RESUMEN
Recent studies have demonstrated that the rat adipose tissue expresses some of the components necessary for the production of angiotensin II (Ang II) and the receptors mediating its actions. The aim of this work is to characterize the expression of the renin-angiotensin system (RAS) components in perivascular adipose tissue and to assess differences in the expression pattern depending on the vascular bed and type of adipose tissue. We analyzed Ang I and Ang II levels as well as mRNA levels of RAS components by a quantitative RT-PCR method in periaortic (PAT) and mesenteric adipose tissue (MAT) of 3-month-old male Wistar-Kyoto rats. PAT was identified as brown adipose tissue expressing uncoupling protein-1 (UCP-1). It had smaller adipocytes than those from MAT, which was identified as white adipose tissue. All RAS components, except renin, were detected in both PAT and MAT. Levels of expression of angiotensinogen, Ang-converting enzyme (ACE), and ACE2 were similar between PAT and MAT. Renin receptor expression was five times higher, whereas expression of chymase, AT(1a), and AT(2) receptors were significantly lower in PAT compared with MAT respectively. In addition, three isoforms of the AT(1a) receptor were found in perivascular adipose tissue. The AT(1b) receptor was found at very a low expression level. Ang II levels were higher in MAT with no differences between tissues in Ang I. The results show that the RAS is differentially expressed in white and brown perivascular adipose tissues implicating a different role for the system depending on the vascular bed and the type of adipose tissue.
Asunto(s)
Tejido Adiposo Pardo/química , Tejido Adiposo Blanco/química , Sistema Renina-Angiotensina , Angiotensina I/análisis , Angiotensina II/análisis , Animales , Canales Iónicos/análisis , Masculino , Proteínas Mitocondriales/análisis , Peptidil-Dipeptidasa A/análisis , Peptidil-Dipeptidasa A/genética , ARN Mensajero/análisis , Ratas , Ratas Endogámicas WKY , Receptor de Angiotensina Tipo 2/análisis , Receptor de Angiotensina Tipo 2/genética , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Proteína Desacopladora 1 , Receptor de ProreninaRESUMEN
BACKGROUND: Apoptosis plays a key role in the pathogenesis of cardiac diseases. We examined the influence of the renin-angiotensin system (RAS) on different regulators of apoptosis using an isolated hemoperfused working porcine heart model of acute ischemia (2 h), followed by reperfusion (4 h). METHODS AND RESULTS: 23 porcine hearts were randomized to 5 groups: hemoperfused non-infarcted hearts (C), infarcted hearts (MI: R. circumflexus), infarcted hearts treated with quinaprilat (Q), infarcted hearts treated with angiotensin-I (Ang I), and infarcted hearts treated with angiotensin-I and quinaprilat (QA). Fas, Bax, bcl-2 and p53 proteins were increased in MI hearts and further elevated by Ang I. Quinaprilat reduced Bax and p53. Bcl-2 was elevated in Q and reduced in QA. An early upregulation of caspase-3 gene and protein expression was detected in MI and Ang I hearts compared to C. Q reduced caspase-3 gene expression, but had no effect on caspase-3 and Fas protein. CONCLUSIONS: These data suggest that the RAS plays a pivotal role in cardiac apoptosis which is the early and predominant form of death in myocardial infarction. Ischemia/reperfusion induces programmed cell death via extrinsic and intrinsic pathways. Early treatment with quinaprilat attenuated cardiomyocyte apoptosis.
Asunto(s)
Apoptosis/fisiología , Corazón/fisiología , Infarto del Miocardio/patología , Miocardio , Sistema Renina-Angiotensina/fisiología , Daño por Reperfusión , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Forma de la Célula , Femenino , Infarto del Miocardio/metabolismo , Miocardio/citología , Miocardio/metabolismo , Miocardio/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Distribución Aleatoria , Porcinos , Tetrahidroisoquinolinas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Receptor fas/metabolismoRESUMEN
The interaction of the two transcriptional regulators RcsA and RcsB with a specific operator is a common mechanism in the activation of capsule biosynthesis in enteric bacteria. We describe RcsAB binding sites in the wza promoter of the operon for colanic acid biosynthesis in Escherichia coli K-12, in the galF promoter of the operon for K2 antigen biosynthesis in Klebsiella pneumoniae, and in the tviA (vipR) promoter of the operon for Vi antigen biosynthesis in Salmonella typhi. We further show the interaction of RcsAB with the rcsA promoters of various species, indicating that rcsA autoregulation also depends on the presence of both proteins. The compilation of all identified RcsAB binding sites revealed the conserved core sequence TaAGaatatTCctA, which we propose to be termed RcsAB box. The RcsAB box is also part of Bordetella pertussis BvgA binding sites and may represent a more distributed recognition motif within the LuxR superfamily of transcriptional regulators. The RcsAB box is essential for the induction of Rcs-regulated promoters. Site-specific mutations of conserved nucleotides in the RcsAB boxes of the E. coli wza and rcsA promoters resulted in an exopolysaccharide-negative phenotype and in the reduction of reporter gene expression.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Operón , Polisacáridos Bacterianos/biosíntesis , Factores de Transcripción , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Secuencia de Bases , Sitios de Unión , Escherichia coli/genética , Homeostasis , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Polisacáridos Bacterianos/genética , Regiones Promotoras GenéticasRESUMEN
The regulation of capsule synthesis (Rcs) regulatory network is responsible for the induction of exopolysaccharide biosynthesis in many enterobacterial species. We have previously shown that two transcriptional regulators, RcsA and RcsB, do bind as a heterodimer to the promoter of amsG, the first reading frame in the operon for amylovoran biosynthesis in the plant pathogenic bacterium Erwinia amylovora. We now identified a 23-base pair fragment from position -555 to -533 upstream of the translational start site of amsG as sufficient for the specific binding of the Rcs proteins. In addition, we could detect an RcsA/RcsB-binding site in a corresponding region of the promoter of cpsA, the homologous counterpart to the E. amylovora amsG gene in the operon for stewartan biosynthesis of Pantoea stewartii. The specificity and characteristic parameters of the protein-DNA interaction were analyzed by DNA retardation, protein-DNA cross-linking, and directed mutagenesis. The central core motif TRVGAAWAWTSYG of the amsG promoter was found to be most important for the specific interaction with RcsA/RcsB, as evaluated by mutational analysis and an in vitro selection approach. The wild type P. stewartii Rcs binding motif is degenerated in two positions and an up-mutation according to our consensus motif resulted in about a 5-fold increased affinity of the RcsA/RcsB proteins.