RESUMO
Bradykinin (BK) is a vasorelaxant, algesic and inflammatory agent. Angiotensin II (AngII) is known to control vascular tone and promote growth, inflammation and artherogenesis. There is evidence for cross talking between BK and AngII receptors. Therefore, the effect of lack of kinin receptors was assessed in mice with genetic disruption of B(1) or B(2) and both receptors. Responsiveness of abdominal aortic rings to BK and AngII as well as the receptor gene expression of both peptides were analysed. Although no specific phenotype was displayed in the normotensive and healthy mice lacking the kinin receptors, a decreased expression level of the remaining kinin receptor mRNA was observed. AT(1) receptor mRNA level was also reduced, indicating that kinin receptors regulate AngII receptors. Downregulation of the receptors was well correlated with reduction in the reactivity of both agonists to induce contraction of aortic rings, but other signal regulations must be sought in these transgenic mice. We conclude that cross talk between kinin and AngII receptors occurs in mouse abdominal aorta and that both peptides may regulate the initiation and progression of important pathophysiological processes, such as hypertension and inflammation.
Assuntos
Aorta Abdominal/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/farmacologia , Animais , Aorta Abdominal/efeitos dos fármacos , Bradicinina/farmacologia , Imidazóis/antagonistas & inibidores , Cininas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , NG-Nitroarginina Metil Éster/farmacologia , Reação em Cadeia da Polimerase , Piridinas/antagonistas & inibidores , Receptor B1 da Bradicinina/genética , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Receptores de Angiotensina/genética , Vasodilatadores/farmacologiaRESUMO
In the kallikrein-kinin and renin-angiotensin systems the main receptors, B1 and B2 (kinin receptors) and AT1 and AT2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B1 agonists Des-Arg9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg9-BK or Des-Arg10-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT1 agonist (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT1 receptor. To investigate this hypothesis, we replaced Arg212 for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B1 receptor by the B2 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg212 in the TM V and a region of TM VI of rat B1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.
Assuntos
Bradicinina/análogos & derivados , Calidina/análogos & derivados , Receptor B1 da Bradicinina/química , Sequência de Aminoácidos , Animais , Bradicinina/química , Bradicinina/metabolismo , Células CHO , Cricetinae , Cricetulus , Células HeLa , Humanos , Calidina/química , Calidina/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Ratos , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/metabolismoRESUMO
Methane production from marine microalgae Isochrysis galbana was assessed before and after mechanical and chemical pretreatments. Mechanical pretreatment resulted in a 61.7% increase in soluble Chemical Oxygen Demand. Different hydrolysis conditions were evaluated by varying temperature - T, sulfuric acid concentration - AC and biomass suspension concentration (measured as particulate COD - CODp) using an experimental design. The most significant interaction occurred between AC and T and the hydrolysis condition that showed the best result in the anaerobic digestion step was the condition at 40°C with addition of 0.2% (v/v) acid for 16h (9.27LCH4/kgVS). The low methane yields were attributed to inhibitory sodium concentrations for anaerobic digestion. Eliminating inhibitory sodium in the anaerobic digestion by biomass prewashing, there was a 71.5% increase in methane yield for biomass after acid hydrolysis, demonstrating the need for pretreatment and reduction in sodium concentration in the anaerobic digestion.
Assuntos
Organismos Aquáticos/metabolismo , Haptófitas/metabolismo , Metano/biossíntese , Microalgas/metabolismo , Anaerobiose/efeitos dos fármacos , Organismos Aquáticos/efeitos dos fármacos , Biodegradação Ambiental/efeitos dos fármacos , Biocombustíveis/microbiologia , Análise da Demanda Biológica de Oxigênio , Biomassa , Dióxido de Carbono/metabolismo , Haptófitas/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Salinidade , Sódio/farmacologia , Ácidos Sulfúricos/farmacologia , TemperaturaRESUMO
Bradykinin (BK) and des-Arg(9)-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B2 (B2R) and B1 (B1R) receptors, respectively. It was already shown that the deletion of kinin B1R or of B2R induces upregulation of the remaining receptor subtype. However studies on overexpression of B1R or B2R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B1R (TGR(Tie2B1)) exclusively in the endothelium. In another study, mice overexpressing B1R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B2R was investigated in the thoracic aorta isolated from TGR(Tie2B1) rats overexpressing the B1R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B2R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie2B1) rats could be due to the enhanced expression of B2R associated with the overexpression of the B1R.
Assuntos
Endotélio Vascular/fisiologia , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Acetilcolinesterase/análise , Acetilcolinesterase/metabolismo , Angiotensina II/farmacologia , Animais , Aorta/efeitos dos fármacos , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina , Regulação da Expressão Gênica , Técnicas In Vitro , Indometacina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/genética , Regulação para Cima , Vasodilatação/efeitos dos fármacosRESUMO
Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.
Assuntos
Angiotensina II/metabolismo , Sinalização do Cálcio/fisiologia , Peptidil Dipeptidase A/metabolismo , Análise de Variância , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Citometria de Fluxo , Lisinopril/farmacologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Previous studies on angiotensin II (AngII) AT(1) receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys(274) residue that supposedly makes a disulfide bond with N-terminal Cys(18). As demonstrated by assays with Del(267-275)AT(1), the role of the Cys(18)-Cys(274) disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT(1) receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S-S disulfide bond was disrupted in (C18S,C274S) AT(1) receptor. The importance of the free N-terminal amino group of Asp(1) and of the Arg(2) guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp(1) for Sar(1) or Arg(2) for Lys(2), as ligands. This study showed that like AngII, [Sar(1)]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys(2)]-AngII binding is still more reduced. Interestingly, when (125)I-AngII instead of (3)H-AngII was used, no significant binding of this mutant was observed although wild type AT(1) receptor was shown to bind all AngII analogues.
Assuntos
Angiotensina II/metabolismo , Cisteína/metabolismo , Radioisótopos do Iodo/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Ligação Proteica , Ensaio RadioliganteRESUMO
OBJECTIVE: Kinins mediate pathophysiological processes related to hypertension, pain, and inflammation through the activation of two G-protein-coupled receptors, named B(1) and B(2). Although these peptides have been related to glucose homeostasis, their effects on energy balance are still unknown. RESEARCH DESIGN AND METHODS: Using genetic and pharmacological strategies to abrogate the kinin B(1) receptor in different animal models of obesity, here we present evidence of a novel role for kinins in the regulation of satiety and adiposity. RESULTS: Kinin B(1) receptor deficiency in mice (B(1)(-/-)) resulted in less fat content, hypoleptinemia, increased leptin sensitivity, and robust protection against high-fat diet-induced weight gain. Under high-fat diet, B(1)(-/-) also exhibited reduced food intake, improved lipid oxidation, and increased energy expenditure. Surprisingly, B(1) receptor deficiency was not able to decrease food intake and adiposity in obese mice lacking leptin (ob/ob-B(1)(-/-)). However, ob/ob-B(1)(-/-) mice were more responsive to the effects of exogenous leptin on body weight and food intake, suggesting that B(1) receptors may be dependent on leptin to display their metabolic roles. Finally, inhibition of weight gain and food intake by B(1) receptor ablation was pharmacologically confirmed by long-term administration of the kinin B(1) receptor antagonist SSR240612 to mice under high-fat diet. CONCLUSIONS: Our data suggest that kinin B(1) receptors participate in the regulation of the energy balance via a mechanism that could involve the modulation of leptin sensitivity.