RESUMEN
The purpose of this study was to assess, in the murine kidney, the mechanisms underlying the endothelium-dependent control of vascular tone and whether or not, in a severe model of hypertension and renal failure, KCa channels contribute to its regulation. Wild-type (BL) and double-transgenic female mice expressing human angiotensinogen and renin (AR) genes received either control or a high-salt diet associated to a nitric oxide (NO) synthase inhibitor treatment (BLSL and ARSL). Changes in renal perfusion pressure (RPP) were measured in isolated perfused kidneys. BLSL and AR were moderately hypertensive without kidney disease while ARSL developed severe hypertension and renal failure. In the four groups, methacholine induced biphasic endothelium-dependent responses, a transient decrease in RPP followed by a cyclooxygenase-dependent increase in RPP. In the presence or not of indomethacin, the vasodilatations were poorly sensitive to NO synthase inhibition. However, in the presence of cyclooxygenase and NO synthase inhibitors, apamin, and/or TRAM-34, blockers of KCa2.3 and KCa3.1, respectively, abolished the decrease in RPP in response to either methacholine or the two activators of KCa2.3/KCa3.1, NS309, and SKA-31. Thus, KCa2/3 channels play a major role in the regulation of murine kidney perfusion and this mechanism is maintained in hypertension, even when severe and associated with kidney damage.
Asunto(s)
Hipertensión Renovascular/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Insuficiencia Renal/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Vasodilatación , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Animales , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Femenino , Humanos , Hipertensión Renovascular/etiología , Hipertensión Renovascular/fisiopatología , Indometacina/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Insuficiencia Renal/etiología , Insuficiencia Renal/fisiopatología , Renina/genética , Renina/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Sodio en la Dieta/efectos adversosRESUMEN
The purpose of the present work was to elucidate the mechanisms underlying the endothelium-dependent and endothelium-independent components of the vascular relaxation induced by a water-soluble and ruthenium-based carbon monoxide (CO)-releasing agent, tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). Changes in isometric tension and cyclic guanosine monophosphate (cGMP) production were measured in isolated aortic rings from normotensive Wistar-Kyoto rats. Nitric oxide (NO) generation was assessed in cultured human umbilical vein endothelial cells (HUVEC) by electron spin resonance. In rat aortic rings, CORM-3, but not the inactivated compound, iCORM, induced relaxations. In rings with but not in those without endothelium relaxations were partially inhibited by L-nitro-arginine (L-NA), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ), or hydroxocobalamin, inhibitors of NO-synthase, soluble guanylyl cyclase, and scavenger of NO, respectively. In rings with and without endothelium, deoxyhemoglobin abolished the relaxations. A combination of potassium channel blockers (barium, glibenclamide, and iberiotoxin) blunted the relaxation in rings without endothelium. CORM-3 produced an endothelium-dependent generation of cGMP that was inhibited by L-NA. CORM-3, but not iCORM, inhibited the endothelium-dependent relaxation to acetylcholine without affecting the response to sodium nitroprusside. In HUVEC, CORM-3 produced a concentration-dependent release of NO. Therefore, CORM-3-induced relaxations involve the soluble guanylyl cyclase-independent activation of smooth muscle potassium channels. Additionally, CO can produce concomitantly activation and inhibition of NO synthase, the former being responsible for the endothelium- and cGMP-dependent effect of CORM-3, the latter for the inhibition of acetylcholine-induced endothelium-dependent relaxations.
Asunto(s)
Monóxido de Carbono/farmacología , Endotelio Vascular/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Compuestos Organometálicos/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Análisis de Varianza , Animales , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/metabolismo , Monóxido de Carbono/química , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Óxido Nítrico/metabolismo , Compuestos Organometálicos/química , Canales de Potasio/metabolismo , Unión Proteica , Ratas , Ratas Endogámicas WKY , Solubilidad , Vasodilatadores/químicaRESUMEN
BACKGROUND AND PURPOSE: The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury. EXPERIMENTAL APPROACH: Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar-Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N(ω) -nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg(-1) ·day(-1) ). KEY RESULTS: Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N(w) -nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca(2+) -activated K(+) channels of intermediate conductance (K(Ca) 3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca(2+) -activated K(+) channels of small conductance (K(Ca) 2.3), was ineffective. Dilatation induced by an opener of K(Ca) 3.1/K(Ca) 2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K(Ca) 3.1, but not K(Ca) 2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR). CONCLUSIONS AND IMPLICATIONS: In SHR kidneys, an EDHF-mediated response, involving activation of K(Ca) 3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.
Asunto(s)
Endotelio Vascular/fisiología , Hipertensión/fisiopatología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Riñón/fisiología , Vasodilatación/fisiología , Acetilcolina/farmacología , Animales , Apamina/farmacología , Factores Biológicos/fisiología , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Indoles/farmacología , Indometacina/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Riñón/efectos de los fármacos , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Nitroprusiato/farmacología , Oximas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
INTRODUCTION: Age and hypertension are two major determinants of arterial stiffness, as well as endothelial dysfunction. The present study was designed to test whether a chronic reduction of endogenous nitric oxide (NO) produces arterial stiffening close to that observed in old spontaneously hypertensive rats (SHR), and also to study the effect of an acute or a chronic decrease in blood pressure (BP) on aortic distensibility. METHODS: BP, aortic stiffness, endothelial dysfunction and remodelling were measured in male adult (20-week-old) SHR, in adult SHR treated with a nonspecific NO synthase inhibitor L-NAME (SHR/L-NAME) for 2 weeks, in adult SHR/L-NAME cotreated with perindopril (1 mg/kg/day) and in old SHR (55-week-old). Age-matched WKY were used as a normotensive group. RESULTS: Aortic endothelial dysfunction, remodelling and stiffening appeared in old SHR. Reduction of NO production in adult SHR caused similar alterations. Acute decreases in BP in SHR/L-NAME did not improve isobaric aortic distensibility but a chronic reduction of BP prevented endothelial dysfunction, aortic remodelling and aortic wall stiffening. CONCLUSION: NO reduction in adult SHR induces aortic alterations similar to those observed during aging, which supports the major role of NO in the development of arterial stiffening. These aortic alterations can be prevented by angiotensin-converting enzyme inhibitor treatment.
Asunto(s)
Envejecimiento/patología , Aorta/fisiopatología , Hipertensión/fisiopatología , Óxido Nítrico/antagonistas & inhibidores , Rigidez Vascular/efectos de los fármacos , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Aorta/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Clonidina/farmacología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Perindopril/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
The (pro)renin receptor (PRR) has recently been demonstrated to bind equally well renin and its precursor, prorenin, leading to a similar intracellular signaling independent of angiotensin II. In this study, we report that human embryonic kidney cells (HEK) exposed to renin or prorenin for 24 h in the presence of a blocking concentration of the angtiotensin-converting enzyme inhibitor perindoprilate increased superoxide anion production as measured by luminescence (lucigenin) and electron spin resonance spectroscopy (hydroxylamine radical transition). Also, both renin and prorenin increased Nox4 expression while Nox2, p47(phox), and p67(phox) remained unchanged. In an investigation of the effects of renin and prorenin on fibrosis genes, it appeared that both proteins stimulated transforming growth factor-ß (TGF-ß), fibronectin, and plasminogen activator inhibitor type 1 (PAI-1) expression and therefore participated to an overall switch toward a profibrotic state of the kidney cells. When the cells were transfected with a siRNA targeting the PRR, Nox4 expression was efficiently prevented as well as the increase in superoxide production, TGF-ß, fibronectin, and PAI-1. Finally, we demonstrated that transfection of the cells with a Nox4-specific small interfering (si) RNA also prevented fibrosis gene expression following treatment with renin or prorenin. The results demonstrate that renin and prorenin, through their specific membrane receptor and independently of angiotensin II, promote fibrosis gene expression via a Nox4-dependent mechanism.
Asunto(s)
Fibrosis/metabolismo , Receptores de Superficie Celular/metabolismo , Renina/farmacología , Superóxidos/metabolismo , Análisis de Varianza , Western Blotting , Células Cultivadas , Fibrosis/genética , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Receptor de ProreninaRESUMEN
Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification, and quantification of growing importance. Among free cysteines, the bulk of modifications occurs on a subset of cysteines that are more reactive. These exist as thiolate anions at physiological pH because of their surrounding electrostatic environment. Reagents with iodoacetamide-active groups can be used to selectively label these reactive thiols with a high degree of selectivity. Thiol adducts can be detected by the failure to label with iodoacetamide or other reagents; restoration of labeling by specific reducing agents (e.g., ascorbate or glutaredoxin) can be used to detect reversible S-nitroso and S-glutathione adducts. These adducts also may be detected with radiolabels and antibodies. S-Glutathiolation in response to physiological stimuli may be detected in cells and tissues with glutathione ester labeled with biotin. Mass spectrometry can identify thiol modifications with precision, and with isotope-coded affinity tags, used to quantify modification of specific thiols. Combinations of these methods increase sensitivity and specificity, and enable quantification and precise identification of thiol modifications that occur under physiological and pathological conditions.
Asunto(s)
Cisteína/metabolismo , Animales , Biotinilación , Glutatión/metabolismo , Yodoacetamida/química , Marcaje Isotópico , Espectrometría de Masas , Nitrosación , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotope-coded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative posttranslational thiol modifications of p21ras caused by peroxynitrite (ONOO(-)) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased after exposure to GSSG, but not to ONOO(-). The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO(-) showed that ICAT labeling of Cys(118) was decreased by 47%, whereas Cys(80) was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys(118) by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras.
Asunto(s)
Cisteína/química , Espectrometría de Masas/métodos , Oxidación-Reducción , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Compuestos de Sulfhidrilo/química , Secuencia de Aminoácidos , Cromatografía Liquida , Radicales Libres , Glutatión/química , Humanos , Datos de Secuencia Molecular , Oxígeno/química , Ácido Peroxinitroso/química , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
OBJECTIVE: To understand the mechanism by which oxidants are linked to insulin resistance, bovine aortic endothelial cells were exposed to oxidized low-density lipoproteins (oxLDL) or peroxynitrite. METHODS AND RESULTS: OxLDL transiently increased phosphorylation of Erk and Akt within 5 minutes, but 60 minutes later, resulted in decreased insulin-induced Akt phosphorylation. OxLDL promoted a 2- to 5-fold increase in oxidant generation as measured by dihydrorhodamine or dihydroethidium oxidation that was ascribed to peroxynitrite. Exogenous peroxynitrite (25 to 100 micromol/L) or oxidized glutathione mimicked the effects of oxLDL. OxLDL increased the S-glutathiolation of p21ras, and adenoviral transfection with either a mutant p21ras (C118S) lacking the predominant site of S-glutathiolation or a dominant-negative mutant restored insulin-induced Akt phosphorylation. The requirement for oxidant-mediated S-glutathiolation and activation of p21ras in mediating insulin resistance was further implicated by showing that insulin signaling was restored by Mek inhibitors or by overexpression of glutaredoxin-1. Furthermore, oxLDL increased Erk-dependent phosphorylation of insulin receptor substrate-1 serine-616 that was prevented by inhibiting oxidant generation, Erk activation, or by the p21ras C118S mutant. CONCLUSIONS: This study provides direct evidence for a novel molecular mechanism by which oxidants can induce insulin resistance via S-glutathiolation of p21ras and Erk-dependent inhibition of insulin signaling.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Glutatión/metabolismo , Resistencia a la Insulina/fisiología , Lipoproteínas LDL/farmacología , Proteína Oncogénica p21(ras)/metabolismo , Ácido Peroxinitroso/farmacología , Alprostadil/análogos & derivados , Alprostadil/farmacología , Animales , Aorta/citología , Bovinos , Dinoprostona/agonistas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Disulfuro de Glutatión/farmacología , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina , Lisofosfatidilcolinas/farmacología , Proteína Oncogénica p21(ras)/efectos de los fármacos , Oxidantes/farmacología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Prostaglandinas E Sintéticas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
P21ras, the translation product of the most commonly mutated oncogene, is a small guanine nucleotide exchange protein. Oxidant-induced post-translational modifications of p21ras including S-nitrosation and S-glutathiolation have been demonstrated to modulate its activity. Structural characterization of this protein is critical to further understanding of the biological functions of p21ras. In this study, high-resolution and high mass accuracy Fourier transform mass spectrometry was utilized to map, in detail, the post-translational modifications of p21ras (H-ras) exposed to oxidants by combining bottom-up and top-down techniques. For peroxynitrite-treated p21ras, five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines (including C118) were identified by "bottom-up" analysis, and the major oxidative modification of C118, Cys118-SO3H, was confirmed by several tandem mass spectrometry experiments. Additionally, "top-down" analysis was conducted on p21ras S-glutathiolated by oxidized glutathione and identified C118 as the major site of glutathiolation among the four surface cysteines. The present study provides a paradigm for an effective and efficient method not only for mapping post-translational modifications of proteins but also for predicting the relative selectivity and specificity of oxidative post-translational modifications, especially using top-down analysis.
Asunto(s)
Análisis de Fourier , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteínas ras/análisis , Proteínas ras/química , Secuencia de Aminoácidos , Glutatión/química , Glutatión/metabolismo , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Ácido Peroxinitroso , Proteínas ras/metabolismoRESUMEN
The highly reactive species, peroxynitrite, is produced in endothelial cells in pathological states in which the production of superoxide anion and NO is increased. Here, we show that peroxynitrite added exogenously or generated endogenously in response to exposure to an NO donor or oxidized low-density lipoproteins (oxLDL) increases p21ras activity in bovine aortic endothelial cells. The activation is not dependent on upstream elements but rather is due to direct targeting of p21ras by reversible S-glutathiolation of cysteine thiols as demonstrated by biotin-labeling techniques. The time course of p21ras S-glutathiolation following peroxynitrite corresponds to the increase in its Raf-1 binding activity and translocation to the membrane. Moreover, p21ras S-glutathiolation and activation can be reversed by dithiothreitol, confirming the importance of a disulfide bond. S-glutathiolation also promoted guanine nucleotide exchange of recombinant p21ras. In addition, the oxidant-induced activation of Mek/Erk and PI3 kinase/Akt was abrogated by dominant-negative and Cys-118 p21ras mutants, and the latter also prevented S-glutathiolation of p21ras. These results indicate that peroxynitrite arising from NO donors or pathological stimuli such as oxLDL triggers direct S-glutathiolation of p21ras Cys-118, which increases p21ras activity and mediates downstream signaling.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Glutatión/metabolismo , Ácido Peroxinitroso/farmacología , Proteínas Proto-Oncogénicas p21(ras)/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Acetofenonas/farmacología , Androstadienos/farmacología , Animales , Aorta , Butadienos/farmacología , Bovinos , Células Cultivadas/metabolismo , Cisteína/metabolismo , Células Endoteliales/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanosina Difosfato/metabolismo , Lipoproteínas LDL/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitrilos/farmacología , Oxidación-Reducción , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , WortmaninaRESUMEN
Polyamines are ubiquitous molecules, which, like iron, are essential for cell growth. All eukaryotic cells are equipped with a specific polyamine transport system (PTS). Polyamines have primary and secondary amino groups which chelate bivalent metal cations such as Fe and Cu. In the present study, we investigated the potential contribution of naturally occurring polyamines and their active transport system to iron uptake. In presence of subtoxic Fe(III) (10microM), treatment of CHO cells with spermine, and to a lesser extent with spermidine (10-100microM), resulted in a marked cytotoxic effect. This cytotoxicity was prevented by the addition of an iron-chelator, deferioxamine, and was not observed in CHO-MG cells, a mutant cell line devoid of polyamine transport activity. Experiments using 14C-polyamines and 55Fe(III) revealed that these toxic effects were related to polyamine-modulation of iron uptake, and were dependent on the presence of the active PTS. These results demonstrated active uptake of polyamine-iron complexes via the PTS. The number of amino groups affected the efficacy of the studied natural polyamines to transport iron via the PTS. Spermine, a tetramine, was more efficient than the triamine spermidine. Co-transport of iron by the diamine putrescine was not observed. These results demonstrate that the cell polyamine transport system is a potential cell entry pathway for iron. The studied polyamines, spermine and spermidine, may be components of the pool of transferrin-independent iron-chelating vectors, which have recently attracted the attention of many investigators.