RESUMEN
Recent studies suggest that mitochondrial aldehyde dehydrogenase (ALDH-2) plays a central role in the process of nitroglycerin (glyceryl trinitrate, GTN) biotransformation in vivo and that its inhibition accounts for mechanism-based tolerance in vitro. The extent to which ALDH-2 contributes to GTN tolerance (impaired relaxation to GTN) and cross-tolerance (impaired endothelium-dependent relaxation) in vivo remain to be elucidated. Rats were treated for three days with GTN. Infusions were accompanied by decreases in vascular ALDH-2 activity, GTN biotransformation, and cGMP-dependent kinase (cGK-I) activity. Further, whereas in control vessels, multiple inhibitors and substrates of ALDH-2 reduced both GTN-stimulation of cGKI and GTN-induced vasodilation, these agents had little effect on tolerant vessels. A state of functional tolerance (in the GTN/cGMP pathway) was recapitulated in cultured endothelial cells by knocking down mitochondrial DNA (rho(0) cells). In addition, GTN increased the production of reactive oxygen species (ROS) by mitochondria, and these increases were associated with impaired relaxation to acetylcholine. Finally, antioxidants/reductants decreased mitochondrial ROS production and restored ALDH-2 activity. These observations suggest that nitrate tolerance is mediated, at least in significant part, by inhibition of vascular ALDH-2 and that mitochondrial ROS contribute to this inhibition. Thus, GTN tolerance may be viewed as a metabolic syndrome characterized by mitochondrial dysfunction.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Nitroglicerina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vasodilatadores/metabolismo , Animales , Aorta/metabolismo , GMP Cíclico/metabolismo , Tolerancia a Medicamentos , Modelos Animales , Miocardio/metabolismo , Ratas , Ratas WistarRESUMEN
Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS-generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO-releasing compound DETA-NO in a concentration- and time-dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL-1beta on nox1 transcription is counteracted by an inhibitory effect of IL-1beta-evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA-NO attenuated platelet-derived growth factor (PDGF)-BB or serum stimulated production of superoxide as assessed by real-time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine-tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.
Asunto(s)
Dominio Catalítico , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Células Mesangiales/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Óxido Nítrico/metabolismo , Animales , Células Cultivadas , Clonación Molecular , Interleucina-1/farmacología , Células Mesangiales/efectos de los fármacos , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Interferente Pequeño , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sitio de Iniciación de la Transcripción , Triazenos/farmacologíaRESUMEN
During the last century, nitroglycerin has been the most commonly used antiischemic and antianginal agent. Unfortunately, after continuous application, its therapeutic efficacy rapidly vanishes. Neurohormonal activation of vasoconstrictor signals and intravascular volume expansion constitute early counter-regulatory responses (pseudotolerance), whereas long-term treatment induces intrinsic vascular changes, eg, a loss of nitrovasodilator-responsiveness (vascular tolerance). This is caused by increased vascular superoxide production and a supersensitivity to vasoconstrictors secondary to a tonic activation of protein kinase C. NADPH oxidase(s) and uncoupled endothelial nitric oxide synthase have been proposed as superoxide sources. Superoxide and vascular NO rapidly form peroxynitrite, which aggravates tolerance by promoting NO synthase uncoupling and inhibition of soluble guanylyl cyclase and prostacyclin synthase. This oxidative stress concept may explain why radical scavengers and substances, which reduce oxidative stress indirectly, are able to relieve tolerance and endothelial dysfunction. Recent work has defined a new tolerance mechanism, ie, an inhibition of mitochondrial aldehyde dehydrogenase, the enzyme that accomplishes bioactivation of nitroglycerin, and has identified mitochondria as an additional source of reactive oxygen species. Nitroglycerin-induced reactive oxygen species inhibit the bioactivation of nitroglycerin by thiol oxidation of aldehyde dehydrogenase. Both mechanisms, increased oxidative stress and impaired bioactivation of nitroglycerin, can be joined to provide a new concept for nitroglycerin tolerance and cross-tolerance. The consequences of these processes for the nitroglycerin downstream targets soluble guanylyl cyclase, cGMP-dependent protein kinase, cGMP-degrading phosphodiesterases, and toxic side effects contributing to endothelial dysfunction, such as inhibition of prostacyclin synthase, are discussed in this review.
Asunto(s)
Nitroglicerina/farmacología , Vasodilatadores/farmacología , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa Mitocondrial , Animales , Biotransformación , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Tolerancia a Medicamentos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Óxido Nítrico/biosíntesis , Nitroglicerina/uso terapéutico , Estrés Oxidativo , Ácido Peroxinitroso/metabolismo , Hidrolasas Diéster Fosfóricas/fisiología , Especies Reactivas de Oxígeno , Transducción de Señal , Superóxidos/metabolismo , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Nitroglycerin (glyceryl trinitrate, GTN) relaxes blood vessels primarily via activation of the soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK-I) pathway. Although the precise mechanism of sGC activation by GTN in the vascular wall is unknown, the mediatory role of nitric oxide (NO) has been postulated. We tested the GTN/NO hypothesis in different types of isolated rat and rabbit blood vessels using two novel approaches: (1) EPR spin trapping using colloid Fe(DETC)2 and (2) analysis of cGK-I-dependent phosphorylation of the vasodilator-stimulated phosphoprotein at Ser239 (P-VASP). For comparison, another organic nitrate, isosorbide dinitrate (ISDN), and endothelium-dependent vasodilator, calcium ionophore A23187, were tested. We found a marked discrepancy between GTN's strong vasoactivity (vasodilation and augmentation of P-VASP) and its poor NO donor properties. In aortas precontracted with phenylephrine, GTN, ISDN, and A23187 induced nearly full relaxations (>80%) and doubling of vascular P-VASP content at concentrations of 100 nmol/L, 100 micromol/L, and 1 micromol/L, respectively. GTN applied in vasorelaxant concentrations (10 to 1000 nmol/L) did not significantly increase the basal vascular NO production, in contrast to ISDN and A23187. The absence of GTN-derived NO was confirmed in rabbit vena cava and renal artery. A significant increase in vascular NO formation was observed only at suprapharmacological GTN concentrations (>10 micromol/L). The concentration dependency of NO formation from GTN was comparable to that of ISDN, although the latter exhibits 100-folds lower vasorelaxant potency. We conclude that GTN activates the sGC/cGMP/cGK-I pathway and induces vasorelaxation without intermediacy of the free radical NO. The full text of this article is available online at http://www.circresaha.org.
Asunto(s)
Óxido Nítrico/metabolismo , Nitroglicerina/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Benomilo/farmacología , Calcimicina/farmacología , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa , Técnicas In Vitro , Ionóforos/farmacología , Dinitrato de Isosorbide/farmacología , Masculino , Proteínas de Microfilamentos , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Conejos , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiología , Guanilil Ciclasa Soluble , Detección de Spin , Vasoconstrictores/farmacología , Venas Cavas/efectos de los fármacos , Venas Cavas/fisiologíaRESUMEN
Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolemia, hypertension, diabetes mellitus, chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species (ROS), such as the superoxide radical, and the subsequent decrease in vascular bioavailability of nitric oxide (NO). Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include the NAD(P)H oxidase, the xanthine oxidase, and mitochondrial superoxide-producing enzymes. Superoxide produced by the NADPH oxidase may react with NO released by endothelial nitric oxide synthase (eNOS), thereby generating peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, thereby switching an antiatherosclerotic NO-producing enzyme to an enzyme that may initiate or even accelerate the atherosclerotic process by producing superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and has also been demonstrated to occur within the smooth muscle cell layer in the setting of hypercholesterolemia, diabetes mellitus, hypertension, congestive heart failure, and nitrate tolerance. Increased superoxide production by the endothelial and/or smooth muscle cells has important consequences with respect to signaling by the soluble guanylyl cyclase (sGC) and the cGMP-dependent protein kinase I (cGK-I), the activity and expression of which has been shown to be regulated in a redox-sensitive fashion. The present review summarizes current concepts concerning eNOS uncoupling and also focuses on the consequences for downstream signaling with respect to activity and expression of the sGC and cGK-I in various diseases.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Endotelio Vascular/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Guanilato Ciclasa , Humanos , Guanilil Ciclasa SolubleRESUMEN
The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Células Endoteliales/enzimología , Miocardio/metabolismo , NADH NADPH Oxidorreductasas/biosíntesis , NADPH Oxidasas/biosíntesis , Acetilcolina/farmacología , Animales , Aorta/enzimología , Western Blotting , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Proteínas de Microfilamentos , Miocardio/química , NADPH Oxidasa 1 , NADPH Oxidasa 4 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Nitroglicerina/farmacología , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Ratas , Ratas Wistar , Superóxidos/metabolismo , Vasodilatadores/farmacología , Xantina Oxidasa/metabolismoRESUMEN
The hemodynamic and anti-ischemic effects of nitroglycerin (NTG) are rapidly blunted as a result of the development of nitrate tolerance. With initiation of NTG therapy, it is possible to detect neurohormonal activation and intravascular volume expansion. These so-called pseudotolerance mechanisms may compromise the vasodilatory effects of NTG. Long-term nitrate treatment also is associated with decreased vascular responsiveness caused by changes in intrinsic mechanisms of the tolerant vasculature itself. According to the oxidative stress concept, increased vascular superoxide (O2-) production and an increased sensitivity to vasoconstrictors secondary to activation of protein kinase C contribute to the development of tolerance. Nicotinamide adenine dinucleotide phosphate oxidase and the uncoupled endothelial nitric oxide synthase may be O2- -producing enzymes. Nitric oxide (NO) and O2-, both derived from NTG and the vessel wall, form peroxynitrite in a diffusion-limited rapid reaction. Peroxynitrite, O2-, or both may be responsible for the development of nitrate tolerance and cross-tolerance to direct NO donors (eg, sodium nitroprusside, sydnonimines) and endothelium-dependent NO synthase-activating vasodilators. Hydralazine is an efficient reactive oxygen species (ROS) scavenger and an inhibitor of O2- generation. When given concomitantly with NTG, hydralazine prevents the development of nitrate tolerance and normalizes endogenous rates of vascular O2- production. Recent experimental work has defined new tolerance mechanisms, including inhibition of the enzyme that bioactivates NTG (ie, mitochondrial aldehyde dehydrogenase isoform 2 [ALDH2]) and mitochondria as potential sources of ROS. NTG-induced ROS inhibit the bioactivation of NTG by ALDH2. Both mechanisms increase oxidative stress and impair NTG bioactivation, and now converge at the level of ALDH2 to support a new theory for NTG tolerance and NTG-induced endothelial dysfunction. The consequences of these processes for NTG downstream targets (eg, soluble guanylyl cyclase, cyclic guanosine monophosphate-dependent protein kinase), toxic effects contributing to endothelial dysfunction (eg, prostacyclin synthase inhibition) and novel applications of the antioxidant properties of hydralazine are discussed.
Asunto(s)
Resistencia a Medicamentos , Hidralazina/administración & dosificación , Isquemia Miocárdica/tratamiento farmacológico , Nitroglicerina/administración & dosificación , Nitroglicerina/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Animales , Ensayos Clínicos como Asunto , Angiografía Coronaria , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Interacciones Farmacológicas , Endotelio Vascular/efectos de los fármacos , Prueba de Esfuerzo , Femenino , Pruebas de Función Cardíaca , Humanos , Hidralazina/efectos adversos , Cuidados a Largo Plazo , Masculino , Dosis Máxima Tolerada , Isquemia Miocárdica/diagnóstico , Isquemia Miocárdica/mortalidad , Índice de Severidad de la Enfermedad , Resistencia Vascular/efectos de los fármacosRESUMEN
OBJECTIVE: The Ca2+ antagonist amlodipine increases the generation of nitric oxide (NO) from native and cultured endothelial cells. The aim of this investigation was to determine whether or not the activation of the endothelial NO synthase (eNOS) by this Ca2+ antagonist is related to alterations in eNOS phosphorylation. METHODS AND RESULTS: In isolated, pre-contracted, endothelium-intact porcine coronary arteries, amlodipine elicited an NO-mediated relaxation and a leftward shift in the concentration-relaxation curve to bradykinin. Moreover, the Ca2+ antagonist increased the generation of NO from native endothelial cells, as detected by electron spin resonance spectroscopy and stimulated an 8-fold increase in cyclic GMP levels in cultured endothelial cells. In unstimulated endothelial cells, eNOS was not phosphorylated on Ser1177 but was phosphorylated on Thr495. Amlodipine elicited the phosphorylation of Ser1177 and attenuated Thr495 phosphorylation, with a time course similar to that of eNOS activation. The amlodipine-induced relaxation of porcine coronary arteries was attenuated by the B2 kinin receptor antagonist, icatibant, but this antagonist did not affect amlodipine-induced changes in eNOS phosphorylation in cultured endothelial cells. Moreover, amlodipine elicited the NO-mediated relaxation of rat aortic rings which do not express the B2 receptor. Amlodipine time-dependently attenuated the phosphorylation of protein kinase C (PKC) in endothelial cells, with a time course similar to the changes in eNOS phosphorylation, and prevented the phorbol-12-myristate-13-acetate-induced activation of PKC. The PKC inhibitor, Ro 31-8220, also elicited the phosphorylation of Ser1177 and the dephosphorylation of Thr495 in cultured cells and induced a leftward shift in the concentration-relaxation curve to bradykinin in rings of porcine coronary artery. CONCLUSION: The Ca2+ antagonist, amlodipine, enhances endothelial NO generation by inducing changes in the phosphorylation of eNOS. Although the activation of eNOS was related to the activation of the B2 kinin receptor in the porcine coronary artery, a B2 receptor-independent mechanism involving the inhibition of PKC appears to account for the effects observed in the rat aorta as well as in cultured endothelial cells.
Asunto(s)
Amlodipino/farmacología , Bradiquinina/análogos & derivados , Bloqueadores de los Canales de Calcio/farmacología , Células Endoteliales/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Aorta , Bradiquinina/farmacología , Antagonistas del Receptor de Bradiquinina B2 , Células Cultivadas , Vasos Coronarios , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Humanos , Técnicas In Vitro , Indoles/farmacología , Óxido Nítrico/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Ratas Endogámicas WKY , Serina/metabolismo , Porcinos , Treonina/metabolismo , Venas Umbilicales , Vasodilatación/efectos de los fármacosAsunto(s)
Plaquetas/metabolismo , Nitroglicerina/farmacología , Superóxidos/metabolismo , Vasodilatadores/farmacología , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Tolerancia a Medicamentos , Cardiopatías/tratamiento farmacológico , Humanos , NADPH Oxidasas/metabolismo , Nitratos/uso terapéutico , Óxido Nítrico/biosíntesis , Nitroglicerina/uso terapéutico , Inhibidores de Agregación Plaquetaria/farmacología , Vasodilatadores/uso terapéuticoAsunto(s)
Vasos Sanguíneos/fisiología , Vasos Sanguíneos/fisiopatología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Transducción de Señal/fisiología , Animales , Plaquetas/metabolismo , Vasos Sanguíneos/enzimología , GMP Cíclico/metabolismo , Endotelio Vascular/enzimología , Endotelio Vascular/fisiología , Endotelio Vascular/fisiopatología , Humanos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/fisiología , Músculo Liso Vascular/fisiopatología , Estrés Oxidativo , Fosforilación , Sistema Vasomotor/fisiologíaRESUMEN
Oxidative stress and protein aggregation are biochemical hallmarks of Parkinson's disease (PD), a frequent sporadic late-onset degenerative disorder particularly of dopaminergic neurons in the substantia nigra, resulting in impaired spontaneous movement. PARK6 is a rare autosomal-recessively inherited disorder, mimicking the clinical picture of PD with earlier onset and slower progression. Genetic data demonstrated PARK6 to be caused by mutations in the protein PINK1, which is localized to mitochondria and has a serine-threonine kinase domain. To study the effect of PINK1 mutations on oxidative stress, we used primary fibroblasts and immortalized lymphoblasts from three patients homozygous for G309D-PINK1. Oxidative stress was evident from increases in lipid peroxidation and in antioxidant defenses by mitochondrial superoxide dismutase and glutathione. Elevated levels of glutathione reductase and glutathione-S-transferase were also observed. As a putative cause of oxidation, a mild decrease in complex I activity and a trend to superoxide elevation were detectable. These data indicate that PINK1 function is critical to prevent oxidative damage and that peripheral cells may be useful for studies of progression and therapy of PARK6.
Asunto(s)
Glutatión/metabolismo , Peroxidación de Lípido/genética , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/metabolismo , Adulto , Línea Celular Transformada , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Femenino , Fibroblastos , Humanos , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/fisiopatología , Proteínas Quinasas/genética , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismoRESUMEN
In the present study we compared the vascular reactivity and integrity of the nitric oxide (NO)-cyclic 3',5'-guanosine monophopsphate (cGMP) pathway in carotid arteries of hyper- and normolipidemic rabbits. Vasodilation to acetylcholine, nitroglycerin, and sodium nitroprusside was desensitized in hyperlipidemia, but the nitroprusside-induced relaxation was normalized by an NO synthase inhibitor in endothelium-intact and -denuded vessels. Hyperlipidemic carotid arteries exhibited increased basal NO (detected by EPR spin-trapping) and reactive oxygen species formation (detected by chemiluminescence), whereas acetylcholine-induced NO formation was nearly abolished. Hyperlipidemia increased NADPH-dependent superoxide formation in carotid membranes, and carotid cryosections stained with the fluorescent dye dihydroethidium revealed increased endothelial and medial reactive oxygen species formation. Hyperlipidemia elicited macrophage invasion into the carotid wall, as detected by a dot-immunoblot. The basal activity of cGMP-dependent proteinkinase, the nitroprusside-stimulated activity of soluble guanylyl cyclase, and its protein expression were decreased by hyperlipidemia. The cGMP phosphodiesterase activity was marginally increased by hyperlipidemia, such that the ratio of cGMP-forming vs. -degrading capacity was decreased by 2-fold. Hyperlipidemia triggers infiltration of macrophages into the carotid wall and endothelial as well as smooth muscle superoxide formation. Consequently, relaxation of the carotid arteries are impaired due to smooth muscle and endothelial dysfunction.
Asunto(s)
Arterias Carótidas/fisiología , Arterias Carótidas/fisiopatología , Hiperlipidemias , Animales , Arterias Carótidas/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2 , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/metabolismo , Macrófagos/metabolismo , Proteínas de Microfilamentos/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Fosfoproteínas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Conejos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/farmacologíaRESUMEN
Nebivolol is a beta(1)-receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of angiotensin II-induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin-NO concentrations. Treatment with the beta-receptor blocker nebivolol but not metoprolol (10 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH oxidase, as well as membrane association of NADPH oxidase subunits (Rac1 and p67(phox)). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67(phox) and Rac1, as well as an inhibition of NADPH oxidase activity assessed in heart membranes from angiotensin II-infused animals, as well as in homogenates of Nox1 and cytosolic subunit-transfected and phorbol ester-stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH oxidase. Thus, inhibitory effects of this beta-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II-induced hypertension.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Angiotensina II/farmacología , Benzopiranos/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Etanolaminas/farmacología , NADPH Oxidasas/metabolismo , Superóxidos/antagonistas & inhibidores , Superóxidos/metabolismo , Acridinas , Animales , Vasos Sanguíneos/metabolismo , Línea Celular , GMP Cíclico/metabolismo , Dicarbetoxidihidrocolidina/análogos & derivados , Fluorescencia , Hemoglobinas/metabolismo , Humanos , Luminiscencia , Sustancias Luminiscentes , Luminol/análogos & derivados , Masculino , Miocardio/metabolismo , Nebivolol , Óxido Nítrico/sangre , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/sangre , Fosfoproteínas/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The heme-enzyme soluble guanylyl cyclase (sGC) is an ubiquitous NO receptor, which mediates NO downstream signaling by the generation of cGMP. We studied the mechanism of action of the anthranilic acid derivatives 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (HMR1766) (proposed international nonproprietary name, ataciguat sodium) and 2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (S3448) as a new class of sGC agonists. Both compounds activated different sGC preparations (purified from bovine lung, or crude from human corpus cavernosum) in a concentration-dependent and quickly reversible fashion (EC50 = 0.5-10 microM), with mixed-type activation kinetics. Activation of sGC by these compounds was additive to activation by NO donors, but instead of being inhibited, it was potentiated by the heme-iron oxidants 1H-[1,2,4]-oxdiazolo[3,4-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), suggesting that the new compounds target the ferric heme sGC isoform. Protoporphyrin IX acted as a competitive activator, and zinc-protoporphyrin IX inhibited activation of heme-oxidized sGC by HMR1766 and S3448, whereas heme depletion of sGC by Tween 20 treatment reduced activation. Both compounds increased cGMP levels in cultured rat aortic smooth muscle cells; induced vasorelaxation of isolated endothelium-denuded rat aorta, porcine coronary arteries, and human corpus cavernosum (EC50 1 to 10 microM); and elicited phosphorylation of the cGMP kinase substrate vasodilator-stimulated phosphoprotein at Ser239. HMR1766 intravenous bolus injection decreased arterial blood pressure in anesthetized pigs. All of these pharmacological responses to the new compounds were enhanced by ODQ and NS2028. Our findings suggest that HMR1766 and S3448 preferentially activate the NO-insensitive heme-oxidized form of sGC, which exists to a variable extent in vascular tissues, and is a pharmacological target for these new vasodilator drugs.
Asunto(s)
Guanilato Ciclasa/metabolismo , Hemo/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , ortoaminobenzoatos/metabolismo , ortoaminobenzoatos/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Bovinos , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Activación Enzimática , Oxidación-Reducción , Vasodilatadores/metabolismoRESUMEN
In aged spontaneously hypertensive rats (SHR), vasorelaxant responses to NO are attenuated compared with normotensive control rats (Wistar-Kyoto [WKY]) because of a decreased expression of the important NO receptor soluble guanylyl cyclase (sGC). Because the expression of sGC subunits alpha1 and beta1 is controlled at the post-transcriptional level by the mRNA-binding protein human-antigen R (HuR), we now assessed whether or not altered expression of HuR could account for downregulation of sGCalpha1 and sGCbeta1 in genetic hypertension. The expression of HuR (and sGCalpha1 and sGCbeta1) in aortas from aged SHR was significantly decreased at the mRNA and protein level compared with age-matched WKY rats, whereas expression of HuR was not different in prehypertensive young (2 months of age) SHR and age-matched WKY rats. The mRNA-binding activity of HuR in native aortic protein extracts from aged SHR was markedly reduced compared with normotensive WKY rats, as detected by RNA electrophoretic mobility shift analysis, using biotin-labeled adenine and uracil (AU)-rich element (ARE)-containing RNA probes from the 3'-untranslated region of sGCalpha1 and sGCbeta1. In contrast, ARE-binding activity was not different between prehypertensive young SHR and young WKY rats. In vitro RNA degradation assays using the same AU-rich sGC mRNA probes revealed an accelerated sGCalpha1 and sGCbeta1 mRNA decay in the presence of native protein extract from hypertensive SHR, which was less rapid with aortic protein from normotensive WKY rats. These findings suggest that in this animal model of genetic hypertension, the reduced expression of sGC subunits is mediated by downregulation of the sGC mRNA-stabilizing protein HuR.
Asunto(s)
Antígenos de Superficie/metabolismo , Regulación hacia Abajo , Hipertensión/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/metabolismo , Envejecimiento/metabolismo , Animales , Antígenos de Superficie/genética , Aorta/química , Aorta/metabolismo , Enfermedad Crónica , Proteínas ELAV , Proteína 1 Similar a ELAV , Electroforesis , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Hipertensión/genética , Técnicas In Vitro , Masculino , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Proteínas Recombinantes/metabolismo , Solubilidad , Extractos de Tejidos/farmacologíaRESUMEN
Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study, we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knock-out mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Vasodilator potency of mouse aorta in response to acetylcholine and GTN was assessed by isometric tension studies. Mitochondrial ROS formation was detected by 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-enhanced chemiluminescence and mitochondrial aldehyde dehydrogenase (ALDH-2) activity was determined by a high-performance liquid chromatography-based assay. Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or long-term GTN infusion caused a marked higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- compared with wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice, correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN treatment. The data presented indicate that deficiency in Mn-SOD leads to a higher degree of tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of ROS to these processes.
Asunto(s)
Heterocigoto , Nitroglicerina/farmacología , Estrés Oxidativo , Superóxido Dismutasa/fisiología , Aldehído Deshidrogenasa/metabolismo , Animales , Aorta/enzimología , Tolerancia a Medicamentos , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/enzimología , Superóxido Dismutasa/genéticaRESUMEN
We analyzed whether the cyclic AMP induced down-regulation of the nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is mediated by the mRNA-protecting protein HuR. Exposure (up to 24 h) of isolated rat aortic segments to the activator of adenylyl cyclase, forskolin (10 microM), and to both activators of cAMP-stimulated protein kinase (PKA), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Spisomer (Sp-5,6-DCl-cBIMPS; 400 nM), and N6-phenyl-cAMP (10 microM), strongly reduced sGCalpha1beta1 and HuR protein and mRNA expression in a time-dependent and actinomycin D (10 microM)-sensitive fashion. In vitro degradation of sGCalpha1 and beta1 poly(A)+ mRNA by native rat aortic protein was markedly increased by pretreatment of intact aortas with forskolin. Native protein extract from rat aorta shifted the electrophoretic mobility of biotin-labeled riboprobes from the 3'-untranslated region of sGCalpha1 and beta1 mRNA, and these bands was supershifted by a monoclonal antibody directed against the mRNA-stabilizing protein HuR. Forskolin decreased the HuR-sGCalpha1 and beta1 mRNA interaction and HuR protein expression in rat aorta, and this was prevented by the PKA inhibitory cAMP analog 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In cultured smooth muscle cells from rat aorta, forskolin induced a rapid increase in Fos/p-Fos protein levels and activator protein 1 (AP-1) binding activity. Inhibition of this transcription factor by an AP-1 decoy prevented the forskolin-induced down-regulation of HuR. We conclude that forskolin/cAMP decrease the expression of heterodimeric sGC in rat aortic smooth muscle cells via activation of Fos/AP-1, which decreases the expression of HuR and thus destabilizes the sGCalpha1 and beta1 mRNA.
Asunto(s)
Antígenos de Superficie/metabolismo , AMP Cíclico/farmacología , Expresión Génica/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Estabilidad del ARN/fisiología , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/efectos de los fármacos , Regiones no Traducidas 3'/metabolismo , Adenilil Ciclasas , Animales , Aorta , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/análogos & derivados , Dactinomicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Interacciones Farmacológicas , Proteínas ELAV , Proteína 1 Similar a ELAV , Regulación de la Expresión Génica , Genes fos , Guanilato Ciclasa/genética , Masculino , Estabilidad del ARN/efectos de los fármacos , Ratas , Ratas Wistar , Factor de Transcripción AP-1/metabolismoRESUMEN
We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.
Asunto(s)
Antígenos de Superficie , Aorta/enzimología , Procesamiento Postranscripcional del ARN , Receptores Citoplasmáticos y Nucleares/biosíntesis , Regiones no Traducidas 3' , Animales , Aorta/metabolismo , Aorta/patología , Secuencia de Bases , Northern Blotting , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Dactinomicina/farmacología , Regulación hacia Abajo , Proteínas ELAV , Proteína 1 Similar a ELAV , Activadores de Enzimas/farmacología , Guanilato Ciclasa , Indazoles/farmacología , Masculino , Datos de Secuencia Molecular , Músculo Liso/citología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oxadiazoles/farmacología , Oxazinas/farmacología , Poli A/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Guanilil Ciclasa Soluble , Factores de Tiempo , Transcripción Genética , TransfecciónRESUMEN
Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN- and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations >1 microM. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.