RESUMO
We previously demonstrated that Ang II inhibits the renal plasma membrane Ca(2+)-ATPase. In the present work we have studied the effect of Ang II, at concentrations similar to those found in the renal interstitium, on the Ca(2+)-ATPase from proximal tubule cells. High Ang II concentration (5 x 10(-7) mol/L) led to the recovery of Ca(2+)-ATPase activity previously inhibited by 50% at low Ang II concentration (10(-10) mol/L). Reactivation occurred in parallel with: (i) formation of only two dead-end metabolites [Ang-(3-4) and Tyr] after incubation of isolated membranes with micromolar Ang II; and (ii) dissociation of constitutive AT(1)R/AT(2)R heterodimers, which are preserved with 10(-10) mol/L Ang II. When the membranes were incubated with 10(-14) mol/L Ang-(3-4), inhibition by 10(-10) mol/L Ang II was no longer observed. The counteracting effect of Ang-(3-4) was abolished by PD123319, an antagonist of AT(2)R, and mimicked by CGP42112A, an agonist of AT(2)R. Ang-(1-7) is an intermediate in the formation of Ang-(3-4) via a pathway involving angiotensin-converting enzyme (ACE), and complete dipeptide breakdown to Tyr and Val is impaired by low Ang II. We conclude that Ang-(3-4) may be a physiological regulator of active Ca(2+) fluxes in renal proximal cells by acting within the renin-angiotensin axis.
Assuntos
Angiotensina II/metabolismo , Angiotensina II/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Rim/enzimologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Antagonistas de Receptores de Angiotensina , Animais , Cromatografia Líquida de Alta Pressão , Ativação Enzimática/efeitos dos fármacos , Imidazóis/farmacologia , Técnicas In Vitro , Oligopeptídeos/farmacologia , Piridinas/farmacologia , Receptores de Angiotensina/agonistas , Receptores de Angiotensina/metabolismo , Ovinos , Espectrometria de Massas por Ionização por Electrospray , Vasoconstritores/farmacologiaRESUMO
BACKGROUND: The circumventricular structures of the central nervous system and nitric oxide are involved in arterial blood pressure control, and general anesthesia may stimulate the central renin-angiotensin system. We therefore investigated the central role of angiotensin II and nitric oxide on the regulation of systemic arterial blood pressure in conscious and anesthetized rats. METHODS: Rats with stainless steel cannulae implanted into their lateral ventricle were studied. We injected the AT1 and AT2 angiotensin II receptor antagonists, losartan and PD123319, L-NAME, 7-nitroindazole (nitric oxide synthetase inhibitors), and FK409 (nitric oxide donor agent) into the lateral ventricles. Mean arterial blood pressure (MAP) was recorded in conscious and zoletil-anesthetized rats. RESULTS: Mean +/- sem baseline MAP was 117.5 +/- 2 mm Hg. Angiotensin II injected into the brain lateral ventricle increased MAP from 136.5 +/- 2 mm Hg to 138.5 +/- 4 mm Hg (Delta16 +/- 3 mm Hg to Delta21 +/- 3 mm Hg) for all experimental groups versus control from 116 +/- 2 mm Hg to 120 +/- 3 mm Hg (Delta3 +/- 1 mm Hg to Delta5 +/- 2 mm Hg) (P < 0.05). L-NAME or 7-nitroindazole enhanced the angiotensin II pressor effect (P < 0.05). Prior injection of losartan and PD123319 decreased the angiotensin II pressor effect and the enhancement effect of L-NAME and 7-nitroindazole (P < 0.05). Zoletil anesthesia did not interfere with the effects of angiotensin II, AT1, AT2 antagonists, or nitric oxide synthetase inhibitors. CONCLUSIONS: Endogenous nitric oxide functions tonically as a central inhibitory modulator of the angiotensinergic system. AT1 and AT2 receptors influence the angiotensin II central control of arterial blood pressure. Zoletil anesthesia did not interfere with these effects.
Assuntos
Angiotensina II/farmacologia , Estado de Consciência/efeitos dos fármacos , Óxido Nítrico/fisiologia , Receptores de Angiotensina/fisiologia , Tiletamina/farmacologia , Vasoconstritores/farmacologia , Zolazepam/farmacologia , Anestesia/métodos , Animais , Estado de Consciência/fisiologia , Combinação de Medicamentos , Ratos , Ratos Sprague-Dawley , Receptores de Angiotensina/agonistasRESUMO
It has been described recently that the nonpeptide AVE 0991 (AVE) mimics the effects of angiotensin-(1-7) [Ang-(1-7)] in bovine endothelial cells. In this study, we tested the possibility that AVE is an agonist of the Ang-(1-7) receptor Mas, in vitro and in vivo. In water-loaded C57BL/6 mice, AVE (0.58 nmol/g body weight) produced a significant reduction in urinary volume (0.06+/-0.03 mL/60 min [n=9] versus 0.27+/-0.05 [n=9]; P<0.01), associated with an increase in urinary osmolality. The Ang-(1-7) antagonist A-779 completely blocked the antidiuretic effect of AVE. As observed previously for Ang-(1-7), the antidiuretic effect of AVE after water load was blunted in Mas-knockout mice (0.37+/-0.10 mL/60 min [n=9] versus 0.27+/-0.03 mL/60 min [n=11] AVE-treated mice). In vitro receptor autoradiography in C57BL/6 mice showed that the specific binding of 125I-Ang-(1-7) to mouse kidney slices was displaced by AVE, whereas no effects were observed in the binding of 125I-angiotensin II or 125I-angiotensin IV. Furthermore, AVE displaced the binding of 125I-Ang-(1-7) in Mas-transfected monkey kidney cells (COS) cells (IC50=4.75x10(-8) mol/L) and of rhodamine-Ang-(1-7) in Mas-transfected Chinese hamster ovary (CHO) cells. It also produced NO release in Mas-transfected CHO cells blocked by A-779 but not by angiotensin II type-1 (AT1) and AT2 antagonists. Contrasting with these data, the antidiuretic effect of AVE was totally blocked by AT2 antagonists and partially blocked (approximately 60%) by AT1 antagonists. The binding data, the results obtained in Mas-knockout mice and in Mas-transfected cells, show that AVE is a Mas receptor agonist. Our data also suggest the involvement of AT2/AT1-related mechanisms, including functional antagonism, oligomerization or cross-talk, in the renal responses to AVE.
Assuntos
Angiotensina I/agonistas , Imidazóis/farmacologia , Rim/fisiologia , Fragmentos de Peptídeos/agonistas , Receptores de Angiotensina/agonistas , Animais , Pressão Sanguínea , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Diurese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas , Receptores Acoplados a Proteínas G , TransfecçãoRESUMO
Angiotensin II (AII) AT(1) receptor mutants with replacements of aliphatic amino acids in the distal region of helix VI and the adjoining region of the third extracellular loop (EC-3) were expressed in Chinese hamster ovary (CHO) cells to determine their role in ligand binding and activation. The triple mutant [L262D, L265D, L268D]AT(1) (L3D) showed a marked reduction in affinity for AII and for non-peptide (losartan) and peptide ([Sar(1)Leu(8) ]AII) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of AII were reduced in L3D. Replacement of Leu(268) (in EC-3) and Leu(262) (in the transmembrane domain) by aspartyl residues did not cause significant changes in the receptor's affinity for the ligands and in IP production. In contrast, the point mutation L265D, at helix VI, markedly decreased affinity and ability to stimulate phosphatidylinositol turnover. Molecular modeling of the AT(1) receptor based on a recent crystal structure of rhodopsin, suggests that the side chain of Leu(265) but not that of Leu(262) is facing a cleft between helices V and VI and interacts with the lipid bilayer, thus helping to stabilize the receptor structure near the Lys(199) residue of helix V in the agonist binding site which is necessary for full activity.
Assuntos
Aminoácidos/química , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Receptores de Angiotensina/agonistas , Receptores de Angiotensina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Feminino , Fosfatos de Inositol/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Receptor Tipo 1 de Angiotensina , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Relação Estrutura-AtividadeRESUMO
To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.
Assuntos
Compostos de Bifenilo/metabolismo , Imidazóis/metabolismo , Mutagênese Sítio-Dirigida , Receptores de Angiotensina/agonistas , Receptores de Angiotensina/metabolismo , Sequência de Aminoácidos/fisiologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Imidazóis/farmacologia , Dados de Sequência Molecular , Ratos , Receptor Tipo 1 de Angiotensina , Receptores de Angiotensina/química , Receptores de Angiotensina/genéticaRESUMO
The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.
Assuntos
Leucina/química , Receptores de Angiotensina/agonistas , Receptores de Angiotensina/química , Sequência de Aminoácidos/fisiologia , Substituição de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Células CHO , Cricetinae , Leucina/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Ratos , Receptor Tipo 1 de Angiotensina , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismoRESUMO
Recently, we demonstrated that the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubules is reversed, in a dose-dependent manner, by Ang-(1-7) [Biochim. Biophys. Acta 1467 (2000) 189]. In the present paper, we characterized the receptor involved in this phenomenon. The preincubation of the Na(+)-ATPase with 10(-8) M Ang II increases the enzyme activity from 7.50+/-0.02 (control) to 12.40+/-1.50 nmol Pi mg(-1) min(-1) (p<0.05). Addition of 10(-9) M Ang-(1-7) completely reverts this effect returning the ATPase activity to the control level. This effect seems to be specific to Ang-(1-7) since Ang III (10(-12)-10(-8) M) does not modify the stimulation of the renal proximal tubule Na(+)-ATPase activity by Ang II. Saralasin abolishes the Ang-(1-7) effect in a dose-dependent manner being the maximal effect obtained at 10(-11) M. The increase in A779 concentration (from 10(-12) to 10(-7) M), a specific Ang-(1-7) antagonist, also abolishes the Ang-(1-7) effect. On the other hand, PD123319 (10(-8)-10(-6) M), an AT(2) antagonist receptor, and losartan (10(-12)-10(-7) M), an AT(1) antagonist receptor, does not modify the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) reverts the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubule through a A779-sensitive receptor.
Assuntos
Angiotensina II/antagonistas & inibidores , Angiotensina II/farmacologia , Angiotensina I/farmacologia , Anti-Hipertensivos/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Angiotensina II/análogos & derivados , Angiotensina III/farmacologia , Antagonistas de Receptores de Angiotensina , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Relação Dose-Resposta a Droga , Imidazóis/farmacologia , Técnicas In Vitro , Túbulos Renais Proximais/enzimologia , Losartan/farmacologia , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/agonistas , Saralasina/farmacologia , SuínosRESUMO
Intracerebroventricular (i.c.v.) injection of serotonin (5-HT) in pigeons dose-dependently evokes a prompt and intense drinking behavior, which resembles that evoked by i.c.v. injections of angiotensin II (ANGII) in the same species. In the present study, we have examined the possible participation of central ANGII receptors in both ANGII- and 5-HT-evoked drinking behavior. The effects of i.c.v. injections of 5-HT (155 nmol), avian ANGII ([Asp(1),Val(5)]-ANGII, 0.1 nmol) or vehicle were studied in pigeons pretreated 20 min before with i.c.v. injections of the nonspecific ANGII receptor antagonist [Sar(1),Ile(8)]-ANGII (SAR; 1, 0.1 or 0.01 nmol), the AT(1) receptor antagonist losartan (2 or 4 nmol), the AT(2) receptor antagonist PD 123,319 (2 or 4 nmol) or vehicle (NaCl 0.15 M, 1 microl, n = 8/group). Immediately after treatment, they were given free access to water and drinking behavior was recorded during the next 60 min. At the doses presently used both 5-HT and ANGII treatments evoked comparable water intake amounts with similar behavioral profiles. While pretreatment with SAR dose-dependently reduced the water intake evoked by both 5-HT and ANGII, neither losartan nor PD 123,319 pretreatment affected the drinking induced by these treatments. The present results indicate that ANGII- and 5-HT-induced drinking in pigeons may be mediated by AT receptors possibly different from mammalian AT(1) and AT(2) receptors and suggest that activation of ANGII central circuits is a necessary step for the intense drinking induced by i.c.v. injections of 5-HT in this species.
Assuntos
Angiotensina II/farmacologia , Columbidae/fisiologia , Comportamento de Ingestão de Líquido/efeitos dos fármacos , Receptores de Angiotensina/metabolismo , Serotonina/farmacologia , 1-Sarcosina-8-Isoleucina Angiotensina II/administração & dosagem , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/administração & dosagem , Angiotensina II/agonistas , Angiotensina II/antagonistas & inibidores , Antagonistas de Receptores de Angiotensina , Animais , Imidazóis/farmacologia , Injeções Intraventriculares , Losartan/farmacologia , Masculino , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/agonistas , Serotonina/administração & dosagemRESUMO
Angiotensin-(1-7) (Ang-(1-7)) modulates the Na+-ATPase, but not the Na+,K+-ATPase activity present in pig kidney proximal tubules. The Na+-ATPase, insensitive to ouabain, but sensitive to furosemide, is stimulated by Ang-(1-7) (68% by 10(-9) M), in a dose-dependent manner. This effect is due to an increase in Vmax, while the apparent affinity of the enzyme for Na+ is not modified. Saralasin, a general angiotensin receptor antagonist, abolishes the stimulation, demonstrating that the Ang-(1-7) effect is mediated by receptor. The Ang-(1-7) stimulatory effect is not changed by either PD 123319, an AT2 receptor antagonist, or A779, an Ang-(1-7) receptor antagonist. On the other hand, increasing the concentration of the AT1 receptor antagonist losartan from 10(-11) to 10(-9) M, reverses the Ang(1-7) stimulation completely. A further increase to 10(-3) M losartan reverses the Na+-ATPase activity to a level similar to that obtained with Ang-(1-7) (10(-9) M) alone. The stimulatory effect of Ang-(1-7) at 10(-9) M is similar to the effect of angiotensin II (AG II) alone. However, when the two peptides are both present, Na+-ATPase activity is restored to control values. These data suggest that Ang-(1-7) selectively modulates the Na+-ATPase activity present in basolateral membranes of kidney proximal tubules through a losartan-sensitive receptor. This receptor is probably different from the receptor involved in the stimulation of the Na+-ATPase activity by angiotensin II.