RESUMEN
Antioxidant enzymes play a key role in plant tolerance to different types of stress, including ultraviolet-B (UV-B) radiation. Here we report that nitric oxide (NO) enhances antioxidant enzymes gene expression and increases the activity of specific isoforms protecting against UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented lipid peroxidation, ion leakage and H2O2 and superoxide anion accumulation in leaves of UV-B-treated soybean plants. Transcripts levels of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were significantly induced by SNP. These data correlated with the enhancement of particular antioxidant enzyme isoforms, such as one CAT isoform and two APX isoforms. Moreover, SNP induced the expression of three new isoforms of SOD, identified as Mn-SOD subclass. Further results showed that total activities of SOD, CAT and APX significantly increased by 2.2-, 1.8- and 2.1-fold in SNP-treated plants compared to controls, respectively. The protective effect of SNP against UV-B radiation was negated by addition of the specific NO scavenger cPTIO, indicating that NO released by SNP mediates the enhancement of antioxidant enzymes activities. In conclusion, NO is involved in the signaling pathway that up-regulates specific isoforms of antioxidant enzymes protecting against UV-B-induced oxidative stress.
Asunto(s)
Glycine max/metabolismo , Óxido Nítrico/metabolismo , Rayos Ultravioleta , Ascorbato Peroxidasas/genética , Ascorbato Peroxidasas/metabolismo , Catalasa/genética , Catalasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Nitroprusiato/química , Nitroprusiato/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Glycine max/efectos de los fármacos , Glycine max/efectos de la radiación , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
Heme oxygenase (HO) has antioxidant properties and is up-regulated by reactive oxygen species (ROS) in ultraviolet-B-irradiated soybean plants. This study shows that nitric oxide (NO) protects against oxidative damage and that nitric oxide synthase (NOS)-like activity is also required for HO-1 induction under UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented chlorophyll loss, H(2)O(2) and O(2)(*-) accumulation, and ion leakage in UV-B-treated plants. HO activity was significantly enhanced by NO and showed a positive correlation with HO-1 transcript levels. In fact, HO-1 mRNA levels were increased 2.1-fold in 0.8 mM SNP-treated plants, whereas subsequent UV-B irradiation augmented this expression up to 3.5-fold with respect to controls. This response was not observed using ferrocyanide, a SNP inactive analog, and was effectively blocked by 2-(4-carboxyphenil)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO-scavenger. In addition, experiments carried out in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) or tungsten, well-known inhibitors of NOS and nitrate reductase, showed that NOS is the endogenous source of NO that mediates HO-1 expression. In summary, we found that NO is involved in the signaling pathway leading to HO-1 up-regulation under UV-B, and that a balance between NO and ROS is important to trigger the antioxidant response against oxidative stress.
Asunto(s)
Glycine max/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/biosíntesis , Rayos Ultravioleta , Clorofila/análisis , Clorofila/metabolismo , Clorofila/efectos de la radiación , Hemo Oxigenasa (Desciclizante)/efectos de la radiación , Peróxido de Hidrógeno/análisis , NG-Nitroarginina Metil Éster/química , Óxido Nítrico Sintasa/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Hojas de la Planta/química , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Glycine max/genética , Glycine max/efectos de la radiación , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
This study was performed to provide insight into the regulatory role of angiotensin II and arterial pressure on the activity of antioxidant enzymes and oxidative stress generation in the hypertensive kidney from an experimental animal model of renovascular hypertension. Aortic coarcted and sham-operated rats received vehicle, losartan or minoxidil in their drinking water. After 7 d of treatment rats were sacrificed; hypertensive kidneys were excised, and the NAD(P)H oxidase subunits expression, TBARS production, glutathione level and the activity of heme oxygenase-1 and classical antioxidant enzymes, were evaluated. Losartan administration significantly reduced oxidative stress generation decreasing NAD(P)H oxidase expression, independently of the drop in arterial pressure. On the other hand, antioxidant enzymes were regulated by arterial pressure and they were not implicated in kidney protection against oxidative damage. Findings here reported strongly suggest that clinical therapeutics with the Ang II type 1 receptor blocker prevents oxidative stress generation and may attenuate the kidney oxidative damage in the renovascular hypertension. We hypothesize that the pathway followed by the Ang II blocker to achieve this renoprotection, though independent of the primary antioxidant enzymatic system, depends on NAD(P)H oxidase downregulation.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Presión Sanguínea/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/metabolismo , Losartán/farmacología , NADPH Oxidasas/metabolismo , Animales , Western Blotting , Glutatión/metabolismo , Hemo-Oxigenasa 1/metabolismo , Peróxido de Hidrógeno/metabolismo , Hipertensión Renovascular/tratamiento farmacológico , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Although interleukins (IL) 8 and 10 predict lung viability in lung transplantation from heart beating donors (HBD) and IL-1beta is a marker of ex vivo performance from after cardiac death donors (ACDD), IL expression in the recipient remains unknown. This study assessed IL-1beta, IL-8 and IL-10 as indicators of functional performance in single-lung transplantation from ACDD pigs. Animals were divided into: (i) HBD: immediate lung excision; (ii) ACDD: fibrillation, 30 min warm ischemia and 3 h topical cooling. Left lungs of both groups were then flushed with Perfadex and stored at 3-4 degrees C for 3 h. IL in bronchoalveolar lavage fluid (BAL) and hemodynamic and graft function were measured in the donor and during the 2 h reperfusion period in the recipient. Myeloperoxidase, nuclear factor kappa beta, wet/dry weight ratio and a histologic injury score were assessed from biopsies in basal conditions in the donor and at the end of reperfusion. Despite similar pulmonary function and histologic markers of injury in both groups and higher IL-1beta in the donor of ACDD, IL-8 during reperfusion was significantly lower in ACDD (119 +/- 33% of basal) than in HBD (306 +/- 238%, P < 0.05) recipients. The paradoxical behavior of IL-8 makes it an unreliable predictor of ACDD early outcome in this transplantation model.
Asunto(s)
Interleucina-8/metabolismo , Trasplante de Pulmón , Disfunción Primaria del Injerto/diagnóstico , Animales , Líquido del Lavado Bronquioalveolar/química , Muerte , Hemodinámica , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Pulmón/patología , Masculino , Preservación de Órganos , Disfunción Primaria del Injerto/metabolismo , Sus scrofa , Donantes de Tejidos , Recolección de Tejidos y ÓrganosRESUMEN
The aim of this study was to provide new insights into the role of angiotensin II and arterial pressure in the regulation of antioxidant enzyme activities in a renovascular model of cardiac hypertrophy. For this purpose, aortic coarcted rats were treated with losartan or minoxidil for 7 days. Angiotensin II induced cardiac hypertrophy and oxidative stress via Nox4, p22(phox) and p47(phox), which are components of the NAD(P)H oxidase. Antioxidant enzymes were regulated by arterial pressure and were not implicated in cardiac hypertrophy. Heme oxygenase-1, the rate-limiting enzyme in heme catabolism, behaved as a catalase and glutathione peroxidase, and is regulated by arterial pressure. In summary, the present report indicates that cardiac hypertrophy, induced by renovascular hypertension, depends on angiotensin II through reactive oxygen species and is not prevented by the action of antioxidant enzymes.
Asunto(s)
Angiotensina II/fisiología , Cardiomegalia/etiología , Hipertensión Renovascular/complicaciones , Estrés Oxidativo , Animales , Presión Sanguínea , Catalasa/metabolismo , Glutatión/análisis , Hipertensión Renovascular/metabolismo , Losartán/farmacología , Masculino , NADPH Oxidasas/metabolismo , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
Heme oxygenase (HO, EC 1.14.99.3) catalyses the oxidative conversion of heme to biliverdin IX alpha (BV) with the concomitant released of carbon monoxide and iron. Recently, plant HOs have been involved in the defence mechanism against oxidative stress. The goal of this study was to evaluate the time-course of HO-1 and catalase (CAT, EC 1.11.1.6) gene expressions in nodules and roots of soybean plants subjected to Cd treatment. No significant changes were observed up to 24 h. After 48 h of 200 microM Cd exposure, an up-regulation of HO-1 mRNA (110%) occurred in nodules. On the other hand, a down-regulation was found in roots (39%). While there was an augmentation in CAT transcript levels (30%) in nodules, an important diminution (52%) was evidenced in roots. Changes observed in gene expression were also found in protein levels and activities. These data suggest that an induction of CAT and HO-1 occurred in nodules as a response of cell protection against oxidative damage. However, after 72 h treatment, a down-regulation of HO-1 mRNA was found either in nodules or in roots (78% and 94%, respectively), while a similar response was evidenced for CAT (40% and 83%, respectively). These results are consistent with our previous findings suggesting that oxidative stress produced by Cd were more pronounced in roots than in nodules of soybean plants. Moreover, this behaviour could explain the major viability observed in nodules respect to roots, and provide a new insight into the processes involved in the antioxidant defence system in plant tissues.
Asunto(s)
Cadmio/farmacología , Catalasa/genética , Glycine max/enzimología , Hemo Oxigenasa (Desciclizante)/genética , Estrés Oxidativo , Proteínas de Plantas/genética , Raíces de Plantas/enzimología , Antioxidantes/metabolismo , Catalasa/metabolismo , Inducción Enzimática , Regulación de la Expresión Génica de las Plantas , Hemo Oxigenasa (Desciclizante)/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Glycine max/anatomía & histología , Glycine max/efectos de los fármacosRESUMEN
Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidative conversion of heme to biliverdin IXalpha with the concomitant release of carbon monoxide and iron. Recently, HO has been involved in the protection against oxidative stress in plants. The fact that nitric oxide (NO), an endogenous signaling molecule in animals and plants mediates responses to abiotic and biotic stresses, prompted us to study whether this molecule could modulate HO-1 gene transcription. To fulfill this objective leaves of soybean (Glycine max L.) plants were stimulated with Cd, employing an acute intoxication model. Cadmium caused dehydration, chlorophyll loss and ion leakage. Semi-quantitative RT-PCR analysis showed no augmentation of HO-1 transcript levels with respect to controls. Pretreatment with 100 microM sodium nitroprussiate (SNP), a well-known NO donor, prevented the effects caused by Cd. When the HO-1 mRNA levels were analyzed, a significant augmentation (54%) was observed with respect to Cd-treated plants. On the other hand, 50 or 300 microM SNP did not fully prevent the effects elicited by Cd. When HO-1 transcript levels were analyzed, no significant enhancement or a down-regulation was observed. The potassium salt of 2-(4-carboxylphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO scavenger, arrested NO-mediated protective effects against to Cd-induced oxidative damage. These data provide an understanding of one of the possible roles that NO can play against an oxidative insult. NO is cytoprotective depending on its concentration, and it was further demonstrated that this protection could be, at least in part, mediated by an enhancement of HO-1 mRNA, as it happens with genes associated with the antioxidant defense system.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glycine max/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/genética , Óxido Nítrico/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glycine max/enzimologíaRESUMEN
The behavior of glutathione reductase (GR, EC 1.6.4.2) activity and isoforms were analyzed in wheat (Triticum aestivum L.) leaves and roots exposed to a chronic treatment with a toxic cadmium (Cd) concentration. A significant growth inhibition (up to 55%) was found in leaves at 7, 14 and 21 days, whereas roots were affected (51%) only after three weeks. Wheat plants grown in the presence of 100microM Cd showed a time-dependent accumulation of this metal, with Cd concentration being 10-fold higher in roots than in leaves. Nevertheless, lipid peroxidation was augmented in leaves in all experiments, but not in roots until 21 days. Cadmium treatment altered neither the GR activity nor the isoform pattern in the leaves. However, GR activity increased 111% and 200% in roots at 7 and 14 days, respectively, returning to control levels after 21 days. Three GR isoforms were found in roots of control and treated plants, two of which were enhanced by Cd treatment at 7 and 14 days, as assessed by activity staining on native gels. The changes in the isoform pattern modified the global kinetic properties of GR, thereby decreasing significantly (2.5-fold) the Michaelis constant (K(m)) value for oxidized glutathione. Isozyme induction was not associated with an enhancement of GR mRNA and protein expression, indicating that post-translational modification could occur. Our data demonstrated that up-regulation of GR activity by the induction of distinctive isoforms occurs as a defense mechanism against Cd-generated oxidative stress in roots.
Asunto(s)
Cadmio/toxicidad , Glutatión Reductasa/metabolismo , Isoenzimas/metabolismo , Hojas de la Planta/enzimología , Raíces de Plantas/enzimología , Triticum/enzimología , Cartilla de ADN , Glutatión Reductasa/genética , Isoenzimas/genética , Cinética , Hojas de la Planta/efectos de los fármacos , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , ARN Mensajero/genética , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Triticum/efectos de los fármacosRESUMEN
We have recently reported that in chronic myocardial ischemia, adult mammalian cardiomyocytes express P-glycoprotein (P-gp). We now investigate if P-gp is also expressed in acute regional ischemia followed by reperfusion. Adult conscious sheep underwent 12-min occlusion of the mid-left anterior descending artery (inflatable cuff). Successful ischemia-reperfusion was confirmed by monitoring percent systolic left ventricular anterior wall thickening (sonomicrometry) during the whole ischemic period and every 10 min over 2 hr following cuff deflation. At 3, 24, and 48 hr after reperfusion, P-gp expression was investigated by immunohistochemistry and Western blot and MDR-1 mRNA by RT-PCR. Cardiomyocytes in the occluded artery territory (but not those in remote areas) consistently expressed P-gp at their sarcolemma. Whereas at 3 and 24 hr P-gp was mainly observed in the T tubules, at 48 hr it predominated in intercalated discs and gap junctions. RT-PCR and Western blot revealed higher expression in ischemic than in control myocardium. We conclude that in adult sheep with acute myocardial ischemia, the MDR-1 gene-encoded P-gp is expressed at the sarcolemma of the cardiomyocytes from 3 hr up to at least 48 hr after reperfusion.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Femenino , Genes MDR , Reperfusión Miocárdica , Miocitos Cardíacos/ultraestructura , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ovinos , Factores de TiempoRESUMEN
OBJECTIVES: This study was designed to investigate the association between hypertension and aortic valve stenosis (AVS) in a rabbit model. BACKGROUND: Degenerative AVS is a prevalent disease in elderly persons. Its molecular mechanisms remain unclear, in part because of the absence of experimental models. Epidemiologic data suggest a link between hypertension and AVS. However, there has been no evidence of a cause-effect relationship. METHODS: New Zealand White rabbits were divided into two groups: 1) animals (n = 20) instrumented according to one-kidney/one-clip hypertensive model; and 2) control animals (n = 10) sham operated. Echocardiography (S12 MHz) was used to assess aortic valve (AV) morphology and function as well as left ventricular mass at baseline and after two and four months of hypertension. RESULTS: Blood pressure and left ventricular mass increase were highly significant in the animal model but not in controls at two months, without noticeable AV function abnormalities. After 4 months, however, 14 hypertensive survived animals showed a 14.6% reduction of AV area (0.240 +/- 0.063 cm2 vs. 0.205 +/- 0.060 cm2, p < 0.05), a 19.6% increase of AV thickness (0.056 +/- 0.011 cm vs. 0.067 +/- 0.010 cm, p < 0.001), a 40.4% increase of transvalvular mean gradient (5.35 +/- 2.26 mm Hg vs. 7.51 +/- 3.73 mm Hg, p < 0.05) and a 63.6% increase of transvalvular maximal gradient (10.56 +/- 3.68 mm Hg vs. 17.28 +/- 10.95 mm Hg, p < 0.05). Control animals did not show significant changes. CONCLUSIONS: We report a novel experimental model of AVS in rabbits that may prove useful in studying the progression of the disease and the efficacy of new treatments. The present findings support the hypothesis of a causal link between hypertension and AVS.
Asunto(s)
Estenosis de la Válvula Aórtica/etiología , Hipertensión/complicaciones , Animales , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Estenosis de la Válvula Aórtica/patología , Presión Sanguínea , Progresión de la Enfermedad , Ecocardiografía , Hipertensión/fisiopatología , Conejos , Índice de Severidad de la EnfermedadRESUMEN
Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and leads to the generation of reactive oxygen species (ROS). Heme oxygenase (HO, EC 1.14.99.3) plays a protective role against oxidative stress in mammals, but little is known about this issue in plants. Here, we report for the first time the response of HO in leaves of soybean (Glycine max L.) plants subjected to UV-B radiation. Under 7.5 and 15 kJ m(-2 )UV-B doses, HO, catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities were increased and the production of thiobarbituric acid reactive substances (TBARS) regain control values after 4 h of plant recuperation. Treatment with 30 kJ m(-2) UV-B provoked a decrease in these antioxidant enzyme activities. Immunoblot analysis showed a 4.3 and 3.7-fold increase in HO-1 protein expression after irradiation with 7.5 and 15 kJ m(-2), respectively. HO-1 transcript levels were enhanced (up to 77%) at these doses, as assessed by semi-quantitative RT-PCR. These data demonstrated that increased HO activity was associated with augmented protein expression and transcript levels. Plants pre-treated with the antioxidant ascorbic acid did not show the UV-B-induced up-regulation of HO-1 mRNA, but hydrogen peroxide treatment could mimic this reaction. Our results indicate that HO is up-regulated in a dose-depending manner as a mechanism of cell protection against oxidative damage and that such response occurred as a consequence of HO-1 mRNA enhancement involving ROS.
Asunto(s)
Glycine max/efectos de la radiación , Hemo Oxigenasa (Desciclizante)/efectos de la radiación , Especies Reactivas de Oxígeno/efectos de la radiación , Rayos Ultravioleta , Regulación hacia Arriba , Ascorbato Peroxidasas , Ácido Ascórbico/farmacología , Catalasa/metabolismo , Catalasa/efectos de la radiación , Expresión Génica , Hemo Oxigenasa (Desciclizante)/metabolismo , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/efectos de la radiación , Peroxidasas/metabolismo , Peroxidasas/efectos de la radiación , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Glycine max/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.