RESUMO
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
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
Human alpha(1b)-adrenoceptors stably expressed (B(max) approximately 800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca(2+) and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the alpha(1b)-adrenergic-mediated actions but did not block the ability of the cells to respond to lysophosphatidic acid. Inhibition or downregulation of protein kinase C also blocked the action of the tumor promoter on the adrenergic effects. Photolabeling experiments indicated that the receptor has an apparent molecular weight of approximately 80 kDa. The receptors were phosphorylated in the basal state and such phosphorylation was increased when the cells were incubated with phorbol myristate acetate or noradrenaline. Incubation of the cells with phorbol myristate acetate or noradrenaline blocked noradrenaline-promoted [35S]GTP-gamma-S binding to membranes, suggesting receptor-G protein uncoupling. The results indicate that activation of protein kinase C blocked/desensitized human alpha(1b)-adrenoceptors and that such effect was associated to receptor phosphorylation.
Assuntos
Proteína Quinase C/fisiologia , Receptores Adrenérgicos alfa 1/metabolismo , Animais , Ligação Competitiva , Cálcio/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Indóis/farmacologia , Fosfatos de Inositol/metabolismo , Membranas/efeitos dos fármacos , Membranas/metabolismo , Norepinefrina/farmacologia , Fentolamina/metabolismo , Fosforilação/efeitos dos fármacos , Piperazinas/metabolismo , Prazosina/metabolismo , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaio Radioligante , Receptores Adrenérgicos alfa 1/genética , Radioisótopos de Enxofre , Acetato de Tetradecanoilforbol/farmacologia , TrítioRESUMO
The subtype selectivity of the alpha 1-adrenoceptor antagonist, tamsulosin, was tested using hepatocytes and liver membranes from guinea pigs and rabbits (expressing alpha 1-adrenoceptors with alpha 1A pharmacology) and rats (alpha 1B-adrenoceptors). Tamsulosin blocked the alpha 1-adrenergic activation of phosphorylase with higher affinity in hepatocytes from guinea pigs and rabbits than in those from rats. [3H]Tamsulosin binding to liver membranes was rapid, reversible and saturable. The Kd values obtained also indicated higher affinity for alpha 1A-adrenoceptors (70 and 140 pM, for liver membranes obtained from guinea pigs and rabbits, respectively) than for those of the alpha 1B-subtype (510 pM). Chloroethylclonidine potently and completely inactivated [3H]tamsulosin binding sites in membranes from rabbit and rat livers, but not those in guinea pig liver membranes. Binding competition and inactivation experiments were performed to further characterize the receptor subtypes present in the livers of these animals. In summary, tamsulosin is a very potent alpha 1-adrenoceptor antagonist that has higher affinity for alpha 1A-adrenoceptors than for those of the alpha 1B-subtype.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/farmacologia , Sulfonamidas/farmacologia , Animais , Cobaias , Técnicas In Vitro , Fígado/metabolismo , Coelhos , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/classificação , Especificidade da Espécie , TansulosinaRESUMO
In guinea pig hepatocytes, histamine increased phosphorylase activity and inositol phosphate production. Similar effects were obtained with 2-(2-aminoethyl)-thiazole, a histamine H1 receptor agonist, but not with dimaprit or impromidine, H2 receptor agonists. These effects of histamine were dose-dependently inhibited by the H1 antihistamines, (+)-chlorpheniramine and mepyramine (pyrilamine) but not by cimetidine or ranitidine, H2 antagonists. (+)-Chlorpheniramine and mepyramine had similar potencies (apparent Ki values approximately 3 nM) when incubated with the cells for 1 min (phosphorylase a assays) but the former was 15-20-fold more potent than the latter at longer incubation times (apparent Ki values approximately 3-4 nM and 45-90 nM, respectively) indicating that mepyramine is actively metabolized by guinea pig hepatocytes. Histamine increased cytosol calcium approximately 2-fold, an effect also mediated through H1 receptors. The actions of histamine were not affected by in vivo ADP-ribosylation by pertussis toxin. Our data clearly indicate that histamine modulates the metabolism of guinea pig hepatocytes via activation of H1 receptors. These receptors are coupled to the phosphoinositide turnover-calcium mobilization signalling pathway through a pertussis toxin-insensitive process.