RESUMO
A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization
Assuntos
Animais , Cricetinae , Ratos , Angiotensina II/fisiologia , Células CHO , Endocitose , Receptores de Angiotensina/fisiologia , Angiotensina II/antagonistas & inibidores , Northern Blotting , Membrana Celular , Endocitose/fisiologia , Ligantes , Microscopia Confocal , Transdução de Sinais , TransfecçãoRESUMO
A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.
Assuntos
Angiotensina II/fisiologia , Células CHO/metabolismo , Endocitose , Membrana Nuclear/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/antagonistas & inibidores , Antagonistas de Receptores de Angiotensina , Animais , Northern Blotting , Membrana Celular , Cricetinae , Endocitose/fisiologia , Ligantes , Microscopia Confocal , Ratos , Receptores de Angiotensina/fisiologia , Transdução de Sinais , TransfecçãoRESUMO
The role of the external third of helix VI of the angiotensin II (AII) AT1 receptor for the interaction with its ligand and for the subsequent signal transduction was investigated by individually replacing residues 252-256 by Ala, and residues 259 or 261 by Tyr, and permanently transfecting the resulting mutants to Chinese hamster ovary (CHO) cells. Binding experiments showed no great changes in affinity of any of the mutants for AII, [Sar1]-AII, or [Sar1, Leu8]-AII, but the affinity for the nonpeptide antagonist DuP753 was significantly decreased. The inositol phosphate response to AII was remarkably decreased in mutants V254A, H256A, and F259Y. These results indicate that AT1 residues Val254, His256, and Phe259 are not involved in ligand binding but participate in signal transduction. Based in these results and in others from the literature, it is suggested that, in addition to the His256 imidazole ring, the Phe259 aromatic ring interacts with the AII's Phe8, thus contributing to the signal-triggering mechanism.
Assuntos
Angiotensina II/metabolismo , Receptores de Angiotensina/fisiologia , Transdução de Sinais/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Ligação ao GTP/fisiologia , Histidina/química , Humanos , Fosfatos de Inositol/fisiologia , Ligantes , Mutagênese Sítio-Dirigida , Fenilalanina/química , Ligação Proteica , Ratos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Relação Estrutura-Atividade , Transfecção , Valina/químicaRESUMO
Angiotensin II (AII) tachyphylaxis occurs in the guinea pig ileum, but is not induced by analogs lacking the N-terminal amino group or the Arg2 guanidino group. Both AII and Lys2AII increased cell inositol trisphoshate content in cultured intestinal smooth muscle cells. Protein kinase C inhibition by staurosporine or downregulation by prolonged incubation with phorbol reverted tachyphylaxis of the inositol trisphoshate response, but not that of the Na+ uptake response, indicating that the uncoupling of the phosphoinositide signal system by protein kinase C did not involve all processes distal to receptor activation. Tachyphylaxis of the Na+ uptake response was prevented when receptor internalization was blocked by reduction of the temperature (4 degrees C) or by pretreatment of the cells with phenylarsine oxide. Acid washings, which prevented tachyphylaxis of the 24Na+ influx response, also prevented tachyphylaxis of the contractile response of the guinea pig ileum to AII. Although these findings suggest that sequestration or internalization of the AII receptor might be involved in AII tachyphylaxis, binding of [125I]AII and of [125I]Lys2AII to the cells was equally unaffected by repeated administrations of the peptides. The results suggest that conformational change of the AII-receptor complex within the plasma membrane, but not internalization, is the most important factor responsible for tachyphylaxis.
Assuntos
Angiotensina II/farmacologia , Íleo/efeitos dos fármacos , Taquifilaxia , Alcaloides/farmacologia , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Animais , Arsenicais/farmacologia , Cálcio/metabolismo , Células Cultivadas , Temperatura Baixa , Feminino , Cobaias , Íleo/citologia , Íleo/metabolismo , Íleo/fisiologia , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Sódio/metabolismo , EstaurosporinaRESUMO
Simultaneous recordings of the tension and intracellular Ca2+ concentration of guinea-pig ileum longitudinal smooth muscle strips, as well as 24Na+ and 45Ca2+ influx measurements in cultured myocytes from the same tissue, were used to investigate the mechanisms underlying angiotensin-induced desensitization and tachyphylaxis. Angiotensin II and [2-lysine]-angiotensin II (Lys2All), incubated for prolonged periods (10 min) with muscle strips, induced fading of the contractile response (desensitization) and reappearance of the intracellular Ca2+ concentration oscillations, which were inhibited during the initial increase in cytosolic Ca2+. The desensitization was paralleled, in cultured myocytes, by inhibition of the 45Ca2+ but not of the 24Na+ influxes which were initially stimulated by the peptides. On the other hand, repeated administrations of angiotensin II (but not of Lys2All) caused gradual reduction of the contractile response and of the 24Na+ influx stimulation evoked by the agonist (tachyphylaxis). Treatment with phorbol 12-13 dibutyrate accelerated the desensitization induced by both angiotensin II and by Lys2All and aggravated the tachyphylaxis to angiotensin II. The results support the hypothesis that activation of protein kinase C is responsible for the desensitization and that tachyphylaxis is due to the slow dissociation of angiotensin II from a postulated Na(+)-dependent regulatory site on the receptor.
Assuntos
Angiotensina II/farmacologia , Íleo/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Proteína Quinase C/fisiologia , Sódio/fisiologia , Taquifilaxia/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Cobaias , Masculino , Contração Muscular/fisiologia , Dibutirato de 12,13-Forbol/farmacologia , Sódio/metabolismoRESUMO
The effects of angiotensin II (ANG) on Na+ and Ca++ fluxes in cultured intestinal smooth muscle cells from the guinea pig ileum were studied and correlated with the contraction and desensitization observed in whole muscles. The effects of ANG were compared with those of acetylcholine (ACh), an agonist that acts at muscarinic receptors in the intestinal smooth muscle and which does not induce desensitization. Both ANG and ACh stimulated 24Na+ influx upon addition to the cells, and this stimulation persisted for at least 30 min. Both agonists also stimulated 45Ca++ uptake but ANG's effect was transient, whereas that of ACh was persistent. Short-term (30 min) treatment with PMA (phorbol-12-myristate-13-acetate) caused a fade of the tonic response of the whole muscle to ANG, and also blocked this hormone's stimulating effect on 45Ca++, but not on 24Na+ influx. Long-term (7 hr) treatment with PMA, which suppresses protein kinase C activity, restored ANG's ability to stimulate 45Ca++ influx. The stimulating effects of ACh on 24Na+ and 45Ca++ influxes were not affected by short- or long-term treatment of the cells with PMA. Our results suggest that ANG desensitization involves protein kinase C inhibition of a step in the stimulus-response chain that is subsequent to phospholipase C-activation.