RESUMEN
INTRODUCTION: Metabolic syndrome induces endothelial dysfunction, a surrogate marker of cardiovascular disease. In parallel, metabolic syndrome is frequently associated with non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. The aim of the present study was to evaluate intrahepatic endothelial dysfunction related to cyclooxygenase end products and oxidative stress as possible mechanisms involved in the pathophysiology of NAFLD. MATERIALS AND METHODS: Sprague-Dawley rats were fed standard diet (control-diet, CD) or high-fat-diet (HFD) for 6 weeks. Metabolic syndrome was assessed by recording arterial pressure, lipids, glycemia and rat body weight. Splanchnic hemodynamics were measured, and endothelial dysfunction was evaluated using concentration-effect curves to acetylcholine. Response was assessed with either vehicle, L-NG-Nitroarginine (L-NNA), indomethacin, tempol, or a thromboxane receptor antagonist, SQ 29548. We quantified inflammation, fibrosis, oxidative stress, nitric oxide (NO) bioavailability and thromboxane B2 levels. RESULTS: HFD rats exhibited metabolic syndrome together with the presence of NAFLD. Compared to control-diet livers, HFD livers showed increased hepatic vascular resistance unrelated to inflammation or fibrosis, but with decreased NO activity and increased oxidative stress. Endothelial dysfunction was observed in HFD livers compared with CD rats and improved after cyclooxygenase inhibition or tempol pre-incubation. However, pre-incubation with SQ 29548 did not modify acetylcholine response. CONCLUSIONS: Our study provides evidence that endothelial dysfunction at an early stage of NAFLD is associated with reduced NO bioavailability together with increased cyclooxygenase end products and oxidative stress, which suggests that both pathways are involved in the pathophysiology and may be worth exploring as therapeutic targets to prevent progression of the disease.
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Endotelio/metabolismo , Óxido Nítrico/sangre , Enfermedad del Hígado Graso no Alcohólico/sangre , Estrés Oxidativo , Circulación Esplácnica , Tromboxano B2/sangre , Acetilcolina/farmacocinética , Acetilcolina/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes , Óxidos N-Cíclicos/farmacocinética , Óxidos N-Cíclicos/farmacología , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Endotelio/patología , Endotelio/fisiopatología , Ácidos Grasos Insaturados , Hidrazinas/farmacocinética , Hidrazinas/farmacología , Indometacina/farmacocinética , Indometacina/farmacología , Masculino , Nitroarginina/farmacocinética , Nitroarginina/farmacología , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Ratas , Ratas Sprague-Dawley , Marcadores de SpinRESUMEN
Introducción: La L-arginina (L-Arg) es un aminoácido semiesencial y precursor de la síntesis de óxido nítrico (NO). Recientemente, los suplementos nutricionales que contiene L-Arg son comercializados con la pretensión de promover vasodilatación, debido al aumento de la producción de NO en músculo. El resultado de la vasodilatación elevaría la perfusión sanguínea, promoviendo un mayor aporte de nutrientes y oxigeno, los cuales pueden mejorar el rendimiento y la recuperación muscular. Propósito: Identificar el efecto agudo de la suplementación con L-Arg sobre la ratio de recuperación del trabajo (WRR), potencia media, trabajo total (TW3S) y los indicadores de producción de NO, nitrito y nitrato plasmático (NOx), durante el ejercicio de contra-resistencia. Métodos: Diecisiete hombres sanos y entrenados, participaron en un estudio, doble ciego, controlado con placebo. Se tomaron muestras de sangre antes y 90 min después de la ingesta de 6 g de LArg o placebo. El protocolo de ejercicio (3 series de 10 repeticiones máximas de extensión de codo isocinético concéntrico en 60º.s-1 con 2 min de descanso entre las series) se inició 80 minutos después de la suplementación. Mediciones de NOx se realizaron por el método de Griess usando un espectrofotómetro de absorción a 540 nm. Resultados: No se encontraron diferencias significativas entre el grupo suplementado con L-Arg vs placebo en lo referente parámetros de WRR, potencia media y TW3S (2630,4 ± 758,0vs 2573,1 ± 669,9 Joules). Además, no se observó diferenciasignificativa en el NOx plasmático, en ningún momento, entre el grupo suplementado con L-Arg vs placebo, antes de la suplementación (9,8 ± 2,3vs 9,5 ± 1,4 micro mol/L) o inmediatamente después del ejercicio (11,9 ± 5,5 vs 10,2 ±2,3 micro mol/L). Conclusión: Nuestros resultados indican que la ingesta aguda de L-Arg no aumenta la producción de NO, ni mejora el rendimiento recuperación muscular. En base a estos resultados, es precipitado recomendar suplementos nutricionales que contienen L-Arg como ayuda ergogénica para optimizar la recuperación muscular después del ejercicio de contra-resistencia en individuos sanos y entrenados (AU)
Introduction: L-arginine (L-Arg) is a semi-essential aminoacid precursor to nitric oxide (NO) synthesis. Recently, nutritional supplements containing L-Arg have been marketed with the purpose of promote vasodilation, due to an increased production of NO in the exercising muscle. The resulting vasodilation would elevate blood perfusion, leading to a higher nutrient and oxygen delivery, which may enhance exercise performance and muscular recovery. Purpose: Identify the acute effect of L-Arg supplementation on work recovery ratio (WRR), average power, total work (TW3S) and indicators of NO production, plasma nitrite and nitrate(NOx), during a resistance exercise protocol. Methods: Seventeen healthy and resistance-trained males participated in a randomized, double-blind, placebo-controlled study. Blood samples were collected before and 90 min (immediately post-exercise) after ingestion of oral 16g of L-Arg or placebo. The exercise protocol (3 sets of 10 maximal voluntary contractions of isokinetic concentric elbow extension at 60 o.s-1with 2-min of rest between sets) was initiated 80 min after supplementation. NOx measurements were made by a traditional Griess reaction colorimetric method using a spectrophotometer monitoring absorbance at 540 nm. Results: No significant difference between L-Arg versus placebo supplemented groups was observed on WRR, average power and TW3S (2630.4 ± 758.0 versus 2573.1 ± 669.9 Joules). Furthermore, no significant difference was observed in plasma NOx at any time point between L-Arg versus placebo supplemented groups at baseline (9.8 ± 2.3 vs. 9.5 ± 1.4 micre mol/L) and immediately post-exercise (11.9 ± 5.5 vs. 10.2 ±2.3 micremol/L). Conclusion: Our data indicates that acute ingestion of L-Arg does not increase NO production nor enhances muscle performance and recovery. Based on this fact, it is still premature to recommend nutritional supplements containing L-Arg as an ergogenic aid to optimize muscle recovery after resistance exercise bouts in healthy and resistance-trained subjects (AU)
Asunto(s)
Humanos , Nitroarginina/farmacocinética , Óxido Nítrico , Relajación Muscular , Músculos/lesiones , Aminoácidos/farmacocinética , Ejercicio Físico/fisiología , Traumatismos en Atletas/rehabilitaciónRESUMEN
La dimetilarginina asimétrica (ADMA) es un inhibidor competitivo delgrupo enzimático óxido nítrico (NO) sintetasa, que cataliza la conversióndel aminoácido L-arginina en L-citrulina y NO. Esta competenciase debe a la estrecha similitud entre la composición química de la Largininay la molécula de ADMA (2 grupos metilos en el grupo aminoterminalde la L-arginina). Concentraciones elevadas de ADMA puedenbloquear la síntesis de NO, molécula antiaterógena endógena porexcelencia, debido a su función reguladora del endotelio vascular. Lasdisminuciones del NO favorecen la disfunción endotelial causada porfactores de riesgo cardiovascular, inflamaciones o alteraciones metabólicas.Se han publicado estudios clínicos prospectivos que señalanque la ADMA es un nuevo marcador de riesgo cardiovascular capaz depredecir de forma independiente nuevos eventos. Más recientementese han descrito variaciones intraindividuales de la ADMA en personassanas que inducen a seguir investigando sobre el valor diagnóstico ypronóstico de este potencial y novedoso marcador de riesgo cardiovascular
The amino acid L-arginine is the substrate for endothelial nitric oxidesynthesis. The endothelium is capable of producing asymmetric dimethylargine(ADMA) (L-arginine methylated). ADMA is able to competewith L-arginine in nitric oxide (NO) production and inhibits oxidenitric synthase activity. Elevated blood levels of ADMA can block thesynthesis of NO, and induce endothelial dysfunction, which may leadto the initiation and progression of atherosclerosic vascular disease.Prospective clinical studies in different patients populations suggestthat ADMA is a new marker in cardiovascular disease and is to able topredict new cardiovascular events. Recently, intraindividual variationsof ADMA in healthy subjects have been described. This fact inducesto continue studying the diagnosis and prognosis value of this potentialand novel marker of cardiovascular risk
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Humanos , Enfermedades Cardiovasculares/epidemiología , Biomarcadores/análisis , Nitroarginina/farmacocinética , Factores de Riesgo , Ajuste de Riesgo , Inhibidores Enzimáticos/farmacocinética , Óxido Nítrico/antagonistas & inhibidores , Endotelio Vascular/fisiopatologíaRESUMEN
The influence of verapamil on stress-induced and histamine-induced gastric ulcers was investigated in rats. The influence of verapamil was also examined on various biochemical parameters that affect the development of these ulcer models. The animals were pretreated with intraperitoneal verapamil (1, 5, 25 mg/kg) by injection 1 h before the induction of experimental ulceration. The gastric lesions were induced by cold-restraint stress or intraperitoneal injection of histamine (300 mg/kg). The gastroprotective effects of verapamil were evaluated by determining the ulcer index, gastric mucus content, free and total acidity, lipid peroxidation and non-protein sulfhydryl content. Verapamil pretreatment at a dose of 25 mg/kg significantly reduced stress-induced ulcers. Verapamil enhanced mucus secretion, reduced total acidity and lipid peroxidation and decreased non-protein sulfhydryl content in a dose-dependent fashion. On the other hand, pretreatment with verapamil at any dose had no significant effect on histamine-induced ulcers. L-Arginine (L-A) (100 mg/kg) or L-nitroarginine (L-NNA) (100 mg/kg) were also injected i.p. to the animals 1 h before stress to test the role of nitric oxide (NO) in the mechanism of the gastroprotective activity of verapamil (25 mg/kg). The results suggested that verapamil stimulates gastric NO production, but the overproduction of NO worsens gastric ulcers. The effects of verapamil on experimentally induced ulcers may be related to its ability to induce biochemical alterations in the parameters measured in gastric tissue.
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Histamina/efectos adversos , Úlcera Gástrica/etiología , Estrés Fisiológico/complicaciones , Verapamilo/uso terapéutico , Animales , Arginina/administración & dosificación , Arginina/farmacocinética , Arginina/uso terapéutico , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Evaluación Preclínica de Medicamentos/métodos , Quimioterapia Combinada , Determinación de la Acidez Gástrica , Mucinas Gástricas/efectos de los fármacos , Mucinas Gástricas/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/fisiopatología , Histamina/administración & dosificación , Inyecciones Intraperitoneales , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Nitroarginina/administración & dosificación , Nitroarginina/farmacocinética , Ratas , Úlcera Gástrica/tratamiento farmacológico , Estrés Fisiológico/fisiopatología , Compuestos de Sulfhidrilo/metabolismo , Verapamilo/farmacologíaRESUMEN
This study was designed to determine whether recombinant human erythropoietin (rHuEpo) administration increases vascular nitric oxide (NO) production in healthy rats. We hypothesized that rHuEpo hypertension is associated with increased endothelial expression of nitric oxide synthase and augmented NO-dependent vasodilation. Male rats were instrumented with pulsed Doppler flow probes around their ascending aorta and with arterial and femoral catheters. Rats were treated for 14 days with rHuEpo (2 U/d) or vehicle. rHuEpo elevated hematocrit and increased mean arterial pressure (142 +/- 3 versus 116 +/- 4 mm Hg). Thoracic aorta segments from rHuEpo rats had a modest increase in NO-dependent relaxation assessed by acetylcholine (10(-10) to 10(-5) mol/L) relaxation of phenylephrine (PE) (10(-6) mol/L) contracted arteries. Relaxation to NO-donor, s-nitrosyl acetylpenicillamine, and PE contraction were not different from control arteries. The NO synthase inhibitor, N-omega-nitro-L-arginine, increased blood pressure and total peripheral resistance more in rHuEpo rats at both 10 and 30 mg/kg. NOS expression in rHuEpo aorta and plasma NOx concentrations were increased compared with control. Thus, it appears that vascular eNOS expression is increased and causes basal vasodilation in rHuEpo hypertensive rats.
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Endotelio Vascular/efectos de los fármacos , Eritropoyetina/administración & dosificación , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/efectos de los fármacos , Animales , Aorta Torácica/química , Aorta Torácica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Eritropoyetina/farmacocinética , Hemodinámica , Riñón/química , Riñón/efectos de los fármacos , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Nitratos/sangre , Óxido Nítrico/biosíntesis , Óxido Nítrico/farmacocinética , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitritos/sangre , Nitroarginina/administración & dosificación , Nitroarginina/farmacocinética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , Vasodilatación/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the involvement of immunoreactive-dynorphin A in the inhibitory effect of N-nitro-L-arginine on the morphine physical dependence in rats. METHODS: The rats were rendered dependent on morphine by subcutaneous administration of morphine solution three times daily in a manner of dose increment of 5 mg.kg(-1) for 6 days. The degree of morphine physical dependence was monitored by scoring the abstinence syndromes precipitated by 5 mg.kg(-1) naloxone of the rats. The expression levels of immunoreactive dynorphin A in tissues were determined using a radioimmunoassay. RESULTS: Intraperitoneal injection of 5 mg.kg(-1) N-nitro-L-arginine suppresses most of the withdrawal symptoms of morphine dependent rats. N-nitro-L-arginine can elevate the expression of immunoreactive dynorphin. CONCLUSIONS: Chronic N-nitro-L-arginine administration can inhibit the development of morphine physical dependence in a manner of dose-dependence, which is significantly related to its role of regulating the endogeneous dynorphin system.
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Dinorfinas/fisiología , Dependencia de Morfina/prevención & control , Nitroarginina/farmacología , Nitroarginina/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Ischaemic stroke is characterised by reduction of blood flow, tissue hypoxia, energy depletion and neuronal death. Drugs causing vasodilatation of cerebral arteries may potentially enhance blood supply to the ischaemic area and improve clinical outcome. However, vasodilators could also reduce cerebral blood flow in the ischaemic region by acting on blood vessels in non-ischaemic tissue, a phenomenon known as blood flow steal. To explore whether these drugs could act selectively on cerebral blood vessels in a hypoxic environment, we examined the effect of hypoxia on vasodilator responses to the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) and the ATP-dependent potassium channel (K(ATP)) opener levcromakalim in guinea-pig basilar arteries contracted by endothelin-1. Hypoxia considerably enhanced the vasodilator responses to SNAP, while those to levcromakalim were unaffected. In the presence of the NO synthase inhibitor N(G)-nitro-L-arginine, hypoxia no longer enhanced the vasodilator response to SNAP and suppressed responses to levcromakalim. The results show that the NO donor SNAP, but not the K(ATP) opener levcromakalim, is a more effective vasodilator of cerebral arteries contracted by endothelin-1 during hypoxia than under control conditions. Hypoxia-induced inhibition of basal NO synthesis could explain this enhancement of the vasodilator response to SNAP. Thus, NO donors may have a selective effect on blood vessels in ischaemic brain areas and therefore warrant further evaluation as therapeutic agents in cerebral ischaemia.
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Arteria Basilar/efectos de los fármacos , Cromakalim/farmacología , Cromakalim/farmacocinética , Hipoxia/fisiopatología , S-Nitroso-N-Acetilpenicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina/farmacocinética , Vasodilatación/efectos de los fármacos , Acetilcolina/administración & dosificación , Acetilcolina/farmacocinética , Animales , Arteria Basilar/fisiopatología , Factores Biológicos/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Cobayas , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Óxido Nítrico/metabolismo , Nitroarginina/administración & dosificación , Nitroarginina/farmacocinéticaRESUMEN
Nitric oxide (NO) is an unconventional intercellular messenger in the brain synthesised from L-arginine by a family of enzimes called NO synthases (NOS). Recent studies have demonstrated that NO plays a role in the control of dopamine release in rat striatum. Presumably, NOS inhibitors could decrease locomotor activity by interfering with striatal dopamine. The aim of this study was to assess the effect of L-NOARG (90 mg/kg), a potent NOS inhibitor, on neuronal protein synthesis activity in striatum of mice after subchronic administration for 4 consecutive days. Neuronal protein synthesis activity was analyzed by quantifyng nuclear areas and number of silver-stained nucleolar organiser regions (Ag-NORs) per nucleus. These Ag-NORs may represent the transcriptional activity of the cell. The sections of striatum examined were silver stained according to the method described by Ploton et al. (1986). The results showed that mean number of Ag-NOR per nucleus significantly increased in the striatum of mice, as compared with the control group (p<0.05). These findings indicate the existence of an increase in transcriptional activity after L-NOARG treatment, suggesting that the neostriatal dopaminergic innervation quickly develops tolerance to the interruption of dopamine transmission by L-NOARG (AU)
El óxido nítrico (ON) es un mensajero intercelular atípico sintetizado a partir de la L-arginina por una familia de enzimas denominadas óxido nítrico sintasas (NOS). Recientes estudios han demostrado que el ON está implicado en el control de la liberación de dopamina en el estriado de la rata. Presumiblemente, los inhibidores de la NOS podrían reducir la actividad locomotora bloqueando a la dopamina estriatal. El objetivo de este trabajo es evaluar el efecto de la administración subcrónica (durante 4 días consecutivos) de L-NOARG (90mg/Kg), un potente inhibidor de la NOS, sobre la actividad de síntesis de proteína neuronal en el estriado de ratones. Dicha actividad fue analizada mediante la cuantificación del área nuclear u el número de regiones organizadoras nucleolares teñidas con plata (Ag-NORs) por núcleo. Estos Ag-NORs pueden representar la actividad transcripcional de la célula. Las secciones del estriado examinadas fueron teñidas con plata de acuerdo con el método descrito por Ploton y cols. (1986). Los resultados mostraron un incremento significativo del número medio de Ag-NOARG, sugiriendo que la inervación dopaminérgica neoestrial desarrolla rápidamente tolerancia al bloqueo de la transmisión dopaminérgica inducido por L-NOARG (AU)
Asunto(s)
Animales , Ratones , Nitroarginina/farmacocinética , Óxido Nítrico/antagonistas & inhibidores , Cuerpo Estriado , Región Organizadora del Nucléolo , Factores de TranscripciónRESUMEN
1. In the present study the highly potent nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was intravenously infused and examined for its efficacy in inhibiting NOS activity and in altering blood flow and oxygen uptake in human skeletal muscle. 2. The plasma concentrations of L-NAME and its active metabolite NG-nitro-L-arginine (L-NA), and the activity of NOS in skeletal muscle were measured in healthy male subjects (n = 6) before (control) and after 60 min of intravenous infusion of L-NAME (4 mg kg(-1)). In another group of healthy males (n = 8), the physiological effects of L-NAME were studied at rest, and during submaximal and exhaustive knee extensor exercise before (control) and 30 min after L-NAME infusion (4 mg kg(-1)). 3. The plasma concentrations of L-NAME and L-NA were highest (8.4 +/- 1.6 and 8.3 +/- 0.8 micromol l(-1)) after 60 min of L-NAME infusion. Ninety minutes later mainly L-NA remained in plasma (5.1 +/- 0.4 micromol l(-1)). Thirty minutes after L-NAME infusion, the muscle L-NA content was 38 +/- 4 micromol (kg dry wt)-1 and muscle NOS activity was reduced by 67 +/- 8 % (P < 0.05). 4. Leg blood flow and leg oxygen uptake during submaximal and exhaustive exercise were similar (P > 0.05) following L-NAME infusion and in control. Blood flow during recovery was lower in the L-NAME condition (P < 0.05). 5. In conclusion, the present study shows for the first time that systemic infusion of L-NAME in humans causes a marked reduction in skeletal muscle NOS activity. Despite this attenuated NOS activity, exercise-induced hyperaemia and oxygen uptake were unaltered. Thus, the data strongly suggest that NO is not essential for the regulation of blood flow or oxygen uptake in contracting human skeletal muscle.
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Inhibidores Enzimáticos/farmacología , Ejercicio Físico/fisiología , Hiperemia/fisiopatología , Pierna/irrigación sanguínea , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Consumo de Oxígeno/fisiología , Adulto , Presión Sanguínea/efectos de los fármacos , Inhibidores Enzimáticos/farmacocinética , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Humanos , Masculino , NG-Nitroarginina Metil Éster/farmacocinética , Nitroarginina/farmacocinética , Nitroarginina/farmacología , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, L-Arg and O(2). The Michaelis-Menten constant (K(m)) of NOS for L-Arg is in the micromolar range; cellular levels of L-Arg are much higher. However, evidence strongly suggests that cellular supply of L-Arg may become limiting and lead to reduced NO and increased superoxide anion (O(-)(2)*) formation, promoting cardiovascular dysfunction. Uptake of L-Arg into cells occurs primarily (approximately 85%) through the actions of a Na(+)-independent, carrier-mediated transporter (system y(+)). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of L-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y(+) transporter activity. A rapidly acting NO donor initially increases L-Arg uptake; however, after longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without L-Arg to substance P and a Ca(2+) ionophore (A-23187) increased O(-)(2)* formation, which was blocked with concurrent presence of L-Arg or the NOS antagonist N(omega)-nitro-L-arginine methyl ester. We conclude that factors including NO itself control y(+) transport function and the production of NO and O(-)(2)*.
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Endotelio Vascular/enzimología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacocinética , Penicilamina/análogos & derivados , Superóxidos/metabolismo , Animales , Aorta/citología , Arginina/farmacocinética , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Bradiquinina/farmacología , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidrazinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III , Penicilamina/farmacología , Sustancia P/farmacología , TritioRESUMEN
PURPOSE: To clarify the transport mechanism of NG-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). METHODS: Dog intestinal BBM vesicles were used. RESULTS: The time course of L-NNA uptake showed a Na+ -dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na+, indicating participation of Na+ -dependent and Na+ -independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na+. N(G)-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. CONCLUSIONS: The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na+ differently from that across plasma membranes of nonepithelial cells, because B0,+ and b0,+ amino acid transporters function partly in L-NNA transport across intestinal BBM.
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Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Inhibidores Enzimáticos/farmacocinética , Intestino Delgado/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacocinética , Sistemas de Transporte de Aminoácidos , Aminoácidos/farmacología , Animales , Transporte Biológico Activo , Perros , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Intestino Delgado/ultraestructura , Microvellosidades/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Sodio/fisiología , Factores de TiempoRESUMEN
Overstimulation of N-methyl-D-aspartate (NMDA) receptors is felt to precipitate the neuronal damage following traumatic brain injury (TBI). NMDA receptor-mediated, glutamate-induced excitotoxicity is thought to be mediated via nitric oxide (NO) formed by neuronal nitric oxide synthase (nNOS). The present study examined the mRNA and protein levels of nNOS in the ipsilateral and contralateral cortex of rats as a function of time (5 minutes to 1 week) after controlled cortical impact (CCI) brain injury. Sham-operated rats served as controls. TBI resulted in a significant increase in the levels of nNOS mRNA (1.5- to 2.8-fold, p < .05) between 2 and 4 hours after the injury. There was also a significant increase in the levels of nNOS protein (by 55% to 90%, p < .05) and binding densities of the nNOS-specific ligand L-[3H]nitroarginine (L-[3H]NOARG) (by 35% to 59%, p < .05) between 2 and 12 hours after the injury. Increased nNOS expression and function may contribute to the concomitant excitotoxic neuronal death after TBI.
Asunto(s)
Lesiones Encefálicas/enzimología , Regulación Enzimológica de la Expresión Génica , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacocinética , Transcripción Genética , Animales , Lesiones Encefálicas/fisiopatología , Lateralidad Funcional , Masculino , Óxido Nítrico Sintasa de Tipo I , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TritioRESUMEN
Cortical spreading depression (CSD) is associated with various short- and long-term physiological and neurochemical changes and has been shown to confer an increased susceptibility to accompanying ischemic injury or provide protection against a subsequent experimental ischemia. Nitric oxide is involved in the processes of ischemic injury and under certain conditions mediates cellular protection. To investigate the possibility that CSD-induced alterations in nitric oxide synthase (NOS) expression and activity occur and might be associated with the time-dependent enhancement or prevention by CSD of ischemic damage, this study examined the spatiotemporal changes in nNOS expression and activity in cerebral cortex following CSD. Anesthetized rats had unilateral CSD induced by a 10-min topical application of KCl and were killed at various times thereafter. CSD increased both nNOS mRNA and protein levels throughout layers II-III of cortex. nNOS mRNA in the affected neocortex was significantly increased by 30-90% at 2, 7, and 14 days (P < or = 0.05) compared with contralateral levels, but was not significantly above control values at 1-6 h, 1 day, and 28 days after CSD induction. Levels of [3H]-L-N(G)-nitroarginine binding to NOS were increased by 40-170% 7, 14, and 28 days (P < or = 0.01) after CSD in a similar, but delayed, profile to nNOS mRNA. Levels of nNOS-immunoreactivity were also increased in both neurons and astrocytes of ipsilateral cortex 7 and 14 days after CSD--confirmed by double-immunofluorescence localization. Ex vivo NOS activity in layers I-III of ipsilateral cortex was also increased by 30-50% (P < or = 0.01) at 7 and 14 days after CSD, times coincident with reported maximal ischemic protection. These results demonstrate that nNOS is up-regulated by cellular depolarization/depression occurring during CSD, or by resultant stimuli and suggest that "CSD-conditioned" cortex may be capable of producing appropriate levels of NO to mediate or contribute to protective/adaptive responses to subsequent physical ischemic injury.
Asunto(s)
Corteza Cerebral/enzimología , Depresión de Propagación Cortical/fisiología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Transcripción Genética , Animales , Astrocitos/citología , Astrocitos/enzimología , Autorradiografía , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Inducción Enzimática , Lateralidad Funcional , Regulación Enzimológica de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/análisis , Cinética , Masculino , Neuronas/citología , Neuronas/enzimología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo I , Nitroarginina/farmacocinética , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacos , TritioAsunto(s)
Neuronas Motoras/metabolismo , Distrofia Muscular Animal/genética , Óxido Nítrico Sintasa/genética , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Médula Espinal/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Distrofia Muscular Animal/metabolismo , Nitroarginina/farmacocinética , Valores de Referencia , Transcripción Genética , alfa-MSH/genética , betaendorfina/genéticaRESUMEN
We investigated the post-ischemic change in [3H]L-N(G)-nitroarginine binding as a marker of nitric oxide (NO) synthase in the animal brain after transient global ischemia or transient focal ischemia. Transient global ischemia in gerbils was induced for 10 min followed by 1 h to 7 days of recirculation. Transient focal ischemia in rats was induced for 45 min followed by 3 days of recirculation. Following transient global ischemia, [3H]L-N(G)-nitroarginine binding showed a significant increase in the striatum (17-18%) and hippocampal CA1 sector (24%) at 48 and 24 h after recirculation, respectively. The hippocampal CA3 sector also showed a significant elevation (32-40%) in [3H]L-N(G)-nitroarginine binding at 24 and 48 h after global ischemia. Furthermore, the dentate gyrus showed a significant increase (30-32%) in [3H]L-N(G)-nitroarginine binding at 5, 24 and 48 h after global ischemia. Thereafter, a significant reduction in [3H]L-N(G)-nitroarginine binding was observed only in the dentate gyrus 7 days after recirculation. In contrast, [3H]L-N(G)-nitroarginine binding was unchanged in the thalamus throughout the recirculation periods. Histological analysis revealed that transient global ischemia caused severe damage or cellular damage in the striatum and the hippocampal CA1 sector. The hippocampal CA3 sector and thalamus were mildly damaged, whereas the dentate gyrus was morphologically intact. Following transient focal ischemia, a marked elevation (50-52%) in [3H]L-N(G)-nitroarginine binding was found in the regions of the ipsilateral striatum in which severe infarction occurred. Our findings suggest that [3H]L-N(G)-nitroarginine binding increases in the striatum and hippocampus after transient global ischemia or transient focal ischemia. This increase in [3H]L-N(G)-nitroarginine binding may play a pivotal role not only in the pathogenesis of ischemic brain damage, but also in the restoration of injury areas after cerebral ischemia.
Asunto(s)
Encéfalo/metabolismo , Ataque Isquémico Transitorio/metabolismo , Nitroarginina/farmacocinética , Animales , Autorradiografía , Encéfalo/diagnóstico por imagen , Encéfalo/enzimología , Encéfalo/patología , Gerbillinae , Ataque Isquémico Transitorio/enzimología , Ataque Isquémico Transitorio/patología , Masculino , Óxido Nítrico Sintasa/metabolismo , Cintigrafía , Radiofármacos/farmacocinética , Ratas , Ratas Wistar , TritioRESUMEN
PURPOSE: Nitric oxide synthase (NOS) inhibitors such as Nitro-L-arginine (L-NA) are being considered for the management of hypotension observed in septic shock. However, little information is available regarding the pharmacokinetic and pharmacodynamic properties of these agents. Our objective was to examine the relationships between L-NA plasma concentration and various hemodynamic effects such as cardiac index (CI), mean arterial pressure (MAP), and heart rate (HR) elicited by L-NA administration in rats. METHODS: L-NA was infused at doses between 2.5-20 mg/kg/hr in anesthetized rats over one hour. Hemodynamic effects and plasma L-NA levels were determined. RESULTS: Infusion of L-NA resulted in dose-dependent increases in MAP and systemic vascular resistance (SVR), decreases in CI, and minimal change in HR. The relationships between the hemodynamic effects and plasma L-NA levels were not monotonic, and hysteresis was observed. Using nonparametric analysis, the equilibration half-time (t1/2,keo) between plasma L-NA and the hypothetical effect site was determined to be 51.5 +/- 6.6 min, 42.4 +/- 10.1 min, 43.4 +/- 9.0 min for MAP, CI, and SVR, respectively (n = 14). The Emax and EC50 values obtained were + 32.5 +/- 8.4 and 2.6 +/- 1.3 microg/ml for MAP and -52.9 +/- 15.6 and 3.7 +/- 1.8 microg/ml for CI, respectively. CONCLUSIONS: Although L-NA can bring about beneficial elevation of MAP, such effect is always accompanied by a stronger effect on CI depression. Dose escalation of L-NA may bring about detrimental negative inotropic effect and loss of therapeutic efficacy.
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Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/farmacocinética , Frecuencia Cardíaca/efectos de los fármacos , Nitroarginina/farmacología , Nitroarginina/farmacocinética , Anestesia , Animales , Peso Corporal/fisiología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/sangre , Infusiones Intravenosas , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
In this study, the transport mechanism of NG-nitro-L-arginine (L-NA), a nitric oxide synthase inhibitor that may be useful for alleviating intraocular inflammation, was characterized in the pigmented rabbit conjunctiva. L-NA, when applied to the mucosal side of the conjunctiva, led to dose-dependent increases in the short-circuit current (Isc) at 37 degrees C but not at 4 degrees C or under the Na+-free condition. Serosally added 1 mM L-NA did not elicit any change in the Isc. Mucosally added 1 mM L-NA elicited a net absorptive Na+ flux of 0.09 microEq/(cm2.hr), comparable with the Isc change. L-NA transport at 0.1 mM in the mucosal-to-serosal (ms) direction was 22 times greater than that in the serosal-to-mucosal direction. There was a good correlation between the ms flux of L-NA and the Isc changes elicited by L-NA under the same experimental conditions. L-NA transport was saturable, with a Km of 0.35 mM and a maximal flux of 290 pmol/(cm2.min). Hill analysis of L-NA flux observed at 0.1 mM L-NA in response to varying Na+ concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.98, suggesting a 1:1 coupling between Na+ and L-NA. Moreover, ms 3H-L-NA transport was inhibited by basic amino acids (L-Arg and L-Lys) and a neutral amino acid (L-Leu), but not by an acidic amino acid (L-Glu) and the D-stereoisomer of L-NA. In the case of L-Arg, inhibition was competitive with a Ki of 0.034 mM. Taken together, the above findings are consistent with the involvement of the L-Arg transport system B0,+ in the conjunctival transport of L-NA.
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Conjuntiva/metabolismo , Inhibidores Enzimáticos/farmacocinética , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacocinética , Sodio/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Inhibidores Enzimáticos/farmacología , Masculino , Nitroarginina/farmacología , Conejos , Sodio/farmacologíaRESUMEN
1. We evaluated the haemodynamic effects and systemic disposition of the nitric oxide synthase (NOS) inhibitor NL-nitro-L-arginine (NOLA) after intravenous (i.v.) administration of two different doses (5 and 20 mg/kg) in awake healthy sheep and awake sheep given a continuous i.v. infusion of endotoxin (lipopolysaccharide, 12 ng/kg per h, i.v., for 18 h). In addition, we determined the systemic disposition of another NOS inhibitor, NL-nitro-L-arginine methylester (L-NAME; 20 mg/kg, i.v.) in awake healthy sheep only. 2. NL-Nitro-L-arginine produced a dose-dependent decrease in heart rate (HR) and cardiac output (CO) together with a dose-dependent increase in mean arterial pressure (MAP) and peripheral vascular resistance (PVR) when compared to baseline. In endotoxic sheep NOLA produced a greater increase in MAP and mean pulmonary arterial pressure (MPAP). 3. In healthy sheep there was a dose-related increase in total body clearance (Cl) of NOLA. The Cl increased from 0.028 L/min after the lower dose to 0.032 L/min after the higher dose. The infusion of endotoxin caused an increase in Cl of NOLA to 0.040 and 0.047 L/min, respectively, and a decrease in plasma slow half-life (t1/2) from 825 to 546 min and from 780 to 453 min, respectively. 4. NL-Nitro-L-arginine methylester was rapidly cleared from the plasma with a slow half-life of approximately 7.5 min and there was a simultaneous appearance of NOLA in the plasma. 5. These results support the view that nitric oxide has a significant role in regulating vascular tone in healthy and endotoxic sheep and indicate that the increases in Cl of NOLA with an increase in its dose and the presence of endotoxin will be important in influencing appropriate dosage regimens in clinical studies.
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Lipopolisacáridos/toxicidad , NG-Nitroarginina Metil Éster/farmacocinética , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Esquema de Medicación , Femenino , Hemodinámica/efectos de los fármacos , Infusiones Intravenosas , Lipopolisacáridos/administración & dosificación , NG-Nitroarginina Metil Éster/administración & dosificación , NG-Nitroarginina Metil Éster/sangre , Nitroarginina/administración & dosificación , Nitroarginina/sangre , Distribución Aleatoria , OvinosRESUMEN
We investigated the density and distribution of nitric oxide synthase (NOS) binding by quantitative autoradiography using [3H]L-NG-nitroarginine ([3H]L-NNA) after transient focal ischemia or intrastriatal injection of N-methyl-D-aspartate (NMDA) in wild-type (SV-129 and C57black/6) and type I (neuronal) and type III (endothelial) NOS-deficient mice. The middle cerebral artery (MCA) was occluded by an intraluminal filament for 3 h followed by 10 min to 7 days of reperfusion. Specific [3H]L-NNA binding, observed in the wild-type and type III mutant mouse at baseline, increased by 50-250% in the MCA territory during ischemia and the first 3 h of reperfusion. The density of binding sites (Bmax), but not the dissociation constant (Kd), increased significantly during the ischemic period as did type I NOS mRNA as detected by quantitative reverse transcription polymerase chain reaction. [3H]L-NNA binding after intrastriatal NMDA injection also increased by 20-230%. In the type I NOS-deficient mouse, [3H]L-NNA binding was low and only a very small increase was observed after ischemia or excitotoxicity. Under conditions of this study, [3H]L-NNA did not bind to type II NOS as there was no difference in the distribution or density of [3H]L-NNA binding in the rat spleen obtained after lipopolysaccharide treatment despite induction of NOS type II catalytic activity. Our data suggest that an ischemic/excitotoxic insult up-regulates type I NOS gene expression and [3H]L-NNA binding and that this up-regulation may play a pivotal role in the pathogenesis of ischemic/excitotoxic diseases.
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Inhibidores Enzimáticos/farmacocinética , Agonistas de Aminoácidos Excitadores/toxicidad , Ataque Isquémico Transitorio/enzimología , N-Metilaspartato/toxicidad , Óxido Nítrico Sintasa/deficiencia , Nitroarginina/farmacocinética , Animales , Autorradiografía , Inhibidores Enzimáticos/administración & dosificación , Agonistas de Aminoácidos Excitadores/administración & dosificación , Femenino , Masculino , Ratones , Ratones Mutantes , N-Metilaspartato/administración & dosificación , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/genética , Nitroarginina/administración & dosificación , Ensayo de Unión Radioligante , Ratas , Distribución Tisular , TritioRESUMEN
Elevated activities of nitric oxide synthase (NOS) have been reported previously in the brains of portacaval-shunted (PCS) rats, a model of chronic hepatic encephalopathy (HE). As L-arginine availability for nitric oxide synthesis depends on a specific uptake mechanism in neurons, we studied the kinetics of L-[3H]-arginine uptake into synaptosomes prepared from the brains of PCS rats. Results demonstrate that L-arginine uptake is significantly increased in cerebellum (60%; p < 0.01), cerebral cortex (42%; p < 0.01), hippocampus (56%; p < 0.01), and striatum (51%; p < 0.01) of PCS rats compared with sham-operated controls. Hyperammonemia in the absence of portacaval shunting also stimulated the transport of L-[3H]arginine; kinetic analysis revealed that the elevated uptake was due to increased uptake capacity (Vmax) without any change in affinity (Km). Incubation of cerebellar synaptosomes with ammonium acetate for 10 min caused a dose-dependent stimulation of L-[3H]arginine uptake. Neither portacaval shunting nor hyperammonemia had any significant effect on the synaptosomal uptake of NG-nitro-L-[3H]arginine. These studies demonstrate that increased NOS activity observed in experimental HE may result from increased availability of L-arginine resulting from a direct stimulatory effect of ammonia on L-arginine transport.