RESUMO
This review focuses on alpha(1)-adrenoceptor phosphorylation and function. Most of what is currently known is based on studies on the hamster alpha(1B)-adrenoceptor. It is known that agonist stimulation leads to homologous desensitization of these receptors and current evidence indicates that such decrease in receptor activity is associated with receptor phosphorylation. Such receptor phosphorylation seems to involve G protein-receptor kinases and the receptor phosphorylation sites have been located in the carboxyl tail (Ser(404), Ser(408), and Ser(410)). There is also evidence showing that in addition to desensitization, receptor phosphorylation is associated with internalization and roles of beta-arrestins have been observed. Direct activation of protein kinase C leads to receptor desensitization/internalization associated with phosphorylation; the protein-kinase-C-catalyzed receptor phosphorylation sites have been also located in the carboxyl tail (Ser(394) and Ser(400)). Activation of G(q)-coupled receptors, such as the endothelin ET(A) receptor induces alpha(1B)-adrenoceptor phosphorylation and desensitization. Such effect involves protein kinase C and a yet unidentified tyrosine kinase. Activation of G(i)-coupled receptors, such as the lysophosphatidic acid receptor, also induces alpha(1B)-adrenoceptor phosphorylation and desensitization. These effects involve protein kinase C and phosphatidyl inositol 3-kinase. Interestingly, activation of epidermal growth factor receptors also induces alpha(1B)-adrenoceptor phosphorylation and desensitization involving protein kinase C and phosphatidyl inositol 3-kinase. A pivotal role of these kinases in heterologous desensitization is evidenced.
Assuntos
Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 1/fisiologia , Animais , Humanos , Fosforilação , Receptores Adrenérgicos alfa 1/metabolismoRESUMO
BACKGROUND: Desensitization of G protein-coupled receptors is associated with receptor phosphorylation. Two groups of kinases seem to participate in such receptor phosphorylation, i.e., second messenger-activated protein kinases and G protein-coupled receptor kinases. Calcium seems to play a role in the phosphorylation of some G protein-coupled receptors. The role of calcium in alpha 1b-adrenoceptor phosphorylation has not been critically assessed. METHODS: Rat-1 fibroblasts stably expressing the hamster alpha 1b-adrenergic receptor were used. To study receptor phosphorylation cells metabolically labeled with [32P]Pi were lysed and the receptor immunoprecipitated using a polyclonal antibody generated against the receptor carboxyl terminal decapeptide. Intracellular calcium was determined by using Fura-2 fluorescence. RESULTS: Norepinephrine, endothelin-1, and lysophosphatidic acid increased intracellular calcium concentration. All these agents and phorbol myristate acetate (PMA) induce alpha 1b-adrenoceptor phosphorylation. The intracellular chelator, BAPTA, abolished the increase in intracellular calcium induced by the previously mentioned agents but did not affect the receptor phosphorylation induced by norepinephrine, PMA, or lysophosphatidic acid. Under these conditions, receptor phosphorylation induced by endothelin was slightly but consistently decreased. Thapsigargin increased intracellular calcium concentration but was unable to induce alpha 1b-adrenoceptor phosphorylation and decreased PMA-induced receptor phosphorylation. No increase in receptor phosphorylation was observed when calcium ionophores were used. CONCLUSIONS: Our data indicate that an increase in [Ca2+]i is not sufficient to induce alpha 1b-adrenoceptor phosphorylation and that buffering of [Ca2+]i does not alter the receptor phosphorylation induced by norepinephrine, lysophosphatidic acid, and PMA. A marginal role of calcium in the alpha 1b-adrenoceptor phosphorylation induced by endothelin-1 cannot be discarded.
Assuntos
Cálcio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Animais , Cricetinae , Fosforilação , RatosRESUMO
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
Alpha 1-adrenoceptors mediate some of the main actions of the natural catecholamines, adrenaline, and noradrenaline. They participate in many essential physiological processes, such as sympathetic neurotransmission, modulation of hepatic metabolism, control of vascular tone, cardiac contraction, and the regulation of smooth muscle activity in the genitourinary system. It is now clear that alpha 1-adrenoceptors mediate, in addition to immediate effects, longer term actions of catecholamines such as cell growth and proliferation. In fact, adrenoceptor genes can be considered as protooncogenes. Over the past years, considerable progress has been achieved in the molecular characterization of different alpha 1-adrenoceptor subtypes. Three main subtypes have been characterized pharmacologically and in molecular terms. Splice variants, truncated isoforms, and polymorphisms have also been detected. Similarly, it is now clear that these receptors are coupled to several classes of G proteins that, therefore, are capable of modulating different signaling pathways. In the present article, some of these aspects are reviewed, together with the distribution of the subtypes in different tissues and some of the known roles of these receptors in health and disease.
Assuntos
Receptores Adrenérgicos alfa 1/classificação , Receptores Adrenérgicos alfa 1/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Cardiomegalia/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Hiperplasia Prostática/metabolismoRESUMO
The action of bradykinin was studied in rat-1 fibroblasts stably expressing alpha1b-adrenoceptors. It was observed that bradykinin and kallidin markedly increase cytosol calcium concentration, but that the B1 agonist, des-Arg9-bradykinin, only mimicked this effect to a minimal extent. Antagonists, selective for the B2 subtype, such as Hoe 140, blocked this effect of bradykinin and kallidin. Similarly, bradykinin and kallidin stimulated the production of inositol phosphates and B2 antagonists blocked their actions. The possibility that bradykinin could modulate alpha1b-adrenoceptors was studied. It was observed that bradykinin and kallidin increased alpha1b-adrenoceptor phosphorylation and that such effect was also blocked by Hoe 140. Interestingly, the ability of norepinephrine to increase intracellular calcium concentration was not altered by pretreatment of the cells with bradykinin, i.e. bradykinin induced alpha1b-adrenoceptor phosphorylation but this did not lead to receptor desensitization.
Assuntos
Receptores Adrenérgicos alfa 1/fisiologia , Receptores da Bradicinina/fisiologia , Transdução de Sinais , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Citosol/metabolismo , Endotelinas/farmacologia , Fosfatos de Inositol/metabolismo , Calidina/farmacologia , Norepinefrina/farmacologia , Fosforilação , Ratos , Receptor B2 da Bradicinina , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores da Bradicinina/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Active migration of Entamoeba histolytica trophozoites through extracellular matrixes might play a role in host tissue destruction. Trophozoites degrade soluble fibronectin (FN) bound to their surface and adhere to substrate-bound FN, producing local degradation. FN proteolytic fragments were used to determine the nature of adhesion and motility-promoting domains within the protein. The 70-kDa fragment (amino-terminal end) promoted the highest adhesion, followed by the 120-kDa fragment, which contains the cell-binding domain. The 25-kDa fragment (carboxy-terminal end of the A chain) promoted half the adhesion, while two Hep II-binding fragments had no effect. The 70- and 120-kDa fragments also stimulated directed migration and chemokinesis. Intact FN and the 25-kDa fragment showed lower stimulation. The Hep II-binding fragments had no activity. Results support previous evidence for distinct cell-surface components as mediators of adhesion to FN and trophozoite motility and the potential importance of cell matrix recognition and degradation in their invasive behavior.
Assuntos
Adesão Celular , Quimiotaxia , Entamoeba histolytica/fisiologia , Fibronectinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Catepsinas/metabolismo , Entamoeba histolytica/metabolismo , Entamoeba histolytica/patogenicidade , Matriz Extracelular/metabolismo , Ligação ProteicaRESUMO
Antibodies directed to immunopurified coagulation protein C (PC) were investigated in serum samples from 108 patients with systemic lupus erythematosus (SLE) and found in 12 of them. However, their presence was not associated with antigenic or functional deficiencies of PC, which were documented in 6 and 17 patients, respectively.
Assuntos
Anticorpos/análise , Coagulação Sanguínea/fisiologia , Lúpus Eritematoso Sistêmico/imunologia , Deficiência de Proteína C , Proteína C/imunologia , Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Proteína C/fisiologiaRESUMO
The association of thrombosis with antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE) could be due to their interference with natural phospholipid dependent anticoagulant mechanisms. We studied antigenic protein C (APC), functional protein C (FPC), free protein S (FPS), protein S bound to C4 binding protein (C4bp-S), antithrombin III (ATIII), as well as IgG and IgM anticardiolipin antibodies (aCL) in 38 patients with SLE with a history of thromboses and 70 patients with SLE without such history. We found a high frequency of deficiencies of natural anticoagulants in both groups of patients with SLE but, because of patient selection, we could not determine the actual prevalence of these defects. Patients having had a venous thrombosis in the previous year had low C4bp-S more frequently than patients with older or no thromboses. When we divided our patients with SLE into those who had a definite, probable, questionable or no antiphospholipid syndrome (aPS) we found the frequency of C4bp-S deficiency to be significantly higher in those with definite aPS than in those without aPS. Intermediate proportions were found in patients with probable and questionable aPS. The levels of C4bp-S decreased as the levels of aCL, particularly IgG, increased. Stepwise discriminant analysis of natural anticoagulants selected deficiencies of C4bp-S and FPC with increased ATIII as a set of variables with highest predictive power for classification of patients with and without aPS. Thus, deficiencies of natural anticoagulants may occur frequently in patients with SLE.(ABSTRACT TRUNCATED AT 250 WORDS)