RESUMEN
Chronic kidney disease (CKD) is associated with decreased renal nitric oxide (NO) production and increased plasma levels of methylarginines. The naturally occurring guanidino-methylated arginines N-monomethyl-l-arginine (l-NMMA) and asymmetric dimethyl-l-arginine (ADMA) inhibit NO synthase activity. We hypothesized that ADMA and l-NMMA compromise the integrity of the glomerular filtration barrier via NO depletion. We studied the effect of ADMA on albumin permeability (P(alb)) in isolated glomeruli and examined whether this effect involves NO- and superoxide (O(2)(*-))-dependent mechanisms. ADMA at concentrations found in circulation of patients with CKD decreased cGMP and increased P(alb) in a dose-dependent manner. A similar increase in P(alb) was caused by l-NMMA but at a concentration two orders of magnitude higher than that of ADMA. NO donor DETA-NONOate or cGMP analog abrogated the effect of ADMA on P(alb). The SOD mimetic tempol or the NAD(P)H oxidase inhibitor apocynin also prevented the ADMA-induced increase in P(alb). The NO-independent soluble guanylyl cyclase (sGC) activator BAY 41-2272, at concentrations that increased glomerular cGMP production, attenuated the ADMA-induced increase in P(alb). Furthermore, sGC incapacitation by the heme site-selective inhibitor ODQ increased P(alb). We conclude that ADMA compromises the integrity of the filtration barrier by altering the bioavailability of NO and O(2)(*-) and that NO-independent activation of sGC preserves the integrity of this barrier under conditions of NO depletion. NO-independent activation of sGS may be a useful pharmacotherapeutic approach for preservation of glomerular function in CKD thereby reducing the risk for cardiovascular events.
Asunto(s)
Antracenos/toxicidad , Arginina/análogos & derivados , Membrana Basal Glomerular/efectos de los fármacos , Membrana Basal Glomerular/metabolismo , Óxido Nítrico/metabolismo , Propano/análogos & derivados , Superóxidos/metabolismo , Albúminas/metabolismo , Animales , Arginina/farmacología , Arginina/toxicidad , Depuradores de Radicales Libres/farmacología , Membrana Basal Glomerular/patología , Guanilato Ciclasa/metabolismo , Masculino , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oxidación-Reducción , Permeabilidad/efectos de los fármacos , Propano/toxicidad , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa Soluble , omega-N-Metilarginina/toxicidadRESUMEN
Endothelial cell dysfunction is associated with bioavailable nitric oxide deficiency and an excessive generation of reactive oxygen species. We modeled this condition by chronically inhibiting nitric oxide generation with subpressor doses of N(G)-monomethyl-L-arginine (L-NMMA) in C57B6 and Tie-2/green fluorescent protein mouse strains. L-NMMA-treated mice exhibited a slight reduction in vasorelaxation ability, as well as detectable abnormalities in soluble adhesion molecules (soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1, and matrix metalloproteinase 9), which represent surrogate indicators of endothelial dysfunction. Proteomic analysis of the isolated microvasculature using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy revealed abnormal expression of a cluster of mitochondrial enzymes, which was confirmed using immunodetection. Aconitase-2 and enoyl-CoA-hydratase-1 expression levels were decreased in L-NMMA-treated animals; this phenotype was absent in nitric oxide synthase-1 and -3 knockout mice. Depletion of aconitase-2 and enoyl-CoA-hydratase-1 resulted in the inhibition of the Krebs cycle and enhanced pyruvate shunting toward the glycolytic pathway. To assess mitochondrial mass in vivo, co-localization of green fluorescent protein and MitoTracker fluorescence was detected by intravital microscopy. Quantitative analysis of fluorescence intensity showed that L-NMMA-treated animals exhibited lower fluorescence of MitoTracker in microvascular endothelia as a result of reduced mitochondrial mass. These findings provide conclusive and unbiased evidence that mitochondriopathy represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward "metabolic hypoxia" through the selective depletion of both aconitase-2 and enoyl-CoA-hydratase-1. These findings may contribute to an early preclinical diagnosis of endothelial dysfunction.
Asunto(s)
Ciclo del Ácido Cítrico/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Mitocondrias/patología , Proteómica , Aconitato Hidratasa/metabolismo , Animales , Enoil-CoA Hidratasa/metabolismo , Inhibidores Enzimáticos/toxicidad , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/metabolismo , Microvasos/patología , Mitocondrias/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Estrés Oxidativo/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Enfermedades Vasculares/metabolismo , omega-N-Metilarginina/toxicidadRESUMEN
Deficiency of nitric oxide (NO) represents a consistent manifestation of endothelial dysfunction (ECD), and the accumulation of asymmetric dimethylarginine occurs early in renal disease. Here, we confirmed in vitro and in vivo the previous finding that a fragment of collagen XVIII, endostatin, was upregulated by chronic inhibition of NO production and sought to support a hypothesis that primary ECD contributes to nephrosclerosis in the absence of other profibrotic factors. To emulate more closely the indolent course of ECD, the study was expanded to an in vivo model with N(G)-monomethyl-L-arginine (L-NMMA; mimics effects of asymmetric dimethylarginine) administered to mice in the drinking water at subpressor doses of 0.3 and 0.8 mg/ml for 3-6 mo. This resulted in subtle but significant morphological alterations detected in kidneys of mice chronically treated with L-NMMA: 1) consistent perivascular expansion of interstitial matrix components at the inner stripe of the outer medulla and 2) collagen XVIII/endostatin abundance. Ultrastructural abnormalities were detected in L-NMMA-treated mice: 1) increased activity of the interstitial fibroblasts; 2) occasional detachment of endothelial cells from the basement membrane; 3) splitting of the vascular basement membrane; 4) focal fibrosis; and 5) accumulation of lipofuscin by interstitial fibroblasts. Preembedding labeling of microvasculature with anti-CD31 antibodies showed infiltrating leukocytes and agglomerating platelets attaching to the visibly intact or denuded capillaries. Collectively, the data indicate that the mouse model of subpressor chronic administration of L-NMMA is not a robust one (endothelial pathology visible only ultrastructurally), and yet it closely resembles the natural progression of endothelial dysfunction, microvascular abnormalities, and associated tubulointerstitial scarring.
Asunto(s)
Modelos Animales de Enfermedad , Endotelio Vascular/patología , Enfermedades Renales/patología , Riñón/patología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , omega-N-Metilarginina/toxicidad , Animales , Células Cultivadas , Endotelio Vascular/ultraestructura , Riñón/ultraestructura , Enfermedades Renales/inducido químicamente , Leucocitos/patología , Ratones , Agregación Plaquetaria , omega-N-Metilarginina/administración & dosificaciónRESUMEN
BACKGROUND: Nitric oxide (NO), produced by the nitric oxide synthase family of enzymes, mediates multiple signaling functions, and when unchecked, NO causes pathological damage. Exposure of embryos to a variety of teratogens, including carbon monoxide (CO), has been shown to increase reactive intermediates, such as NO, and recent work showed that either the excess or absence of NO caused morphological defects. While endogenous NO is known to regulate many adult tissues, its role during embryonic organogenesis and/or in mediating responses to teratogen exposure has not been explored. METHODS: We have examined here the presence of NO during normal chick embryonic organogenesis, and investigated the teratogenicity of NO through the application of sodium nitroprusside (SNP), which mimics NO overproduction, and NG-monomethyl-L-arginine (L-NMMA), which inhibits endogenous NOS activity. RESULTS: Topical treatment with SNP or L-NMMA for 18 h resulted in morphological defects, specifically in the neural tube and somites, which corresponded to sites of altered apoptosis. The location of NO was histochemically correlated with the observed morphological defects. Coadministration of SNP or L-NMMA with CO showed functional coregulation and interaction between NO and CO in chick embryonic development. CONCLUSIONS: Our results showed that regulation of NO is essential for normal axial development, that sites of altered NO expression correlate to those of altered apoptosis and dysmorphogenesis, and that CO coadministration resulted in a rectification of normal NO expression. Collectively, these results suggest that alteration in endogenous NO/CO signaling is responsible, at least in part, for the observed NO-induced teratogenesis.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Defectos del Tubo Neural/inducido químicamente , Óxido Nítrico/metabolismo , Organogénesis/fisiología , Animales , Apoptosis/efectos de los fármacos , Monóxido de Carbono/toxicidad , Embrión de Pollo , Combinación de Medicamentos , Pérdida del Embrión/inducido químicamente , Pérdida del Embrión/patología , Desarrollo Embrionario/fisiología , Inhibidores Enzimáticos/toxicidad , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/patología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Nitroprusiato/toxicidad , Organogénesis/efectos de los fármacos , Somitos/efectos de los fármacos , Somitos/metabolismo , Somitos/patología , Teratógenos/toxicidad , omega-N-Metilarginina/toxicidadRESUMEN
OBJECTIVE: To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms. METHODS: Apoptosis of gastric cancer cells was detected by flow cytometry. Expression of phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 in gastric cancer cells was analyzed using Western blotting. Immunofluorescence assay was performed to localize the intracellular phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1. Transfection of FKHRL1-HA wild type and mutant FKHRL1-HA T32A constructs was performed by lipofectamine plus reagent. NO generation was determined by Griess reaction. RESULTS: Gastric cancer cells were significantly apoptotic after treatment with N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), compared with the control (P<0.01). The apoptosis of gastric cancer cells induced by L-NMMA was dose-dependent and time-independent. However, the Z-DEVD-fmk, a caspase-3, 6, 7, 8, 10 inhibitor, did not prevent the apoptosis. The immunofluorescence assays showed that FKHRL1 protein was strongly expressed in the nucleu and p-FKHRL1 thr-32 protein was strongly expressed in the cytoplasm of SGC-7901 cells when endogenous nitric oxide generation was blocked by L-NMMA, but no change in FKHRL1 ser-253 phosphorylation. Nevertheless, ROCK protein was strongly expressed in p-FKHRL1 thr-32-positive SGC-7901 cells. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase (PI3K), did not block the phosphorylated FKHRL1 thr-32 protein induced by L-NMMA. However, Y-27632, a specific inhibitor of the protein kinase ROCK, significantly blocked apoptosis induced by phosphorylated FKHRL1 thr-32 (P < 0.01), which was mediated by L-NMMA. A significant decrease in NO generation (P < 0.01) and a significant increase in apoptosis (P < 0.01) were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation. CONCLUSIONS: L-NMMA triggers gastric carcinoma cell apoptosis, possibly by promoting FKHRL1 thr-32 phosphorylation and initiating signal of FKHRL1 to ROCK kinase. This apoptotic signaling process is PI3K/Akt as well as caspase-3 independent.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis , Factores de Transcripción Forkhead/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , omega-N-Metilarginina/toxicidad , Caspasa 3/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/toxicidad , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/análisis , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Transfección , Quinasas Asociadas a rhoRESUMEN
Previously, we have shown that C6 glial cells enriched in hexacosenoic acid (HA) incubated with oxidative stressors released higher amounts of nitric oxide (NO) products and superoxide (O2(-)), compared to native C6 cells. In the present study, we examined the effects of pretreatment with some of free radical release inhibitors. The aim was to determine the origin of the enhanced generation of NO and superoxide, and to test the possibility of preventing it. Pre-treatment with L-mono-methyl-arginine and N-acetyl-cysteine in oxidized low-density lipoprotein (ox-LDL) exposed HA cells, inhibited not only nitrite but also superoxide production suggesting that O2(-) anion could partially derive from inducible NO synthase. We also observed that ox-LDL treatment of HA cells reduced the intracellular glutathione levels and activated extracellular signal-related kinases. Since this signalling is related to neurotoxic effect, our data substantiate the role of the free radicals in X-linked adrenoleukodystrophy pathogenesis, as HA cells have been used as an in vitro model for this disease.
Asunto(s)
Acetilcisteína/toxicidad , Inhibidores Enzimáticos/toxicidad , Ácidos Grasos Monoinsaturados/farmacología , Depuradores de Radicales Libres/farmacología , Radicales Libres/metabolismo , Neuroglía/metabolismo , Compuestos Onio/farmacología , omega-N-Metilarginina/toxicidad , Animales , Western Blotting , Línea Celular , Ácidos Grasos/metabolismo , Glutatión/metabolismo , Lipopolisacáridos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuroglía/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Oxidación-Reducción , Fosforilación , Ratas , Superóxidos/metabolismoRESUMEN
OBJECTIVE: Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducible nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells. METHODS: A colonic cell line (HT29) was stimulated for 1-10 weeks with interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) or both and compared with unstimulated cells or cells stimulated for 48 h. Cells were co-incubated with a selective iNOS inhibitor (N-monomethyl-L-arginine (L-NMMA)) in some experiments. Viability was assessed by the dimethylthiazol diphenyl tetrazolium bromide (MTT) test. Production of ROS was determined by the oxidation of 2',7'-dichlorodihydrofluorescein to a fluorescent 2',7'-dichlorofluorescein and measured by fluorescence reading and visualized by fluorescence microscopy. DNA stability was determined by single cell gel electrophoresis. RESULTS: Continuously stimulated colonic cells had increased ROS production, especially those stimulated with TNF-alpha or IFN-gamma/TNF-alpha (P<0.001). The ROS production could be inhibited by L-NMMA co-incubation, indicating that iNOS is responsible for the up-regulation (P<0.05). Continuously stimulated cells had increased DNA instability (P<0.002), whereas short-term stimulated cells did not. The DNA instability was inhibited by L-NMMA co-incubation (P<0.05). CONCLUSIONS: Continuous cytokine exposure induces an iNOS dependent up-regulation of ROS production and DNA instability. This mechanism could be involved in carcinogenesis in chronic inflammatory diseases of the intestinal tract.
Asunto(s)
Colon/efectos de los fármacos , Citocinas/toxicidad , Daño del ADN , ADN/efectos de los fármacos , Antineoplásicos/toxicidad , Muerte Celular/efectos de los fármacos , Colon/patología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Inhibidores Enzimáticos/toxicidad , Células Epiteliales/efectos de los fármacos , Células HT29 , Humanos , Interferón gamma/toxicidad , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/toxicidad , Regulación hacia Arriba/efectos de los fármacos , omega-N-Metilarginina/toxicidadRESUMEN
OBJECTIVE: Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. METHODS AND RESULTS: ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of l-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT(1) receptor antagonist), which simultaneously suppressed vascular lesion formation. CONCLUSIONS: These results provide the first direct evidence that the long-term vascular effects of ADMA are not solely mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of ACE and increased oxidative stress through AT(1) receptor appear to be involved in the long-term vascular effects of ADMA in vivo. This study demonstrates that asymmetrical dimethylarginine (ADMA) causes arteriosclerotic coronary lesions in mice in vivo through mechanisms other than simple inhibition of endothelial NO synthesis. Our findings should contribute to a better understanding of the pathophysiological role of ADMA in arteriosclerosis.
Asunto(s)
Arginina/análogos & derivados , Arginina/toxicidad , Enfermedad Coronaria/inducido químicamente , Óxido Nítrico Sintasa/deficiencia , Peptidil-Dipeptidasa A/biosíntesis , Sistema Renina-Angiotensina/fisiología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Arginina/sangre , Arginina/farmacología , Enfermedad Coronaria/tratamiento farmacológico , Enfermedad Coronaria/enzimología , Enfermedad Coronaria/patología , Inducción Enzimática/efectos de los fármacos , Homocisteína/sangre , Imidazoles/farmacología , Imidazoles/uso terapéutico , Bombas de Infusión Implantables , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , NG-Nitroarginina Metil Éster/farmacología , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Nitritos/metabolismo , Olmesartán Medoxomilo , Estrés Oxidativo , Peptidil-Dipeptidasa A/genética , Superóxidos/metabolismo , Tetrazoles/farmacología , Tetrazoles/uso terapéutico , Tiazepinas/farmacología , Tiazepinas/uso terapéutico , omega-N-Metilarginina/toxicidadRESUMEN
OBJECTIVE: To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers. SAMPLE POPULATION: Polymorphonuclear neutrophils from 3 lactating cows. PROCEDURE: Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots. RESULTS: Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner. CONCLUSIONS AND CLINICAL RELEVANCE: Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.
Asunto(s)
Bovinos/fisiología , Glándulas Mamarias Animales/fisiología , Neutrófilos/inmunología , Nitratos/toxicidad , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oxidantes/toxicidad , Peroxidasa/antagonistas & inhibidores , Superóxido Dismutasa/farmacología , Tirosina/análogos & derivados , Compuestos de Anilina/toxicidad , Animales , Antioxidantes/farmacología , Western Blotting/veterinaria , Muerte Celular/efectos de los fármacos , Inhibidores Enzimáticos/toxicidad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/fisiología , Femenino , Depuradores de Radicales Libres/toxicidad , Histidina/toxicidad , L-Lactato Deshidrogenasa/análisis , Lipopolisacáridos/toxicidad , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/enzimología , Molsidomina/análogos & derivados , Molsidomina/farmacocinética , Molsidomina/toxicidad , Activación Neutrófila/inmunología , Nitratos/farmacocinética , Óxido Nítrico/análisis , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/farmacología , Oxidantes/farmacocinética , Peroxidasa/farmacología , Acetato de Tetradecanoilforbol/toxicidad , Tirosina/análisis , omega-N-Metilarginina/toxicidadRESUMEN
The objective of this study was to investigate the site of action of dipyrone in rat paw prostaglandin-induced hyperalgesia. The intracerebroventricular (i.c.v.) injection of dipyrone had no effect on the hyperalgesic response to prostaglandins. In contrast, intraplantar (i.pl.) and intrathecal (i.t.) injections produced dose-dependent analgesic effects. The analgesia observed following the intraperitoneal (i.p.), i.t., i.pl. or combined i.t. and i.pl. administration of dipyrone was abolished by pretreating the paws with L-NMMA (a nitric oxide synthase inhibitor) or methylene blue (MB, an inhibitor of soluble guanylate cyclase). These results support the suggestion that dipyrone-mediated antinociception results from a combined spinal and peripheral effect in the primary peripheral sensory neuron via stimulation of the arginine/cGMP pathway.
Asunto(s)
Anestesia Raquidea , Antiinflamatorios no Esteroideos/uso terapéutico , Dinoprostona/toxicidad , Dipirona/uso terapéutico , Neuronas Aferentes/efectos de los fármacos , Dolor/tratamiento farmacológico , Análisis de Varianza , Animales , Antiinfecciosos Urinarios/administración & dosificación , Antiinfecciosos Urinarios/toxicidad , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Arginina/metabolismo , Dinoprostona/administración & dosificación , Dipirona/administración & dosificación , Dipirona/farmacología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/toxicidad , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Inyecciones Espinales , Masculino , Azul de Metileno/administración & dosificación , Azul de Metileno/toxicidad , Neuronas Aferentes/metabolismo , Óxido Nítrico/metabolismo , Dolor/inducido químicamente , Ratas , Ratas Wistar , omega-N-Metilarginina/administración & dosificación , omega-N-Metilarginina/toxicidadAsunto(s)
Aminoácidos/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Inhibidores Enzimáticos/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Aminoácidos/toxicidad , Animales , Arginina/toxicidad , Citrulina/análogos & derivados , Citrulina/farmacología , Citrulina/toxicidad , Inhibidores Enzimáticos/toxicidad , Isoenzimas/antagonistas & inhibidores , Cinética , Macrófagos/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , NG-Nitroarginina Metil Éster/toxicidad , Nitroarginina/farmacología , Nitroarginina/toxicidad , Roedores , Relación Estructura-Actividad , Tiourea/análogos & derivados , Tiourea/farmacología , Tiourea/toxicidad , omega-N-Metilarginina/farmacología , omega-N-Metilarginina/toxicidadRESUMEN
The ability of the naturally occurring non-protein toxic amino acids indospicine and canavanine to inhibit nitric oxide synthesis was tested in isolated rat aorta and cultured rat peritoneal macrophages. Both compounds inhibited acetylcholine induced relaxation of rat aorta contracted with noradrenaline, a process mediated by nitric oxide generated in vascular endothelium. Nitric oxide is generated in vascular endothelium from arginine by a constitutive nitric oxide synthase. Indospicine and canavanine also increased superoxide mediated reduction of cytochrome c by phorbol myristate acetate stimulated rat peritoneal macrophages. The increase in superoxide under these conditions was due to decreased nitric oxide synthesis. Macrophage synthesis of nitric oxide is mediated by an inducible form of nitric oxide synthase. It is concluded that indospicine and canavanine are inhibitors of constitutive and inducible nitric oxide synthases and it is suggested that the toxicity associated with these compounds could be related to this activity.
Asunto(s)
Canavanina/toxicidad , Endotelio Vascular/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Norleucina/análogos & derivados , Animales , Aorta , Arginina/análogos & derivados , Arginina/toxicidad , Vacuna BCG/administración & dosificación , Células Cultivadas , Grupo Citocromo c/biosíntesis , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/toxicidad , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/metabolismo , Masculino , NG-Nitroarginina Metil Éster/toxicidad , Óxido Nítrico Sintasa/antagonistas & inhibidores , Norleucina/toxicidad , Oxidación-Reducción , Ratas , Ratas Wistar , Acetato de Tetradecanoilforbol/toxicidad , omega-N-Metilarginina/toxicidadRESUMEN
The current study was designed to characterize nitric oxide (NO.) synthesis during interleukin-2 (IL-2) treatment of mice, and to determine whether NO. mediated IL-2-induced "vascular leak." We developed a technique for chronic subcutaneous infusion of the NO. synthase inhibitor N omega monomethyl-L-arginine (MLA) via osmotic minipump to aid in further study of these processes. After IL-2 administration to C3H/HeN mice (180,000 IU i.p. b.i.d. for 5 days), NO. synthesis increased two-to-three fold, peaking on days 5-8. Administration of MLA reduced NO. synthesis in both IL-2-treated mice (from 2.7 to 1 microM/mouse/day), and normal mice (from 1 to 0.5 microM/mouse/day). This agent decreased IL-2-induced radiolabeled albumin accumulation in the liver after i.p. IL-2 administration (p < 0.02). MLA infusions resulted in minimal systemic toxicity in mice, as reflected by complete blood counts or serum chemistries. MLA also did not impair lymphokine-activated killer cell induction in vitro or in vivo, or alter IL-2-induced tumor responses in a 3-day pulmonary metastasis model. These experiments demonstrated that NO. is a mediator involved in the genesis of vascular permeability induced by IL-2 treatment. Studies designed to further evaluate the toxicity and usefulness of MLA infusions to modify this IL-2 induced toxicity appear to be warranted.