RESUMO
Mast cells (MCs) are important effectors in allergic reactions since they produce a number of pre-formed and de novo synthesized pro-inflammatory compounds in response to the high affinity IgE receptor (FcεRI) crosslinking. IgE/Antigen-dependent degranulation and cytokine synthesis in MCs have been recognized as relevant pharmacological targets for the control of deleterious inflammatory reactions. Despite the relevance of allergic diseases worldwide, efficient pharmacological control of mast cell degranulation has been elusive. In this work, the xanthone jacareubin was isolated from the heartwood of the tropical tree Callophyllum brasilense, and its tridimensional structure was determined for the first time by X-ray diffraction. Also, its effects on the main activation parameters of bone marrow-derived mast cells (BMMCs) were evaluated. Jacareubin inhibited IgE/Ag-induced degranulation in a dose-response manner with an IC50â¯=â¯46â¯nM. It also blocked extracellular calcium influx triggered by IgE/Ag complexes and by the SERCA ATPase inhibitor thapsigargin (Thap). Inhibition of calcium entry correlated with a blockage on the reactive oxygen species (ROS) accumulation. Antioxidant capacity of jacareubin was higher than the showed by α-tocopherol and caffeic acid, but similar to trolox. Jacareubin shown inhibitory actions on xanthine oxidase, but not on NADPH oxidase (NOX) activities. In vivo, jacareubin inhibited passive anaphylactic reactions and TPA-induced edema in mice. Our data demonstrate that jacareubin is a potent natural compound able to inhibit anaphylactic degranualtion in mast cells by blunting FcεRI-induced calcium flux needed for secretion of granule content, and suggest that xanthones could be efficient anti-oxidant, antiallergic, and antiinflammatory molecules.
Assuntos
Anafilaxia/metabolismo , Cálcio/metabolismo , Mastócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgE/antagonistas & inibidores , Xantonas/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Difração de Raios X , Xantonas/isolamento & purificaçãoRESUMO
The cholinergic anti-inflammatory pathway is recognized as one of the main mechanisms of neuromodulation of the immune system. Activation of the α7 nicotinic acetylcholine receptor (nAchRα7) suppresses cytokine synthesis in distinct immune cells but the molecular mechanisms behind this effect remain to be fully described. Mast cells (MCs) are essential players of allergic reactions and innate immunity responses related to chronic inflammation. Activation of TLR4 receptor in MCs leads to the rapid secretion of pre-synthesized TNF from intracellular pools and to the activation of NFκB, necessary for de novo synthesis of TNF and other cytokines. Here we report that the nAchRα7 receptor specific agonist GTS-21 inhibits TLR4-induced secretion of preformed TNF from MCs in vivo and in vitro. Utilizing bone marrow-derived mast cells (BMMCs) it was found that GTS-21 also diminished secretion of de novo synthesized TNF, TNF mRNA accumulation and IKK-dependent p65-NFκB phosphorylation in response to LPS. nAchRα7 triggering prevented TLR4-induced ERK1/2 phosphorylation, which resulted an essential step for TNF secretion due to the phosphorylation of the metallopeptidase responsible for TNF maturation (TACE). Main inhibitory actions of GTS-21 were prevented by AG490, an inhibitor of JAK-2 kinase. Our results show for the first time, that besides the prevention of NFκB-dependent transcription, inhibitory actions of nAchRα7 triggering include the blockade of pathways leading to exocytosis of granule-stored cytokines in MCs.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteína ADAM17/imunologia , Proteína ADAM17/metabolismo , Animais , Ativação Enzimática/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/imunologia , Receptor Nicotínico de Acetilcolina alfa7/imunologiaRESUMO
OBJECTIVE AND DESIGN: To characterize the effects of swim stress on the early mast cell (MC)-dependent peritoneal production of TNF in response to lipopolysaccharide (LPS) administration in mice, identifying the neuroendocrine mediators involved. SUBJECTS: Ten to twelve-week-old Swiss Webster, C57BL/6 J or c-Kit (Wsh/Wsh) mice were used. TREATMENT: Animals were intraperitoneally challenged with LPS at different times after forced swimming (FS) and peak TNF production was determined in peritoneal washes at optimal time after LPS administration. Selective blockage of main neuroendocrine pathways was performed before swim stress. METHODS: TNF concentrations were determined by ELISA. RESULTS: FS provoked an immediate and transient inhibition of LPS-elicited, MC-dependent TNF accumulation in peritoneum, which lasted around 30 min. Suppresive effects of FS were absent on MC-deficient c-Kit (Wsh/Wsh) mice but were recovered after reconstitution with MC. Adrenalectomy or DSP4 administration increased basal ip TNF levels and enhanced LPS-induced TNF release without any effect on stress-induced inhibitory effects, mifepristone did not produce any change on stress-induced inhibition, whereas mecamylamine administration increased basals and attenuated stress effects. CONCLUSIONS: Swim stress transiently inhibits the canonical MC-dependent response of TNF production in response to LPS in murine peritoneal cavity with the main participation of the cholinergic anti-inflammatory reflex.
Assuntos
Mastócitos/imunologia , Peritônio/imunologia , Estresse Fisiológico/imunologia , Fator de Necrose Tumoral alfa/imunologia , Glândulas Suprarrenais/imunologia , Animais , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-kit/genética , NataçãoRESUMO
Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues.
Assuntos
Amnésia/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Memória/efeitos dos fármacos , Memória/fisiologia , Receptores de Serotonina/metabolismo , Amnésia/induzido quimicamente , Animais , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Maleato de Dizocilpina/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Piperazinas/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Psicotrópicos/farmacologia , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Escopolamina/farmacologia , Antagonistas da Serotonina/farmacologia , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
Activation of the high affinity IgE receptor (Fc epsilonRI) through IgE-antigen complexes induces mast cell degranulation, synthesis of lipid mediators and cytokine production. These effects are involved in Type I hypersensitivity reactions and controlling them has been the main objective of many anti-allergic therapies. Here we report that pretreatment of murine bone marrow derived mast cells (BMMC) with super-oxidized solution (SOS) inhibits Fc epsilonRI dependent-beta hexosaminidase and cytokine release. This effect is exerted without altering total protein tyrosine phosphorylation, MAPK activation, cytokine mRNA accumulation or calcium mobilization after Fc epsilonRI triggering. Our data suggest that this neutral pH-SOS acts like a mast cell-membrane stabilizer inhibiting the cell machinery for granule secretion without altering the signal transduction pathways induced by IgE-antigen receptor crosslinking.
Assuntos
Degranulação Celular/efeitos dos fármacos , Citocinas/metabolismo , Peróxido de Hidrogênio/toxicidade , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Animais , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Degranulação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Desinfetantes/toxicidade , Ensaio de Imunoadsorção Enzimática/métodos , Mastócitos/metabolismo , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de IgE/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hipoclorito de Sódio/toxicidade , Tirosina/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
The ectopic spleen characterizes for absence of its suspensory ligaments and a long pedicle that are predisposed to complicate it for a torsion with commitment of the venous drainage at first and arterial at a later time himself, producing increase of its volume and infarct. This anomalous situation, it can be had to a congenital malformation of the development of its suspensory elements and fall toward the inferior abdomen or else to an inferior growth of the mesodermic yolk of that this organ originates itself. The suitable treatment is the laparoscopic splenopexy, but when it exists infarction, it is no possible avoid the splenectomy.
Assuntos
Baço/anormalidades , Esplenopatias/cirurgia , Criança , Feminino , Humanos , Laparoscopia , Radiografia , Baço/embriologia , Esplenectomia , Esplenopatias/diagnóstico por imagem , Infarto do Baço/cirurgia , Anormalidade Torcional/diagnóstico por imagem , Anormalidade Torcional/cirurgiaRESUMO
El bazo ectópico se caracteriza por ausencia de sus ligamentos suspensorios y un pedículo largo, que le predisponen a complicarse por una torsión, con compromiso del drenaje venoso en un principio y posteriormente arterial, produciendo aumento de su volumen e infarto. Esta situación anómala puede ser debida a una malformación congénita del desarrollo de sus elementos suspensorios y caída hacia el abdomen inferior, o bien a un crecimiento inferior de la yema mesodérmica de la que se origina este órgano. El tratamiento idóneo es la esplenopexia vía laparoscópica, pero cuando existe infarto no podemos evitar la esplenectomía (AU)
The ectopic spleen characterizes for absence of its suspensory ligaments and a long pedicle that are predisposed to complicate it for a torsion with commitment of the venous drainage at first and arterial at a later time himself, producing increase of its volume and infarct. This anomalous situation, it can be had to a congenital malformation of the development of its suspensory elements and fall toward the inferior abdomen or else to an inferior growth of the mesodermic yolk of that this organ originates itself. The suitable treatment is the laparoscopic splenopexy, but when it exists infarction, it is no possible avoid the splenectomy (AU)
Assuntos
Feminino , Criança , Humanos , Baço/anormalidades , Baço/fisiologia , Laparoscopia/métodos , Anormalidade Torcional/embriologia , Anormalidade Torcional/fisiopatologia , Omento/fisiologia , Omento/cirurgia , Baço/lesões , Omento/lesões , Ultrassonografia/métodosRESUMO
In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >>epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.
Assuntos
Receptores Adrenérgicos alfa 1/genética , Agonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Células COS , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Epinefrina/farmacologia , Expressão Gênica , Cobaias , Metoxamina/farmacologia , Dados de Sequência Molecular , Norepinefrina/farmacologia , Oxati-Inas/farmacologia , Oximetazolina/farmacologia , Fentolamina/farmacologia , Piperazinas/farmacologia , Prazosina/metabolismo , Prazosina/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sulfonamidas/metabolismo , Tansulosina , TrítioRESUMO
The hematopoietic cell-specific protein Vav1 is a substrate of tyrosine kinases activated following engagement of many receptors, including FcepsilonRI. Vav1-deficient mice contain normal numbers of mast cells but respond more weakly than their normal counterparts to a passive systemic anaphylaxis challenge. Vav1-deficient bone marrow-derived mast cells also exhibited reduced degranulation and cytokine production, although tyrosine phosphorylation of FcepsilonRI, Syk, and LAT (linker for activation of T cells) was normal. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 and calcium mobilization were markedly inhibited. Reconstitution of deficient mast cells with Vav1 restored normal tyrosine phosphorylation of PLCgamma1 and PLCgamma2 and calcium responses. Thus, Vav1 is essential to FcepsilonRI-mediated activation of PLCgamma and calcium mobilization in mast cells. In addition to its known role as an activator of Rac1 GTPases, these findings demonstrate a novel function for Vav1 as a regulator of PLCgamma-activated calcium signals.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Cálcio/metabolismo , Proteínas de Ciclo Celular , Isoenzimas/metabolismo , Mastócitos/metabolismo , Proteínas de Membrana , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Fosfolipases Tipo C/metabolismo , Animais , Proteínas de Transporte/metabolismo , Citocinas/genética , Ativação Enzimática , Expressão Gênica , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-vav , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de IgE/metabolismoRESUMO
BACKGROUND: To understand how the high-affinity IgE receptor (FcepsilonRI) communicates with downstream effectors, we focused on exploring the functional importance of the FcepsilonRI-mediated formation and localization of a signaling complex that contains the hematopoietic cell-specific scaffolding protein linker for activation of T cells (LAT) and the guanine nucleotide exchange factor Vav1. METHODS: Using the mast cell line RBL-2H3, we explored the localization of these proteins by confocal microscopy and cell fractionation. Additionally, the mechanism of function and the importance of LAT and Vav1 to mast cells was studied in genetically disrupted mice and in mast cells derived from their bone marrow. RESULTS: We found that LAT, Vav1 and the adapter molecule SLP-76 associated in detergent-resistant microdomains (lipid rafts) found in the plasma membrane upon FcepsilonRI stimulation. In the absence of LAT, mast cells showed a remarkable loss of the secretory response and reduced cytokine responses. Vav1 deficiency also affected secretion, although not to the extent of LAT deficiency, and inhibited IL-2 and IFN-gamma production. LAT- and Vav1-deficient mice showed reduced blood histamine levels after a systemic anaphylaxis challenge as compared to their normal counterparts. CONCLUSIONS: The results demonstrate that LAT is a central mediator in IgE receptor signaling by regulating multiple signaling pathways that affect mast cell degranulation and cytokine production. Vav1, a component of this LAT-containing signaling complex, regulates a specific subset of these responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular , Mastócitos/imunologia , Fosfoproteínas/fisiologia , Receptores de IgE/metabolismo , Transdução de Sinais , Animais , Citocinas/biossíntese , Citocinas/genética , Exocitose , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Camundongos , Modelos Imunológicos , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-vav , RatosRESUMO
Objetivos: Estudiar la mineralización ósea en la población infantil, con el fin de valorar si las características climatológicas y nutricionales canarias influyen en la población infantil diferenciándola de la de otras regiones. Material y métodos: Se han estudiado un total de 30 niños con edades comprendidas entre los 4 y 14 años. En todos ellos se determinó peso, talla, calcio, fósforo, fosfatasas alcalinas, edad ósea y contenido mineral óseo (CMO). Resultados: No encontramos diferencias estadísticamente significativas al comparar las cifras obtenidas para los niños y las niñas en cada una de las variables. Establecimos una correlación estadísticamente significativa entre la edad, peso, talla y edad ósea. Los valores de los parámetros de mineralización ósea, concretamente la osteocalcina, las fosfatasas alcalinas y el contenido mineral óseo, obtenidos en nuestra población infantil canaria sana fueron superiores a los aportados por otros autores. Conclusiones: En la población infantil canaria, los valores obtenidos para la mineralización ósea (fosfatasas alcalinas, osteocalcina y contenido mineral óseo) fueron superiores a las cifras establecidas en otras regiones (AU)
Assuntos
Adolescente , Feminino , Pré-Escolar , Masculino , Criança , Humanos , Calcificação Fisiológica/fisiologia , Densidade Óssea/fisiologia , Estado Nutricional , Canais de Cálcio/sangue , Fosfatos/sangue , Fosfatase Alcalina/sangue , Osteocalcina/sangue , Comportamento Alimentar , Peso-Idade , Peso-EstaturaRESUMO
Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.
Assuntos
Norepinefrina/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Fosforilação , Marcadores de Fotoafinidade , Testes de Precipitina , Proteína Quinase C/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
It is now well documented that changes in gene expression take place during cell isolation and culture. Here, we report the change in the expression of the mRNAs for alpha(1)-adrenoceptor subtypes, during dissociation of guinea pig liver cells with collagenase. Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, it was observed that during the isolation procedure, the mRNA for the alpha(1A)-adrenoceptor, normally expressed in whole liver, was degraded and the mRNA for alpha(1D) subtype, barely expressed in whole liver, increased in an actinomycin D-sensitive manner. When the isolation procedure was performed in the presence of cycloheximide, the mRNA for the alpha(1A)-adrenoceptor did not diminish and the induction of the alpha(1D)-adrenoceptor mRNA was even more evident. Our data indicate that cell isolation alters alpha(1)-adrenoceptor mRNA expression.
Assuntos
Fígado/metabolismo , RNA Mensageiro/metabolismo , Receptores Adrenérgicos alfa 1/genética , Animais , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica , Cobaias , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Inibidores da Síntese de Proteínas/farmacologia , RNA/efeitos dos fármacos , RNA/genética , RNA/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In C9 (Clone 9) liver cells, angiotensin 11 increased the intracellular Ca2+ content, inositol phosphate production and c-fos mRNA expression. Other angiotensins were also active with the order of potency being angiotensin II = angiotensin III >> angiotensin I > angiotensin IV. Losartan, but not PD 123177 (1-(4-amino-3-methyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imida zo [4,5c]pyridine-6-carboxylic acid), blocked the effects of angiotensin II. Pertussis toxin did not alter these actions of angiotensin II. These data indicate that the effects were mediated through angiotensin AT1 receptors involving pertussis toxin-insensitive G-proteins. Phorbol myristate acetate was also able to increase c-fos mRNA expression. The action of angiotensin II was consistently greater than that of the active phorbol ester. Staurosporine but not genistein inhibited this effect of angiotensin II. Angiotensin II- and phorbol myristate acetate-induced proto-oncogene mRNA expression was attenuated in cells incubated overnight with the active phorbol ester, which suggests a major role of protein kinase C.
Assuntos
Angiotensina II/farmacologia , Cálcio/metabolismo , Fígado/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de Angiotensina/fisiologia , Angiotensina II/efeitos dos fármacos , Animais , Antiarrítmicos/farmacologia , Linhagem Celular , Células Cultivadas , Células Clonais , Interações Medicamentosas , Expressão Gênica/efeitos dos fármacos , Fosfatos de Inositol/biossíntese , Fígado/citologia , Losartan/farmacologia , Toxina Pertussis , RNA Mensageiro/genética , Ratos , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
In guinea pig hepatocytes angiotensin II induced phosphorylase a activation. This effect was mimicked by other angiotensins with the potency order: angiotensin II (EC50 approximately 1 nM) > angiotensin III (EC50 approximately nM) > angiotensin I (EC50 approximately 300 nM). The effect of 10 nM angiotensin II was blocked by the angiotensin II receptor AT1-selective antagonists irbesartan and losartan (Ki values of approximately 1 nM and approximately 10 nM for irbesartan and losartan respectively) but not by the AT2-selective antagonist PD123177. Similar data were obtained when the production of [3H]IP3 from [3H]myo-inositol-labeled cells was studied Angiotensin II induced a dose-dependent increase in [3H]IP3 production; the maximal effect (approximately 3-fold) was observed at a concentration of 10 microM. This effect of angiotensin II was completely blocked by the AT1-selective antagonists irbesartan and losartan, but only in a very limited fashion by PD123177. [125I][Sar1-Ile8]angiotensin II bound with high affinity (approximately 3.8 nM) to a moderately abundant number of sites (approximately 660 fmol/mg protein) in guinea pig liver membranes. Binding competition experiments indicate the following orders of potency for agonists: angiotensin II (approximately 1.5 nM) > angiotensin III (approximately 7 nM) > angiotensin I (approximately 176 nM), and for antagonists: irbesartan (approximately 0.5 nM) > losartan (approximately 36 nM) > > PD123177 (> > 10000 nM). The functional and binding data strongly indicate that the effects of angiotensin II were mediated through AT1 receptors. Expression of the mRNA for these receptors was confirmed by RT-PCR and hybridization of the reaction product with a radiolabeled rat AT1 receptor cDNA probe.
Assuntos
Angiotensina II/metabolismo , Anti-Hipertensivos/farmacologia , Compostos de Bifenilo/farmacologia , Imidazóis/farmacologia , Fígado/metabolismo , Receptores de Angiotensina/metabolismo , Tetrazóis/farmacologia , Angiotensina I/metabolismo , Angiotensina III/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Ligação Competitiva , Ativação Enzimática , Cobaias , Irbesartana , Losartan , Masculino , Reação em Cadeia da Polimerase , Piridinas/farmacologia , RNA Mensageiro/análise , Receptores de Angiotensina/genéticaRESUMO
The alpha 1-adrenoceptors present in the liver of rhesus monkeys was characterized using [3H]prazosin. This radioligand binds to monkey liver membranes with high affinity (KD 0.33 nM) to a moderately abundant number of sites (97 fmol/mg of protein). These sites were characterized pharmacologically, by binding competition, observing two affinities for most ligands. The order of potency for agonists was: (a) for the high affinity sites: oximetazoline > epinephrine = norepinephrine > methoxamine; and (b) for the other sites (low affinity for the alpha 1A-adrenoceptor-selective agonists): oximetazoline > or = epinephrine = norepinephrine > > methoxamine. For antagonists the orders of potency were: (a) for the high affinity sites: R-(-)-5[2-[[2-(ethoxyphenoxy)ethyl]amino]propyl]-2-metoxybenzen esulfonamide HCl (tamsulosin) > or = 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane (WB4101) > or = prazosin > or = (+)-niguldipine > 5-methylurapidil = benoxathian > phentolamine > 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]deca ne-7,9- dione dihydrochloride (BMY 7378); (b) for the other sites (low affinity for the alpha 1A-adrenoceptor-selective antagonists): prazosin > tamsulosin > phentolamine = WB4101 > (+)-niguldipine > or = 5-methyl-urapidil = benoxathian > BMY 7378. These data strongly suggest that Macaca mulatta liver cells coexpress alpha 1A- and alpha 1B-adrenoceptors. Expression of the mRNA for these receptors was confirmed by reverse transcriptase-polymerase chain reactions.
Assuntos
Antagonistas Adrenérgicos alfa/metabolismo , Fígado/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Feminino , Expressão Gênica , Macaca mulatta , Masculino , Prazosina/metabolismo , Ensaio Radioligante , Receptores Adrenérgicos alfa 1/genéticaRESUMO
It has been shown that angiotensin II and PMA increase the expression of proto-oncogenes (c-fos, c-myc and c-mos) in liver cells. In this study the effects of angiotensin II and PMA on c-fos transcription and mRNA stability were investigated. Using nuclear run-off transcription assays, it was observed that PMA and angiotensin II induced a rapid increase in c-fos transcription. The transcription rate of the GAPDH gene did not change, indicating that the effects were not general on gene transcription. The ability of these agents to modulate proto-oncogene mRNA stability was tested by measuring c-fos mRNA half-life. It was observed that c-fos mRNA half-life was relatively short (approximately 14-18 min) and that angiotensin II and PMA markedly stabilized mRNA, increasing its half-life (approximately 4-fold and approximately 2-fold, respectively). The protein synthesis inhibitor cycloheximide increased mRNA stability to a much greater extent. Our results clearly demonstrate that angiotensin II and PMA increased c-fos mRNA accumulation in liver cells through two actions: induction of c-fos gene transcription and increase in mRNA stability.
Assuntos
Angiotensina II/farmacologia , Genes fos , Fígado/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Feminino , Regulação da Expressão Gênica , Fígado/citologia , RNA Mensageiro/genética , Ratos , Transcrição Gênica/efeitos dos fármacosRESUMO
The alpha 1-adrenoceptors present in the liver of cats were characterized using [3H]prazosin. This radioligand binds to cat liver membranes with high affinity ((KD 0.79 nM) to a moderately abundant number of sites (160 fmol/mg of protein). This sites were characterized pharmacologically, by binding competition, observing the following orders of potency: a) for agonists: oxymetazoline > epinephrine = norepinephrine >> methoxamine, and b) for antagonists: WB4101 > or = prazosin > or = (+) niguldipine > or = benoxathian > or = spiperone = 5-methyl-urapidil > phentolamine > BMY 7378. These data suggested that cat liver expresses alpha 1A-adrenoceptors. Expression of the mRNA for this receptor was confirmed by RT-PCR.
Assuntos
Fígado/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Animais , Ligação Competitiva , Gatos , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Reação em Cadeia da Polimerase , Prazosina/farmacologia , Ensaio Radioligante , Receptores Adrenérgicos alfa 1/classificaçãoRESUMO
Using dog liver membranes we observed that [125I]HEAT ((+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone) binds with high affinity (KD 97 pM) to a discrete number of sites (Bmax 40 fmol/mg protein) with the pharmacological characteristics expected for alpha 1-adrenoceptors. Such sites were inactivated by pretreatment with chloroethylclonidine. Binding competition experiments indicated the following order of potency: (a) for agonists: oxymetazoline > epinephrine > or = norepinephrine > methoxamine and (b) for antagonists: WB4101 > or = 5-methyl-urapidil = prazosin > or = benoxathian > or = (+)-niguldipine > phentolamine. Northern analysis indicated that total RNA isolated from dog liver hybridized with an alpha 1c selective probe (bovine brain). The orders of potency for agonists and antagonists, their Ki values and the Northern analysis suggest that dog liver expresses alpha 1A-adrenoceptors.
Assuntos
Fígado/química , Receptores Adrenérgicos alfa 1/classificação , Tetralonas , Animais , Ligação Competitiva , Clonidina/análogos & derivados , Clonidina/farmacologia , Cães , Fenetilaminas/metabolismo , Receptores Adrenérgicos alfa 1/metabolismoRESUMO
Angiotensin II stimulates phosphorylase a activity and c-fos expression in isolated rat hepatocytes. Both effects are mediated through AT1 receptors. However, the time-courses and the dose-dependencies of these responses were different. These effects of angiotensin II were blocked by Losartan when the antagonist was added to the cells before the hormone or when both the hormone and the antagonist were added simultaneously. Interestingly, when the antagonist was added 2 or 3 min after the peptide hormone, the action on phosphorylase a activity was markedly decreased but, in contrast, that on c-fos expression was not affected. The data suggest that angiotensin II-mediated activation of phosphorylase a was reversible and dependent on continuous receptor activation by the hormone whereas c-fos expression did not seem to have these characteristics, i.e., our data suggest that once protooncogene expression is triggered, the process becomes independent of the hormone-receptor interaction.