RESUMEN
Diabetes mellitus (DM) is associated with increased risk of impaired cognitive function. Diabetic neuropathy is one of the most common and important complications of DM. Estrogens prevent neuronal loss in experimental models of neurodegeneration and accelerate nerve regeneration. Aromatase catalyzes the conversion of androgens to estrogens and expressed in a variety of tissues including neurons. Although insulin is known to regulate the activity of aromatase there is no study about the effects of diabetes on this enzyme. Present study was designed to investigate the effects of experimental diabetes on aromatase expression in nervous system. Gender-based differences were also investigated. Rats were injected with streptozotocin to induce diabetes. At the end of 4 and 12 weeks sciatic nerve and hippocampus homogenates were prepared and evaluated for aromatase proteins. Aromatase expressions in sciatic nerves of both genders were decreased in 4 weeks of diabetes, but in 12 weeks the enzyme levels were increased in females and reached to control levels in male animals. Aromatase levels were not altered in hippocampus at 4 weeks but increased at 12 weeks in female diabetic rats. No significant differences were observed at enzyme levels of hippocampus in male diabetic rats. Insulin therapy prevented all diabetes-induced changes. In conclusion, these results indicated for the first time that, DM altered the expression of aromatase both in central and peripheral nervous systems. Peripheral nervous system is more vulnerable to damage than central nervous system in diabetes. These effects of diabetes differ with gender and compensatory neuroprotective mechanisms are more efficient in female rats.
Asunto(s)
Aromatasa/metabolismo , Encefalopatías Metabólicas/enzimología , Trastornos del Conocimiento/enzimología , Citoprotección/fisiología , Complicaciones de la Diabetes/enzimología , Estrógenos/biosíntesis , Animales , Aromatasa/análisis , Encefalopatías Metabólicas/etiología , Encefalopatías Metabólicas/fisiopatología , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/fisiopatología , Complicaciones de la Diabetes/fisiopatología , Diabetes Mellitus Experimental , Neuropatías Diabéticas/enzimología , Neuropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Femenino , Hipocampo/enzimología , Hipocampo/fisiopatología , Insulina/farmacología , Masculino , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/etiología , Degeneración Nerviosa/fisiopatología , Regeneración Nerviosa/fisiología , Fármacos Neuroprotectores/metabolismo , Ratas , Ratas Sprague-Dawley , Nervio Ciático/enzimología , Nervio Ciático/fisiopatología , Caracteres SexualesRESUMEN
Disturbance in cholesterol homeostasis appears to be an important factor in the pathogenesis of neurodegenerative disorders. The aim of the present study was to investigate sterol regulatory element binding protein (SREBP) levels in the nuclear extracts of human neuroblastoma cells and the possible interaction of beta-amyloid peptide (Abeta) and cholesterol with this transcription factor. In this study, cultured human neuroblastoma cells (SHSY-5Y) were incubated in serum-deprived media in the presence or absence of Abeta((25-35)) (1 microM) or cholesterol (300 microM) for 24 h. Nuclear extracts were subjected to SDS-PAGE, and SREBP cleavage product (68 kDa) was detected by immunoblotting. SREBP levels were elevated in the cells incubated 24 h in serum-deprived experimental media and were attenuated by Abeta or cholesterol-supplementation. It is likely that the ability of Abeta to release cholesterol into the medium and downregulate SREBP is due to a feedback mechanism.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Núcleo Celular/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Extractos Celulares , Línea Celular Tumoral , Supervivencia Celular , Colesterol/farmacología , Medio de Cultivo Libre de Suero , Humanos , NeuroblastomaRESUMEN
1. The analogies between the processing of amyloid precursor protein (APP) and other transmembrane sterol regulatory element binding proteins (SREBPs) inspired us to conduct further studies on whether beta-amyloid (Abeta) affects aromatase by interacting with APP and SREBP. 2. In this study, cultured human neuroblastoma cells (SHSY-5Y) were incubated in experimental media (media without FBS, the main cholesterol source) in the presence or absence of Abeta (1 microM) for 24 h. 3. Cellular extracts were subjected to immunoblot analysis using anti-APP, anti-aromatase and anti-SREBP-1. In these cell lines, we detected aromatase (55 kDa), SREBP cleavage product (68 kDa) and APP precursor (100-95 kDa) and cleavage product (60 kDa) by immunoblotting. Aromatase and SREBP levels were elevated in the cells incubated 24 h in experimental media and were attenuated in Abeta-supplemented experimental media. 4. The disturbance of cholesterol homeostasis appears to be an important factor in the pathogenesis of Alzheimer's disease. These findings may have important implications for understanding the mechanisms of the aromatase enzyme gene in disease states such as Alzheimer's.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Aromatasa/metabolismo , Retroalimentación Fisiológica , Neuroblastoma/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Aromatasa/química , Línea Celular Tumoral , Medios de Cultivo/química , Humanos , Peso Molecular , Proteínas de Unión a los Elementos Reguladores de Esteroles/químicaRESUMEN
Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.
Asunto(s)
Astrocitos/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Citocromo P-450 CYP2E1/biosíntesis , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 3 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , RatasRESUMEN
Structural modification at one of the guanidine nitrogens of L-arginine has led to the development of a number of compounds N(G)-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine (L-NOARG), N(G)-nitro-L-arginine methyl ester (L-NAME) that competitively inhibit nitric oxide synthase (NOS). It was reported that another chemically related compound known as a glycation inhibitor, aminoguanidine also inhibits NOS. Recently, two new glycation inhibitors, structurally related to aminoguanidine (AG), pyridoxal aminoguanidine (PLAG) and 8-quinoline carboxylic hydrazide (8Q) were synthesized. In this study, the effects of these two inhibitors on responses mediated by constitutive nitric oxide (NO) were investigated in vitro. For this purpose, in the present study vascular responses to phenylephrine and acetylcholine in isolated aortas were evaluated. Incubation (15 min) with PLAG and 8Q (10(-4)M for each) induced potentiation of phenylephrine-induced contraction in endothelium intact but not in endothelium denuded rings of rat aorta. The ability of PLAG or 8Q to augment phenylephrine-induced tone in endothelium containing rings was completely prevented by preincubation with L-arginine (1mM), but not with D-arginine. Both compounds (PLAG, 8Q) did not affect acetylcholine-induced relaxation. These results suggest that both of the new compounds produced a selective inhibition of basal but not agonist stimulated production of nitric oxide in rat aorta.
Asunto(s)
Aorta Torácica/metabolismo , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Quinolinas/farmacología , Acetilcolina/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/agonistas , Nitroarginina/farmacología , Fenilefrina/farmacología , Ratas , Ratas Wistar , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
We investigated the effect of omeprazole (1 x 10(-5)-3 x 10(-4)M), an inhibitor of H(+),K(+)-ATPase, on rat aortic rings pre-contracted with phenylephrine (10(-6)M). Omeprazole relaxed the tissue in a concentration-dependent manner. Either removal of the endothelium or incubation with nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5)M) significantly attenuated the relaxations. Pre-treatment with L-arginine (10(-3)M), but not with D-arginine, reversed the inhibitory action of L-NAME. Indomethacin (10(-6)M) and tetraethylammonium (TEA, 10(-2)M) did not affect the relaxant responses to omeprazole indicating the lack of involvement of cyclooxygenase products and K(+) channels, respectively. These results suggest a role of NO in the mechanism of action of omeprazole.
Asunto(s)
Endotelio Vascular/fisiología , Inhibidores Enzimáticos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Omeprazol/farmacología , Inhibidores de la Bomba de Protones , Acetilcolina/farmacología , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Técnicas In Vitro , Indometacina/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Papaverina/farmacología , Fenilefrina/antagonistas & inhibidores , Fenilefrina/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Tetraetilamonio/farmacología , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
The spin-trap, alpha-phenyl-N-tert-butylnitrone (PBN) has been shown to have neuroprotective properties and may prevent oxidative injury in vivo and in cultured cells. Although PBN quenches reactive oxygen species, the direct mechanism of neuroprotective action is unknown. In the present study, we examined the effects of PBN on the regulation of the mitogen activated kinase Erk and as well as Src family tyrosine kinases, enzymes known to be activated by oxygen species such as H2O2. In SH-SY5Y human neuroblastoma cells, H2O2 induced activation of Erk and Src kinases was markedly potentiated by treatment with PBN. The potentiation by PBN of the Erk and Src kinase activation by H2O2 required extracellular Ca2+ and appeared dependent on voltage sensitive Ca2+ channels. In contrast, PBN did not affect depolarization-dependent or growth factor-dependent Erk and Src kinase phosphorylation. Our results suggest that PBN might have a protective effect on cells by potentiating the anti-apoptotic Erk and Src kinase pathways responding to H2O2, an effect apparently distinct from its ability to trap oxygen free radicals.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Óxidos de Nitrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Familia-src Quinasas/efectos de los fármacos , Calcio/metabolismo , Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/enzimología , Sistema Nervioso Central/fisiopatología , Óxidos N-Cíclicos , Interacciones Farmacológicas/fisiología , Factor de Crecimiento Epidérmico/farmacología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroblastoma , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Estrés Oxidativo/fisiología , Cloruro de Potasio/farmacología , Células Tumorales Cultivadas , Familia-src Quinasas/metabolismoRESUMEN
Sixteen 1-(2-naphthyloxyacetyl)-4-substituted-3-thiosemicarbazide, 2-(2-naphthyloxymethyl)-5-substitutedamino-1,3,4-oxadiazole, 2-(2-naphthyloxymethyl)-5-substitutedamino-1,3,4-thiadiazole and 5-(2-naphthyloxymethyl)-4-substituted-1,2,4-triazole-3thione derivatives have been prepared and evaluated as orally active anti-inflammatory agents with reduced side-effects. The structures of the compounds were confirmed by IR and 1H NMR spectral data and microanalysis. The anti-inflammatory and ulcerogenic activities of the compounds were compared with naproxen, indomethacin and phenylbutazone. In carrageenan-induced foot pad edema assay, 2-(2-naphthyloxymethyl)-5-methylamino-1,3,4-oxadiazole, 5-(2-naphthyloxymethyl)-4-methyl-1,2,4-triazole-3-thione and 5-(2-naphthyloxymethyl)-4-ethyl-1,2,4-triazole-3-thione showed an interesting anti-inflammatory activity. In the air-pouch test, 1,3,4-oxadiazole and 1,2,4-triazole-3-thione derivatives reduced total number of leukocytes of the exudate that indicates excellent inhibition of prostaglandin production. Side effects of the compounds were examined on gastric mucosa, liver and stomach and none of the compounds showed significant side effects compared with reference nonsteroidal anti-inflammatory drugs (NSAIDs).