/química , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sequências Repetitivas de Ácido Nucleico
12.
Pharmacol Biochem Behav ; 53(1): 155-61, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8848445

RESUMO

Flunarizine (35 mg/kg), but not haloperidol and trifluperazine, counteracted the initial indole depletion induced by D-fenfluramine (dF) in vivo (5 mg/kg), without affecting ex vivo [3H]-serotonin (5-HT) uptake by synaptosomes or changing the brain concentrations of the parent drug and its main active metabolite, D-norfenfluramine (dNF). The long-term indole depletion induced by repeated doses of dF (5 mg/kg, b.i.d. for 4 days) was also reversed by flunarizine pretreatment. Flunarizine, methiothepin, and trifluperazine, but not haloperidol, reduced in vitro the Ca(2+)-dependent [3H]5-HT release stimulated by 0.5 microM dF and dNF from superfused synaptosomes. At the concentrations used in release experiments the drugs were not active on [3H]5-HT uptake nor on the calcium-calmodulin protein kinase activity, thus excluding an effect on the uptake carrier or on phosphorylation of synaptic proteins involved in exocytosis, respectively. The drugs did not consistently affect [3H]5-HT release induced by depolarization, or dNF-induced [3H]dopamine release in vitro. The fact that flunarizine, as methiothepin and 5-HT uptake inhibitors, counteract dF-induced indole depletion in vivo suggests a relation between the reduction of the Ca(2+)-dependent release of [3H]5-HT induced by dF in vitro and the protective effect on the short- and long-lasting depletion of indoles induced in vivo by high doses of dF.


Assuntos
Fenfluramina/antagonistas & inibidores , Fenfluramina/farmacologia , Flunarizina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Dopamina/metabolismo , Interações Medicamentosas , Ácido Hidroxi-Indolacético/metabolismo , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Serotonina/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/enzimologia , Sinaptossomos/metabolismo
13.
J Immunol ; 155(12): 5836-46, 1995 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-7499873

RESUMO

We were interested in studying the lung allergen-specific T cell repertoire in different conditions of allergen exposure in subjects with atopic asthma. Twenty-one allergic individuals were studied: 17 subjects suffering mainly from asthma and 4 from rhinitis. They all performed spirometry and methacholine challenge. All patients were subjected to bronchoalveolar lavage (BAL), either at base line (no challenge) or after allergen or histamine challenge, and the TCR repertoire of their lung T cells was studied with heteroduplex analysis. Expansion of single T cell clones was observed in one (of seven) asthmatic subject that reported a recent exposure to allergen and had high bronchial hypersensitivity to methacholine, and in seven/seven asthmatic subjects who underwent BAL after they suffered an early asthmatic reaction to experimental allergen inhalation. Remarkably, ex vivo expanded clones included allergen-specific T cells. In two of the seven subjects who underwent BAL after allergen challenge, two different lung segments were lavaged. A strikingly symmetrical distribution of the expanded clones was found in these samples. Control subjects and six of seven asthmatic patients studied at base line showed polyclonality of lung T cells. In conclusion, T lymphocytes are clonally expanded in the lower respiratory tract only in asthmatic subjects exposed to allergen. These results suggest that in allergic asthma, the inhalation of sensitizing allergens can recruit to the lung T lymphocytes that include allergen-specific T cell clones.


Assuntos
Alérgenos/imunologia , Asma/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Sequência de Bases , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Testes de Função Respiratória , Rinite/imunologia
14.
Neuropharmacology ; 33(6): 833-5, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7936121

RESUMO

The Ca2(+)-dependent [3H]5-HT release induced by depolarization or by 0.5 microM d-fenfluramine in rat hippocampal synaptosomes, was significantly reduced (35-42%) by three different P-type Ca2+ channels blockers (omega-Agatoxin-IVA, 100 nM, funnel-web spider toxin, FTX, 0.05 microliters/ml, and its synthetic analogue, sFTX, 1 mM), indicating the major role of these channels in the Ca2+ influx preceding neurotransmitter release.


Assuntos
Canais de Cálcio/fisiologia , Fenfluramina/farmacologia , Hipocampo/metabolismo , Potássio/farmacologia , Serotonina/metabolismo , Sinaptossomos/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Poliaminas/farmacologia , Ratos , Ratos Sprague-Dawley , Venenos de Aranha/farmacologia , Sinaptossomos/efeitos dos fármacos , ômega-Agatoxina IVA
15.
J Pharm Pharmacol ; 45(9): 811-4, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7903370

RESUMO

Three selective alpha 2A- or alpha 2B-adrenergic antagonists (BRL-44408, BRL-41992 and imiloxan) were used in the present study designed to classify the presynaptic alpha 2-auto- and heteroreceptors in the rat brain cortex. The rank order of potency in antagonizing the inhibitory effect of (-)-noradrenaline or clonidine on the K(+)-induced [3H]noradrenaline and [3H]5-hydroxytryptamine (5-HT) release from superfused synaptosomes was BRL-44408 > or = BRL-41992 >> imiloxan. The same rank order was found for the affinities of these compounds for [3H]yohimbine binding in human platelet membranes, containing only alpha 2A-adrenoceptors, but does not correlate with the known affinities for alpha 2B-adrenoceptors (BRL-41992 > or = imiloxan > BRL-44408). These data support the conclusion that presynaptic alpha 2-auto- and heteroreceptors in rat brain cortex do not belong to the alpha 2B-subtype and suggest that the modulation of noradrenaline and 5-HT release may be mediated by the alpha 2A-subtype.


Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Córtex Cerebral/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Serotonina/metabolismo , Sinaptossomos/metabolismo , Antagonistas Adrenérgicos alfa/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Dibenzazepinas/metabolismo , Dibenzazepinas/farmacologia , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Técnicas In Vitro , Indóis/metabolismo , Indóis/farmacologia , Isoindóis , Masculino , Norepinefrina/farmacologia , Coelhos , Ratos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos
16.
Eur J Pharmacol ; 238(1): 9-17, 1993 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-8405087

RESUMO

The monoamine releasing activity of d-fenfluramine was investigated with an in vitro model consisting of synaptosomes preloaded with the 3H-neurotransmitter and extensively washed in a superfusion apparatus before a 3-min exposure to d-fenfluramine. With this model, the drug-induced release is real and is not confused by inhibition of reuptake by the drug. d-Fenfluramine (0.5 microM) induced only [3H]5-hydroxytryptamine ([3H]5-HT) release from hippocampal synaptosomes whereas 10 microM also induced some overflow from hippocampal synaptosomes preloaded with [3H]noradrenaline or from striatal synaptosomes preloaded with [3H]dopamine, although the overflow was much lower than from 5-HTergic synaptosomes. We then focused on the [3H]5-HT release induced by 0.5 microM d-fenfluramine, which was previously shown to be Ca2+ dependent. The same finding was confirmed in the present study with other experimental protocols, indicating the requirement for extracellular Ca2+ ions. By measuring [3H]5-HT uptake into rat hippocampal synaptosomes we confirmed that Ca(2+)-ions are not required for the function of the 5-HT uptake carrier or for its interaction with d-fenfluramine. d-Fenfluramine-induced [3H]5-HT release was not altered by 1 microM nitrendipine (blocking the L-type Ca2+ channels) but was slightly decreased (20%) by 0.5 microM omega-conotoxin (blocking the N-type Ca2+ channels). It was also inhibited by 0.5 microM clonidine, interacting with alpha 2-adrenergic heteroreceptors, and by 10 nM tetanus toxin, known to affect the exocytosis of different neurotransmitters including 5-HT. These compounds had very similar effects on the Ca(2+)-dependent, exocytotic release of [3H]5-HT induced by depolarization, i.e. by 15 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Cálcio/metabolismo , Fenfluramina/farmacologia , Hipocampo/efeitos dos fármacos , Serotonina/metabolismo , Sinaptossomos/efeitos dos fármacos , Animais , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Dopamina/farmacologia , Fenfluramina/administração & dosagem , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Norepinefrina/farmacologia , Ratos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Receptores Adrenérgicos alfa/metabolismo , Sinaptossomos/metabolismo , Toxina Tetânica/farmacologia
17.
Neurosci Lett ; 151(2): 205-8, 1993 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-8506082

RESUMO

The effects of tetanus toxin on depolarization-induced [3H]serotonin release from superfused rat brain cortex synaptosomes was investigated. Two hours' preincubation of the synaptosomes with tetanus toxin resulted in a concentration-dependent decrease of K(+)-stimulated release, with an IC50 of about 30 nM (4.5 micrograms/ml); this inhibitory effect was blocked by a previous incubation of the tetanus toxin with antitoxin serum. Tetanus toxin had no effect on reserpine-induced release, a model of Ca(2+)-independent release. These results indicate that tetanus toxin is able to alter the exocytotic machinery of serotoninergic terminals, in agreement with results obtained with other neurotransmitters. They also indicate that serotoninergic terminals possess the receptor for tetanus toxin. These findings are in line with in vivo observations suggesting a role for serotoninergic system in tetanus intoxication.


Assuntos
Córtex Cerebral/metabolismo , Serotonina/metabolismo , Sinaptossomos/metabolismo , Toxina Tetânica/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Técnicas In Vitro , Masculino , Ratos , Reserpina/farmacologia , Sinaptossomos/efeitos dos fármacos
18.
Naunyn Schmiedebergs Arch Pharmacol ; 347(3): 306-12, 1993 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8479546

RESUMO

The present study investigated the anorectic activity of d-fenfluramine (d-F) and the relationship with brain levels of unchanged drug and its metabolite d-norfenfluramine (d-NF) in marmosets, relating them to neurochemical effects on the serotoninergic system. d-F and d-NF were equally active in reducing food intake (ED50 about 3 mg/kg, p.o.). However, the brain concentrations of the metabolite required to reduce food intake after synthetic d-NF were more than twice those after d-F, indicating that d-NF contributes to but does not completely explain the anorectic effect of d-F. At this dose d-F did not appreciably modify the serotonin (5-HT) and 5-hydroxyindoleacetic (5-HIAA) contents of the brain regions examined, except for a slight enhancement of 5-HIAA in hippocampus. In vitro in brain cortical synaptosomes d-F inhibited [3H]5-HT uptake more potently than d-NF, as in other species. d-F and d-NF showed similar potency in stimulating [3H]5-HT release, in a Ca++ dependent manner. The tritium released by d-F and d-NF appeared to be mainly unmetabolized [3H]5-HT. Like in other species the marmoset too has saturable and specific [3H]d-F binding sites, for which d-NF has lower affinity. d-F and d-NF have low affinities for 5-HT receptor subtypes, except that d-NF has appreciable affinity for 5-HT1C and 5-HT1D receptors. Unlike in rodents but similarly to primates in the striatum the pharmacology of 5-HT receptors seems to correspond to the 5-HT1D subtype.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Encéfalo/metabolismo , Fenfluramina/farmacologia , Fenfluramina/farmacocinética , Receptores de Serotonina/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Callithrix , Ingestão de Alimentos/efeitos dos fármacos , Técnicas In Vitro , Masculino , Norfenfluramina/farmacocinética , Norfenfluramina/farmacologia , Ensaio Radioligante , Serotonina/metabolismo
19.
J Pharm Pharmacol ; 44(3): 250-4, 1992 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1354734

RESUMO

The present study was aimed at establishing the importance of brain monoamine uptake and release mechanisms in the anorectic activity of fluoxetine, relating them to the actual brain concentrations of the parent drug and its metabolite norfluoxetine after anorectic doses in rats. Both compounds showed anorectic activity when administered intraperitoneally, norfluoxetine being slightly more active (ED50 = 22.9 mumol kg-1) than fluoxetine (ED50 = 35.0 mumol kg-1) despite the fact that the metabolite is about ten times less potent than the parent drug in inhibiting 5-hydroxytryptamine (5-HT) uptake. Comparing the brain concentrations of norfluoxetine, in terms of maximum concentrations (Cmax) and area under the curve (AUC), after the ED50 of fluoxetine or synthetic norfluoxetine, it also appeared that the metabolite plays a major role in the anorectic effect of the parent drug in rats. Brain Cmax of fluoxetine (48.7 microM) and norfluoxetine (21.7 and 27.3 microM after metabolite and drug, respectively) were several times those blocking 5-HT uptake in-vitro (0.5 microM), making it unlikely that fluoxetine (directly or through its metabolite) reduces food intake by specifically blocking 5-HT neuronal uptake. Brain Cmax of fluoxetine but particularly norfluoxetine were more compatible with those capable in-vitro of affecting catecholaminergic mechanisms, such as inhibition of dopamine and noradrenaline uptake and enhancement of dopamine release. These results together with recent in-vitro findings that the parent compound and its active metabolite induce tritium release from hippocampal synaptosomes previously loaded with [3H]5-HT suggest that mechanisms other than inhibition of 5-HT uptake are involved in the anorectic action of these compounds in rats.


Assuntos
Depressores do Apetite/farmacologia , Monoaminas Biogênicas/fisiologia , Química Encefálica/efeitos dos fármacos , Fluoxetina/análogos & derivados , Fluoxetina/farmacologia , Animais , Fluoxetina/farmacocinética , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
20.
Naunyn Schmiedebergs Arch Pharmacol ; 345(1): 1-6, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1538789

RESUMO

Rat hippocampal synaptosomes preloaded with [3H]serotonin and maintained in a superfusion apparatus were exposed for 3 min to d-fenfluramine or fluoxetine. Both drugs evoked a tritium overflow which was reserpine-sensitive requiring the presence of intact synaptic vesicles. However the two drugs displayed different characteristics: 1) the overflow was immediate with d-fenfluramine whereas the releasing activity of fluoxetine showed a delay of about 2 min; 2) d-fenfluramine-induced overflow was already apparent at 0.15 mumol/l whereas the minimal effective concentration of fluoxetine was 2.5 mumol/l. Their concentration-effect curves were differently shaped, the effect of d-fenfluramine being saturable at 5-20 mumol/l (EC50 about 1 mumol/l) while no saturation was observed with fluoxetine up to 10 mumol/l; 3) only 19% of the tritium overflow evoked by fluoxetine (2.5-10 mumol/l) consisted of true [3H]serotonin, compared with 70% when 0.5 mumol/l d-fenfluramine was used; 4) the releasing action of 0.5 mumol/l d-fenfluramine was completely Ca(++)-dependent, while at higher d-fenfluramine concentrations the Ca(++)-independent overflow became more important. The fluoxetine induced overflow was mainly (70%) Ca(++)-independent; 5) the releasing activity of d-fenfluramine was mainly (80%) blocked by the serotonin uptake blockers indalpine, midalcipram and also fluoxetine whereas fluoxetine-induced overflow was insensitive to inhibition of the serotonin carrier. In conclusion, the releasing activity of d-fenfluramine is already present at a very low concentration (0.5 mumol/l) and at this concentration its mechanism of action was Ca(++)-dependent, together with the requirement of a functional serotonin carrier.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Fenfluramina/farmacologia , Fluoxetina/farmacologia , Hipocampo/efeitos dos fármacos , Serotonina/metabolismo , Sinaptossomos/efeitos dos fármacos , Animais , Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Hipocampo/metabolismo , Masculino , Ratos , Reserpina/farmacologia , Antagonistas da Serotonina/farmacologia , Sinaptossomos/metabolismo
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