RESUMEN
AIMS/HYPOTHESIS: Poly(ADP-ribose) polymerase activation depletes NAD+ and high-energy phosphates, activates protein kinase C, and affects gene expression in various tissues. This study was designed to characterise the effects of the potent, orally active poly(ADP-ribose) polymerase inhibitor PJ34 in the Wistar rat model of early diabetic neuropathy. METHODS: Control and streptozotocin-diabetic rats were maintained with or without PJ34 treatment (30 mg x kg(-1) x day(-1)) for two weeks, after two weeks without treatment. Endoneurial blood flow was assessed by hydrogen clearance; metabolites and high-energy phosphates were assayed by enzymatic spectrofluorometric methods; and poly(ADP-ribose) was detected by immunohistochemistry. RESULTS: Blood glucose concentrations were increased to a similar extent in untreated and PJ34-treated diabetic rats compared with controls. Intense poly(ADP-ribose) immunostaining was observed in the sciatic nerve of diabetic rats, but not in other groups. Final sciatic motor nerve conduction velocity and digital sensory nerve conduction velocity were reduced by 24% and 22% respectively in diabetic rats compared with controls (p<0.01 for both), and both were 98% corrected by PJ34 (p<0.01 vs diabetic group for both). In contrast, with PJ34 treatment, nerve blood flow showed a modest (17%) increase, and vascular conductance showed a tendency to increase. Free mitochondrial and cytosolic NAD+:NADH ratios, assessed from the glutamate and lactate dehydrogenase systems, phosphocreatine concentrations, and phosphocreatine:creatine ratios were decreased in diabetic rats and essentially normalised by PJ34. In both untreated and PJ34-treated diabetic rats, nerve glucose, sorbitol and fructose were increased to a similar extent. PJ34 did not affect any variables in control rats. CONCLUSIONS/INTERPRETATION: Short-term poly(ADP-ribose) polymerase inhibitor treatment reverses functional and metabolic abnormalities of early diabetic neuropathy. Complete normalisation of nerve blood flow is not required for correction of motor or sensory nerve conduction velocities, provided that a therapeutic agent can restore nerve energy state via direct action on Schwann cells.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Neuropatías Diabéticas/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Fenantrenos/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Experimental/patología , Neuropatías Diabéticas/patología , Diuréticos/metabolismo , Masculino , Neuronas Motoras/fisiología , NAD/metabolismo , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Neuronas Aferentes/fisiología , Ratas , Ratas Wistar , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/enzimología , Neuropatía Ciática/patología , Sorbitol/metabolismoRESUMEN
The aim of the present study was to test the effects of a topical administration of a novel nitric oxide donor, linear polyethylenimine-nitric oxide/nucleophile adduct (DS1), on vaginal blood flow and hemodynamics in rats. Laser Doppler flowmetry was used to measure blood flow changes following topical application of DS1 (0.3 or 1.5 mg in 0.15 ml saline) into the vagina of anesthetized Wistar rats. In vivo hemodynamic parameters were measured with Millar-tip-catheter placed in the left ventricle. DS1 (1.5 mg) increased vaginal blood flow by 191+/-24, 226+/-22 and 166+/-23% of the baseline value (at 5, 15 and 30 min, respectively, after application) without affecting systemic blood pressure, heart rate and cardiac function. The increased vaginal blood flow following DS1 application returned to baseline between 45 and 60 min. Thus, topical application of nitric oxide donors such as DS1 may be useful for the treatment of female sexual dysfunction that develops due to an impairment of local blood flow supply to the vaginal tissue.
Asunto(s)
Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Polietileneimina/farmacología , Flujo Sanguíneo Regional/efectos de los fármacos , Vagina/irrigación sanguínea , Administración Tópica , Anestesia , Animales , Femenino , Ratas , Ratas WistarRESUMEN
Insulin-dependent diabetes mellitus (IDDM) is a disease characterized by the autoimmune destruction of the pancreatic beta-cells, which requires the expression of a number of immune-related genes including major histocompatibility complex proteins, cytokines, chemokines, and cytotoxic enzymes, many of which are regulated by the transcription factor, NFkappaB. Inhibition of the entire NFkappaB family of transcription factors may be harmful, as these factors are involved in many normal physiological processes. However, identifying and targeting specific NFkappaB subunits critical for the pathogenesis of disease may prove to be valuable in designing new therapeutic strategies. To assess the potential role of the NFkappaB subunit, p50, in the development of IDDM, mice with gene disruption for NFkappaB (p50) were investigated for susceptibility to IDDM. We found that p50-deficient mice were fully resistant against multiple low-dose streptozotocin-induced diabetes, a model of diabetes with a strong autoimmune component. The site of involvement of NFkappaB (p50) lies at an early, critical juncture of immune activation and proinflammatory mediator production, because: (1) isolated islets of Langerhans from NFkappaB (p50)-deficient mice were not protected from the islet dysfunction induced by in vitro application of proinflammatory cytokines; (2) p50-deficient mice were not resistant to diabetes induced by a single high dose of streptozotocin, a model with a large oxidant component and no autoimmune involvement; and (3) diabetes induced up-regulation of nitric oxide and interleukin-12 was blocked in the p50-deficient mice. Our data suggest that NFkappaB (p50) has an essential role in the development of autoimmune diabetes. Selective therapeutic blockade of this subunit may be beneficial in preventing IDDM.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , FN-kappa B/metabolismo , Animales , Citocinas/farmacología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Esquema de Medicación , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/genética , Subunidades de ProteínaRESUMEN
Nicotine has been previously shown to have immunosuppressive actions. Type I diabetes is an autoimmune disease resulting from the specific destruction of the insulin-producing pancreatic beta-cells. Thus, we hypothesized that nicotine may exert protective effects against type I diabetes. The multiple low-dose streptozotocin (MLDS)-induced model and spontaneous nonobese diabetic (NOD) mouse model of type I diabetes were used to assess whether nicotine could prevent this autoimmune disease. Blood glucose levels, diabetes incidence, pancreas insulin content, and cytokine levels were measured in both models of diabetes, both to asses the level of protection exerted by nicotine and to further investigate its mechanism of action. Nicotine treatment reduced the hyperglycemia and incidence of disease in both the MLDS and NOD mouse models of diabetes. Nicotine also protected against the diabetes-induced decrease in pancreatic insulin content observed in both animal models. The pancreatic levels of the Th1 cytokines interleukin (IL)-12, IL-1, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma were increased in both MLDS-induced and spontaneous NOD diabetes, an effect prevented by nicotine treatment. Nicotine treatment increased the pancreatic levels of the Th2 cytokines IL-4 and IL-10. Nicotine treatment reduces the incidence of type I diabetes in two animal models by changing the profile of pancreatic cytokine expression from Th1 to Th2.
Asunto(s)
Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Edad de Inicio , Animales , Glucemia/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/genética , Relación Dosis-Respuesta a Droga , Femenino , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Nitratos/sangre , Nitritos/sangre , Páncreas/metabolismoRESUMEN
Nitric oxide is generated under normal and pathophysiological conditions by three distinct isoforms of nitric oxide synthase (NOS). A small-molecule inhibitor of NOS (3-Br-7-nitroindazole, 7-NIBr) is profoundly neuroprotective in mouse models of stroke and Parkinson's disease. We report the crystal structure of the catalytic heme domain of endothelial NOS complexed with 7-NIBr at 1.65 A resolution. Critical to the binding of 7-NIBr at the substrate site is the adoption by eNOS of an altered conformation, in which a key glutamate residue swings out toward one of the heme propionate groups. Perturbation of the heme propionate ensues and eliminates the cofactor tetrahydrobiopterin-heme interaction. We also present three crystal structures that reveal how alterations at the substrate site facilitate 7-NIBr and structurally dissimilar ligands to occupy the cofactor site.
Asunto(s)
Biopterinas/análogos & derivados , Inhibidores Enzimáticos/química , Indazoles/química , Óxido Nítrico Sintasa/química , Nitroarginina/química , Sitios de Unión , Biopterinas/química , Cristalización , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Indazoles/farmacología , Modelos Moleculares , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Conformación Proteica , Especificidad por SustratoRESUMEN
Peroxynitrite has been implicated in the pathogenesis of multiple sclerosis and its animal counterpart experimental allergic encephalomyelitis (EAE). Here we have examined the effects of the novel peroxynitrite scavengers, mercaptoethylguanidine (MEG) and guanidinoethyldisulphide (GED), on the development of EAE. Both MEG and GED delayed EAE onset and decreased the number of animals displaying disease signs. However, when EAE developed, its severity was not significantly abrogated by drug administration. These results suggest that while MEG and GED protect against the induction phase of EAE, they do not prevent disease progression. This may be due to the inability of MEG and GED to efficiently scavenge peroxynitrite or result from their capacity to inhibit inducible nitric oxide synthase. Therefore, the development of more potent and selective scavengers of peroxynitrite is necessary for use in EAE.
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Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Ácido Peroxinitroso/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Mutantes , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Óxido Nítrico/metabolismoRESUMEN
We investigated the effect of mercaptoethylguanidine (MEG, 3 mg kg(-1)h(-1)), a combined selective inducible nitric oxide synthase (iNOS) inhibitor, a peroxynitrite and oxygen free radical scavenger with cyclooxygenase-inhibitor properties on intestinal and hepatic perfusion, O2 exchange, and metabolism during long-term hyperdynamic porcine endotoxemia. MEG was started 12 h after onset of endotoxemia. At baseline and after 12, 18, and 24 h of endotoxemia, hepatic arterial and portal venous blood flow, ileal mucosal-arterial PCO2 gap, portal and hepatic venous lactate/pyruvate ratio, free glutathione (GSH), and 8-isoprostanes were measured. Expired NO and plasma nitrate levels were assessed as well. MEG blunted the endotoxin-induced increase in expired NO and prevented the progressive fall in blood pressure without affecting cardiac output. It attenuated both systemic and regional venous acidosis without influencing the impairment of hepatosplanchnic metabolism nor counteracting the increase in GSH levels. In our model MEG failed to beneficially affect variables of oxidative stress.
Asunto(s)
Endotoxemia/fisiopatología , Hemodinámica/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ácido Peroxinitroso/antagonistas & inhibidores , 6-Cetoprostaglandina F1 alfa/sangre , Animales , Gasto Cardíaco , Endotoxemia/sangre , Escherichia coli , Femenino , Glutatión/sangre , Hemoglobinas/metabolismo , Lipopolisacáridos/toxicidad , Masculino , Óxido Nítrico/análisis , Óxido Nítrico Sintasa de Tipo II , Consumo de Oxígeno , Mecánica Respiratoria , Porcinos , Tromboxano B2/sangre , Resistencia VascularRESUMEN
Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-kappaB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Endotelio Vascular/fisiopatología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Diabetes Mellitus Experimental/enzimología , Endotelio Vascular/enzimología , Activación Enzimática , Glucosa/farmacología , Inmunohistoquímica , Técnicas In Vitro , Ratones , FN-kappa B/metabolismoRESUMEN
OBJECTIVE AND DESIGN: Oxygen- and nitrogen-derived free radicals and oxidants play an important role in the pathogenesis of various forms of inflammation. Recent work emphasizes the importance of oxidant-induced DNA strand breakage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in the pathogenesis of various inflammatory diseases. We have recently demonstrated the efficacy of PJ34, a novel, potent phenanthridinone derivative PARP inhibitor, in rodent models of diabetic vascular dysfunction and stroke. Here we tested the efficacy of PARP inhibition in various models of local inflammation in rodents. MATERIALS AND METHODS: PJ34 (at doses of 0.03-30 mg/kg) was tested in rats and mice subjected to standard models of inflammation, with relevant parameters of inflammation measured using standard methods. RESULTS: PJ34 treatment (s.c, i.p. and i.v.) dose-dependently suppressed neutrophil infiltration and nitric oxide (but not KC and IL-1beta) production in peritonitis. In a model of systemic endotoxemia, PJ34 pretreatment significantly reduced plasma levels of TNF-alpha, IL-1beta and nitrite/nitrate (breakdown products of nitric oxide) production. PJ34 treatment (oral gavage) induced a significant suppression of the inflammatory response in dextran sulfate colitis, multiple low dose streptozotocin diabetes and cyclophosphamide-accelerated autoimmune diabetes in the non-obese diabetic mice, and reduced the degree of mononuclear cell infiltration into the iris in an endotoxin-induced uveitis model. Delaying the start of PJ34 administration in the colitis model conferred significant protective effects, while in the arthritis model the post-treatment paradigm lacked protective effects. CONCLUSIONS: PJ34 provides significant, dose-dependent, anti-inflammatory effects in a variety of local inflammation models. Some of its actions are maintained in the post-treatment regimen and/or after discontinuation of treatment. We conclude that PARP inhibition offers a powerful means for reducing the severity of various forms of local inflammatory responses.
Asunto(s)
Antiinflamatorios/farmacología , Inhibidores Enzimáticos/farmacología , Fenantrenos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Adenosina Trifosfato/metabolismo , Animales , Artritis/tratamiento farmacológico , Colitis/tratamiento farmacológico , Colágeno/inmunología , Diabetes Mellitus Experimental/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Endotoxemia/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos DBA , NAD/metabolismo , Peritonitis/tratamiento farmacológico , Uveítis/tratamiento farmacológicoRESUMEN
BACKGROUND: In previous studies of transgenic sickle cell mice, increased renal expression of inducible nitric oxide synthase (iNOS) and endothelial cell isoform of NOS (EcNOS) was found by Western blot and immunohistochemistry. In addition, putative evidence of peroxynitrite (ONOO-) formation was found in the form of positive immunostaining and immunoblot for nitrotyrosine. Apoptosis was also detected by DNA strand breakage and TUNEL assay. The present study was carried out to examine the role of NO/ONOO- in mediating renal tubular cell apoptosis in sickle cell mouse kidneys. METHODS: Mercaptoethylguanidine (MEG), a compound that selectively inhibits iNOS and also is a scavenger of ONOO-, was administered intraperitoneally over a five-day period to control and betas mice. Immunohistochemistry of iNOS and nitrotyrosine, DNA electrophoresis, ApoTACS assay for apoptosis, and Western blot of poly(ADP-ribose) polymerase (PARP) were carried out. RESULTS: MEG administration virtually eliminated renal immunostaining of iNOS and nitrotyrosine and prevented DNA strand breakage. In addition, Western blot analysis of PARP, a nuclear DNA-reparative enzyme activated in response to DNA strand breakage, was found to be cleavaged in hypoxic betas mice, but was partially protected in MEG-treated betas hypoxic mice. Finally, apoptosis was markedly reduced by MEG in betas hypoxic mice. CONCLUSIONS: These observations provide evidence that NO and/or ONOO- are responsible for initiating cell damage, which leads to apoptosis in sickle cell mouse kidneys.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Alanina Transaminasa/sangre , Anemia de Células Falciformes/complicaciones , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Aspartato Aminotransferasas/sangre , Western Blotting , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/fisiología , Electroforesis en Gel de Agar , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Enfermedades Renales/etiología , Túbulos Renales/enzimología , Túbulos Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nitratos/metabolismo , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tirosina/análogos & derivados , Tirosina/análisis , Tirosina/metabolismoRESUMEN
Extracellular purines, including adenosine and ATP, are potent endogenous immunomodulatory molecules. Inosine, a degradation product of these purines, can reach high concentrations in the extracellular space under conditions associated with cellular metabolic stress such as inflammation or ischemia. In the present study, we investigated whether extracellular inosine can affect inflammatory/immune processes. In immunostimulated macrophages and spleen cells, inosine potently inhibited the production of the proinflammatory cytokines TNF-alpha, IL-1, IL-12, macrophage-inflammatory protein-1alpha, and IFN-gamma, but failed to alter the production of the anti-inflammatory cytokine IL-10. The effect of inosine did not require cellular uptake by nucleoside transporters and was partially reversed by blockade of adenosine A1 and A2 receptors. Inosine inhibited cytokine production by a posttranscriptional mechanism. The activity of inosine was independent of activation of the p38 and p42/p44 mitogen-activated protein kinases, the phosphorylation of the c-Jun terminal kinase, the degradation of inhibitory factor kappaB, and elevation of intracellular cAMP. Inosine suppressed proinflammatory cytokine production and mortality in a mouse endotoxemic model. Taken together, inosine has multiple anti-inflammatory effects. These findings, coupled with the fact that inosine has very low toxicity, suggest that this agent may be useful in the treatment of inflammatory/ischemic diseases.
Asunto(s)
Citocinas/antagonistas & inhibidores , Inmunosupresores/farmacología , Mediadores de Inflamación/antagonistas & inhibidores , Inosina/farmacología , Lipopolisacáridos/toxicidad , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Choque Séptico/inmunología , Choque Séptico/prevención & control , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Quimiocinas/antagonistas & inhibidores , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Proteínas I-kappa B/metabolismo , Mediadores de Inflamación/metabolismo , Inyecciones Intraperitoneales , Inosina/administración & dosificación , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Procesamiento Proteico-Postraduccional/inmunología , Agonistas del Receptor Purinérgico P1 , Receptores Purinérgicos P1/fisiología , Choque Séptico/etiología , Choque Séptico/patología , Células TH1/efectos de los fármacos , Células TH1/metabolismoRESUMEN
Chronic airway inflammation induced by Pseudomonas aeruginosa is the eventual cause of respiratory failure in most people affected by cystic fibrosis. Recent evidence implicates the involvement of free radical and oxidant stress in the pathogenesis of the inflammatory injury. Here we report the efficacy of a novel experimental therapeutic, mercaptoethylguanidine (MEG), which has combined actions as a selective inhibitor of the inducible nitric oxide synthase and as a scavenger of peroxynitrite, a potent oxidant formed in the reaction of nitric oxide and superoxide radical. Chronic pulmonary infection was established in FVB/N mice by intratracheal administration of 10(5) colony-forming units of P. aeruginosa in agar beads. Treatment with MEG (10 mg/kg/dose every 8 h i.p.) inhibited weight loss in the first 3 days and reduced histologic injury at 8 days postinfection. MEG also reduced myeloperoxidase activity, a marker of neutrophil infiltration, at 8 days and concentrations of the proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and macrophage inflammatory protein 2 in whole lung homogenates. MEG-treated animals and controls had similar perioperative mortality and comparable colony counts of P. aeruginosa at 8 days, indicating that MEG did not exacerbate infection. Our data suggest that MEG may be an effective immunomodulatory therapy of pulmonary inflammation induced by chronic infection.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Guanidinas/uso terapéutico , Inflamación/tratamiento farmacológico , Neumonía Bacteriana/tratamiento farmacológico , Neumonía Bacteriana/inmunología , Infecciones por Pseudomonas/inmunología , Anestesia , Animales , Biomarcadores , Peso Corporal/efectos de los fármacos , Citocinas/metabolismo , Femenino , Inflamación/microbiología , Inflamación/mortalidad , Intubación Intratraqueal , Ratones , Infiltración Neutrófila , Nitratos/farmacocinética , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Oxidantes/metabolismo , Peroxidasa/metabolismo , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/patología , Factores de TiempoRESUMEN
Michael reaction of the methoxymethyl-protected monodiazeniumdiolate of piperazine (MOM-PIPERAZI/NO) with 4-maleimidobutyric acid followed by its conversion to the N-hydroxy-succinimido ester produces a reagent capable of transferring the nitric oxide (NO)-donating diazeniumdiolate group to the terminal amines of the lysine residues contained in proteins. The reagent has been used to produce diazeniumdiolated bovine serum albumin (D-BSA) and diazeniumdiolated human serum albumin (D-HSA) containing 22 and 19 modified lysyl groups, respectively. Upon dissolution in pH 7.4 phosphate buffer at 37 degrees C, these albumin derivatives gradually released all of their contained NO (approximately 40 mol/mol of protein) with initial rates of about 30-40 pmol/min/mg and half-lives on the order of 3 weeks. This methodology is now available for use in exploiting the unique specific metabolic interactions of proteins to target NO therapy to specific physiological processes in vivo.
Asunto(s)
Hidrazinas/química , Donantes de Óxido Nítrico/síntesis química , Albúmina Sérica/química , Reactivos de Enlaces Cruzados/química , Humanos , Espectrometría de Masas , Óxido Nítrico/química , Donantes de Óxido Nítrico/química , Piperazinas/química , Albúmina Sérica Bovina/química , Succinimidas/químicaRESUMEN
The involvement of nitric oxide in numerous biological functions has led to the intense study of nitric oxide (NO) generation by the nitric oxide synthases (NOS) responsible. In addition to NO, nitric oxide synthases produce N(G)-hydroxy-L-arginine, superoxide anion and, indirectly, NOx species such as peroxynitrite and, possibly, nitrous oxide (N2O). Consequently, the interactions of N(G)-hydroxy-L-arginine with NO and other oxides of nitrogen (NOx) are of considerable interest. N(G)-Hydroxy-L-arginine and other monosubstituted hydroxyguanidines react with aqueous aerobic NO, peroxynitrite, and various NOx and nitrosating agents to form compounds that subsequently release NO and N2O. Spectrometric data indicate that the nitrosation product of N(G)-hydroxy-L-arginine is of the same N-nitroso-N-hydroxy/diazeniumdiolate (formerly "NONOate") structure as previously found for the nitrosation products of other model hydroxyguanidines. These decompose in aqueous solution in a pH-dependent manner to yield mainly NO and ureas at low pH, N2O and cyanamides at basic pH, and what appear to be primary nitrosamines/ nitrosoimines. Studies on purified iNOS using a mass spectrometer with a gas-permeable membrane inlet identified both NO and N2O (or 15NO and 15N15NO with 15N-labeled L-arginine as substrate) as products of NOS activity. These experiments suggest that much more NO than N2O is produced under the conditions studied and that N2O formation can be rationalized via the reaction of NOx species with N(G)-hydroxy-L-arginine.
Asunto(s)
Guanidinas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/metabolismo , Óxido Nitroso/metabolismo , Arginina/análogos & derivados , Arginina/metabolismo , Cromatografía Líquida de Alta Presión , Guanidina/análogos & derivados , Hidroxilaminas , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , EspectrofotometríaRESUMEN
Hydroxyguanidines (OHGs), including the endogenously formed NG-hydroxy-L-arginine (OH-arg), can react with nitric oxide (NO) and nitrogen oxides (NOx) in vitro. Therefore, we have tested OHGs and related compounds for their ability to scavenge peroxynitrite and to protect against peroxynitrite-induced oxidative processes in cells. Hydroxyguanidine, NG-hydroxy-L-arginine and other N-substituted OHGs, dose-dependently inhibited the in vitro oxidation of dihydrorhodamine (DHR) by peroxynitrite (PN), with similar or better efficacy than glutathione or cysteine. Amidoximes, aminoguanidines and O-substituted OHGs were less effective, and guanidines were without effect. In contrast to their effects on DHR oxidation, OHGs exerted only minimal inhibitory effects on the hydroxylation of benzoate by PN, suggesting that OHGs do not react with the activated isomer of peroxynitrous acid. Selected compounds were tested for protection against PN-induced suppression of mitochondrial respiration and protein oxidation in cultured J774 murine macrophages. Aminoguanidines afforded some protection against the effects of PN, but substituted-phenyl OHGs were considerably more effective. Analysis of the products of the reaction of 4-methoxybenzyl-OHG with PN showed rapid formation of nitrosated derivatives, as well as 4-methoxybenzylcyanamide and a small amount of 4-methoxybenzylurea. Nitric oxide and nitrous oxide were also evolved, but indirectly, arising from the decomposition of one of the nitrosation products. The current results demonstrate that hydroxyguanidines react with PN to protect cells against PN-mediated injury and may be more effective than the endogenous antioxidants cysteine and glutathione.
Asunto(s)
Depuradores de Radicales Libres , Guanidinas/farmacología , Nitratos/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Ácido Benzoico/química , Línea Celular , Guanidinas/química , Hidroxilaminas , Hidroxilación , Macrófagos/efectos de los fármacos , Nitratos/química , Oxidación-Reducción , Rodaminas/químicaRESUMEN
A series of substituted aminoguanidines and amino-substituted isothioureas have been examined as inhibitors of nitric oxide (NO) synthase (NOS) isoforms. Each of the agents produced a time- and concentration-dependent inactivation of the NO-forming activity of the affinity-purified NOS isoforms. These inactivations required exposure of NOS to the drug under conditions that supported catalysis, consistent with the proposal that they act as alternate substrate, mechanism-based inactivators. Of the aminoguanidines examined, 2-ethylaminoguanidine was the most efficient inactivator, exhibiting vs. iNOS an apparent KI value of 120 microM as measured at 100 microM arginine and a k(inact max) value of 0.48 min(-1) and thus an apparent second-order rate constant for inactivation of 4.0 mM(-1)min(-1). 2-Ethylaminoguanidine displayed a high isoform selectivity for the iNOS compared with the nNOS and eNOS isoforms. 2-Ethylaminoguanidine inactivated NO synthetic activity in cytokine-induced RAW 264.7 cells as measured at 100 microM extracellular arginine with an apparent KI value of 55 microM and a k(inact max) value of 0.09 min(-1). The inactivated RAW 264.7 cell NO synthetic capability was restored over a 3-hr period after drug removal to a value 60% of its pretreatment value. This recovery occurred despite the presence of cycloheximide sufficient to inhibit protein synthesis by >99%. 1-Amino-S-methylisothiourea by contrast with the aminoguanidines was identified as a mechanism-based inactivator selective for the nNOS isoform. In contrast to S-isopropylisothiourea, which was found to be both cell penetrant and reversible, 1-amino-S-methylisothiourea appeared cell impermeable and inhibited NOS enzyme "irreversibly."
Asunto(s)
Guanidinas/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Tiourea/farmacología , Animales , Bovinos , Citocinas/farmacología , Cinética , Ratones , NADPH Oxidasas/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Relación Estructura-ActividadRESUMEN
Guanidines, amidines, S-alkylisothioureas, and other compounds containing the amidine function (-C(=NH)NH2) have been described as inhibitors of the generation of nitric oxide (NO) by NO synthase (NOS). Here we report on the inhibition of the activity of NOS isoforms by compounds in which the amidine function is attached to a nitrogen of 1,2-diazo heterocycles to form N-carboxamidines and related compounds. 1H-Pyrazole-1-carboxamidine HCl (PCA) inhibited the activity of purified inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) isoforms to a similar extent (IC50 = 0.2 microM). 3-Methyl-PCA and 4-methyl-PCA showed reduced potencies, but a preference for iNOS [IC50 = 5 and 2.4 microM, respectively; cf. N(G)-methyl-L-arginine (NMA) IC50 = 6 microM]. Inhibition of purified iNOS by PCAs could be reversed completely by excess L-arginine, while their inhibition of NO production by stimulated RAW macrophages could be reversed by transfer to a drug-free medium. This suggests a competitive mode of inhibition. PCA caused potent concentration-dependent inhibition of the acetylcholine-induced, endothelium-dependent relaxations of precontracted rat thoracic aorta (IC50 = 30 microM). 4-Methyl-PCA inhibited the relaxations only at > or = 300 microM. In contrast, 4-methyl-PCA was more effective than both PCA and NMA in restoring the ex vivo contractility of aortic rings taken from lipopolysaccharide-treated rats. PCA and NMA, but not 4-methyl-PCA, caused marked increases in mean arterial pressure when administered i.v. to anesthetized rats. In conclusion, PCA and related compounds caused potent inhibition of NOS. Substitution of the pyrazole ring reduced potency, but improved selectivity towards iNOS as exemplified by 4-methyl-PCA.
Asunto(s)
Óxido Nítrico Sintasa/antagonistas & inhibidores , Pirazoles/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Sitios de Unión , Presión Sanguínea/efectos de los fármacos , Bovinos , Línea Celular , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Ratones , Óxido Nítrico/análisis , Óxido Nítrico Sintasa/aislamiento & purificación , Pirazoles/administración & dosificación , Pirazoles/síntesis química , RatasRESUMEN
Nitric oxide (NO) produced by the inducible nitric-oxide synthase (iNOS) is responsible for some of the pathophysiological alterations during inflammation. Part of NO-related cytotoxicity is mediated by peroxynitrite, an oxidant species produced from NO and superoxide. Aminoguanidine and mercaptoethylguanidine (MEG) are inhibitors of iNOS and have anti-inflammatory properties. Here we demonstrate that MEG and related compounds are scavengers of peroxynitrite. MEG caused a dose-dependent inhibition of the peroxynitrite-induced oxidation of cytochrome c2+, hydroxylation of benzoate, and nitration of 4-hydroxyphenylacetic acid. MEG reacts with peroxynitrite with a second-order rate constant of 1900 +/- 64 M-1 s-1 at 37 degrees C. In cultured macrophages, MEG reduced the suppression of mitochondrial respiration and DNA single strand breakage in response to peroxynitrite. MEG also reduced the degree of vascular hyporeactivity in rat thoracic aortic rings exposed to peroxynitrite. The free thiol plays an important role in the scavenging effect of MEG. Aminoguanidine neither affected the oxidation of cytochrome c2+ nor reacted with ground state peroxynitrite, but inhibited the peroxynitrite-induced benzoate hydroxylation and 4-hydroxyphenylacetic acid nitration, indicating that it reacts with activated peroxynitrous acid or nitrogen dioxide. Compounds that act both as iNOS inhibitors and peroxynitrite scavengers may be useful anti-inflammatory agents.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Nitratos/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Línea Celular , Cinética , Modelos Químicos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Norepinefrina/farmacología , Oxidación-Reducción , Consumo de Oxígeno , Ratas , Vasoconstrictores/farmacologíaRESUMEN
1. It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes. Aminoguanidine, NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes. 2. Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 micrograms ml-1) and interferon gamma (IFN gamma, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 microM) for 6 h. This treatment induced a significant production of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF1 alpha, with an IC50 of 20 microM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF1 alpha in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 microM) for 30 min; this also induced a significant production of 6-keto-PGF1 alpha and MEG (1 microM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF1 alpha. Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF1 alpha production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 microM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins.5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF1alpha production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF1alpha production in the HUVECs. 6.The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33microM and 36microM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3mM) decreased COX-1 activity only, while L-NMA (up to 3mM) had no effect on the activity of either enzyme. 7.These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.
Asunto(s)
Inhibidores de la Ciclooxigenasa/síntesis química , Guanidinas/síntesis química , 6-Cetoprostaglandina F1 alfa/metabolismo , Ácido Araquidónico/farmacología , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Activación Enzimática/efectos de los fármacos , Guanidinas/farmacología , Humanos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitritos/metabolismo , Estimulación Química , Especificidad por Sustrato , Tromboxano B2/metabolismoRESUMEN
Novel, non-arginine based compounds have been identified as potent inhibitors of nitric oxide synthase (NOS). Members of the isothiourea and mercapto-alkylguanidine classes have generated much interest, as some members of these classes show selectivity towards the inducible isoform of NOS (iNOS), which plays a role in inflammation and shock. Here we compared the effect of a number of these compounds as well as L-arginine based NOS inhibitor reference compounds on macrophage-derived and liver arginase and macrophage iNOS activities. From the non-arginine based NOS inhibitors studied only S-aminoethyl-isothiourea (AETU) caused a slight inhibition of arginase activity. This inhibition was kinetically competitive and due to the rearrangement of AETU to mercapto-ethylguanidine (MEG). The weak inhibitory effect of non-arginine based iNOS inhibitors on arginase activity further supports the view that such compounds may be of practical use for inhibition of NO production in cells simultaneously expressing iNOS and arginase.