RESUMEN
Biosynthesis of Ds(+)-threo-isocitric acid from ethanol in the Yarrowia lipolytica batch and repeated-batch cultures was studied. Repeated-batch cultivation was found to provide for a good biosynthetic efficiency of the producer for as long as 748 h, probably due to maintenance of high activities of enzymes involved in the biosynthesis of isocitric acid. Under optimal repeated-batch cultivation conditions, the producer accumulated 109.6 g/L Ds(+)-threo-isocitric acid with a production rate of 1.346 g/L h. The monopotassium salt of isocitric acid isolated from the culture liquid and purified to 99.9% was found to remove neurointoxication, to restore memory, and to improve the learning of laboratory rats intoxicated with lead and molybdenum salts. Taking into account the fact that the neurotoxic effect of heavy metals is mainly determined by oxidative stress, the aforementioned favorable action of isocitric acid on the intoxicated rats can be explained by its antioxidant activity among other pharmacological effects.
Asunto(s)
Antioxidantes/metabolismo , Antioxidantes/farmacología , Técnicas de Cultivo Celular por Lotes/métodos , Isocitratos/metabolismo , Isocitratos/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/aislamiento & purificación , Reactores Biológicos/microbiología , Etanol/metabolismo , Isocitratos/aislamiento & purificación , Masculino , Metales Pesados/toxicidad , Ratas , Yarrowia/metabolismoRESUMEN
The effect of biologically active form (threo-Ds-) of isocitric acid (ICA) on oxidative stress was studied using the infusorian Paramecium caudatum stressed by hydrogen peroxide and salts of some heavy metals (Cu, Pb, Zn, and Cd). ICA at concentrations between 0.5 and 10 mM favorably influenced the infusorian cells with oxidative stress induced by the toxicants studied. The maximal antioxidant effect of ICA was observed at its concentration 10 mM irrespective of the toxicant used (either H2O2 or heavy metal ions). ICA was found to be a more active antioxidant than ascorbic acid. Biologically active pharmaceutically pure threo-Ds-ICA was produced through cultivation of the yeast Yarrowia lipolytica and isolated from the culture liquid in the form of crystalline monopotassium salt with a purity of 99.9%.
Asunto(s)
Isocitratos/metabolismo , Paramecium caudatum/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacología , Medios de Cultivo/metabolismo , Peróxido de Hidrógeno/metabolismo , Isocitratos/farmacología , Metales Pesados/metabolismo , Estrés Oxidativo/efectos de los fármacos , Paramecium caudatum/efectos de los fármacos , Yarrowia/metabolismoRESUMEN
A new drimane-type sesquiterpene with an isocitric acid moiety, cryptoporic acid S (1), together with six known compounds, cryptoporic acid D (2), ß-sitosterol (3), ß-daucosterol (4), stigmast-4-en-3-one (5), ergosterol (6), and (22E,24R)-ergosta-7,22-diene-3ß,5α,6ß-triol (7), was isolated from the fruiting bodies of Cryptoporus volvatus. The structures of these compounds were established on the basis of UV, IR, MS, 1D and 2D NMR analysis. In the meanwhile, compounds 1 and 2 were evaluated for antioxidant activity using the methods of 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity (DPPH-RSA) and ferric reducing antioxidant power (FRAP) assay, and they exhibited moderate antioxidant activities.
Asunto(s)
Antioxidantes/aislamiento & purificación , Coriolaceae/química , Isocitratos/aislamiento & purificación , Sesquiterpenos/aislamiento & purificación , Antioxidantes/química , Antioxidantes/farmacología , Compuestos de Bifenilo/farmacología , China , Ergosterol/química , Éteres , Cuerpos Fructíferos de los Hongos/química , Isocitratos/química , Isocitratos/farmacología , Estructura Molecular , Picratos/farmacología , Sesquiterpenos Policíclicos , Sesquiterpenos/química , Sesquiterpenos/farmacología , Sitoesteroles/química , Estigmasterol/análogos & derivados , Estigmasterol/química , Estigmasterol/aislamiento & purificaciónRESUMEN
Breathing is a vital function generated and controlled by a brainstem neural network, which is able to adjust its function to fit different metabolic demands. For instance, the pre-Bötzinger complex (preBötC) can respond to low oxygen availability (hypoxia) by an initial increase in rhythm frequency followed by a decrease in respiratory efforts that leads to gasping generation. Gasping is essential for autoresuscitation, which has motivated studies of the cellular mechanisms involved in these processes. Hypoxia has different effects on enzymes that participate in the Krebs cycle. In particular, aconitase is downregulated, whereas isocitrate dehydrogenase is unaffected or upregulated under hypoxic conditions. We hypothesized that the application of isocitrate, the product of aconitase and the substrate of isocitrate dehydrogenase as well as an alternative metabolic substrate, might enhance breathing and render it more resistant to hypoxic insult. We tested the effects of isocitrate applied on brainstem slices containing the preBötC as well as its central effects in vivo using plethysmography. Our results show that isocitrate increases the frequency of fictive eupnea and fictive gasping produced by the preBötC in vitro. Moreover, isocitrate increases the amplitude of ventilation in vivo in normoxia, increases ventilation during gasping, and favors autoresuscitation when animals were subjected to asphyxiation. In conclusion, we have found that isocitrate improves ventilation under both normoxic and hypoxic conditions through a mechanism that involves the preBötC and possibly other respiratory neural networks. Thus, isocitrate would be useful to avoid the failure of gasping generation and autoresuscitation in pathological conditions.
Asunto(s)
Asfixia/prevención & control , Isocitratos/farmacología , Recuperación de la Función/efectos de los fármacos , Respiración/efectos de los fármacos , Centro Respiratorio/efectos de los fármacos , Animales , Animales Recién Nacidos , Hipoxia/tratamiento farmacológico , Hipoxia/fisiopatología , Técnicas In Vitro , Ratones , Oxígeno/administración & dosificación , Pletismografía , Centro Respiratorio/metabolismo , Factores de TiempoRESUMEN
Phosphofructokinase (PFK-1) activity was examined in L(3) and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4±0.2 to 1.7±0.2 in L(3)s and 2.0±0.3 to 2.4±0.4 in adults) and the K(½) for fructose 6 phosphate (from 0.35±0.02 to 0.75±0.05mM in L(3)s and 0.40±0.03 to 0.65±0.05mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.
Asunto(s)
Fosfofructoquinasa-1/metabolismo , Trichostrongyloidea/enzimología , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Ácido Cítrico/farmacología , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Fructosadifosfatos/farmacología , Fructosafosfatos/metabolismo , Isocitratos/farmacología , Cinética , Larva/enzimología , Malatos/farmacología , Fosfofructoquinasa-1/antagonistas & inhibidores , Fosforilación , OvinosRESUMEN
For centuries, Amaranthus sp. were used as food, ornamentals, and medication. Molecular mechanisms, explaining the health beneficial properties of amaranth, are not yet understood, but have been attributed to secondary metabolites, such as phenolic compounds. One of the most abundant phenolic compounds in amaranth leaves is 2-caffeoylisocitric acid (C-IA) and regarding food occurrence, C-IA is exclusively found in various amaranth species. In the present study, the anti-inflammatory activity of C-IA, chlorogenic acid, and caffeic acid in LPS-challenged macrophages (RAW 264.7) has been investigated and cellular contents of the caffeic acid derivatives (CADs) were quantified in the cells and media. The CADs were quantified in the cell lysates in nanomolar concentrations, indicating a cellular uptake. Treatment of LPS-challenged RAW 264.7 cells with 10 µM of CADs counteracted the LPS effects and led to significantly lower mRNA and protein levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin 6, by directly decreasing the translocation of the nuclear factor κB/Rel-like containing protein 65 into the nucleus. This work provides new insights into the molecular mechanisms that attribute to amaranth's anti-inflammatory properties and highlights C-IA's potential as a health-beneficial compound for future research.
Asunto(s)
Amaranthus/química , Antiinflamatorios/farmacología , Ácidos Cafeicos/farmacología , Isocitratos/farmacología , FN-kappa B/metabolismo , Animales , Ácidos Cafeicos/química , Citocinas/metabolismo , Isocitratos/química , Lipopolisacáridos/efectos adversos , Ratones , Extractos Vegetales/química , Extractos Vegetales/farmacología , Células RAW 264.7 , Transducción de Señal/efectos de los fármacosRESUMEN
Iron-restricted human anemias are associated with the acquisition of marrow resistance to the hematopoietic cytokine erythropoietin (Epo). Regulation of Epo responsiveness by iron availability serves as the basis for intravenous iron therapy in anemias of chronic disease. Epo engagement of its receptor normally promotes survival, proliferation, and differentiation of erythroid progenitors. However, Epo resistance caused by iron restriction selectively impairs proliferation and differentiation while preserving viability. Our results reveal that iron restriction limits surface display of Epo receptor in primary progenitors and that mice with enforced surface retention of the receptor fail to develop anemia with iron deprivation. A mechanistic pathway is identified in which erythroid iron restriction down-regulates a receptor control element, Scribble, through the mediation of the iron-sensing transferrin receptor 2. Scribble deficiency reduces surface expression of Epo receptor but selectively retains survival signaling via Akt. This mechanism integrates nutrient sensing with receptor function to permit modulation of progenitor expansion without compromising survival.
Asunto(s)
Eritropoyesis/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hierro/farmacología , Proteínas de la Membrana/metabolismo , Receptores de Eritropoyetina/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Catepsinas/metabolismo , Línea Celular , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/ultraestructura , Humanos , Isocitratos/farmacología , Ratones Endogámicos C57BL , Modelos Biológicos , Estabilidad Proteica/efectos de los fármacos , Receptores de Transferrina/metabolismoRESUMEN
Intracellular accumulation of free zinc contributes to neuronal death in brain injuries such as ischemia and epilepsy. Pyruvate, a glucose metabolite, has been shown to block zinc neurotoxicity. However, it is largely unknown how pyruvate shows such a selective and remarkable protective effect. In this study, we sought to find a plausible mechanism of pyruvate protection against zinc toxicity. Pyruvate almost completely blocked cortical neuronal death induced by zinc, yet showed no protective effects against death induced by calcium (ionomycin, NMDA) or ferrous iron. Of the TCA cycle intermediates, citrate, isocitrate, and to a lesser extent oxaloacetate, protected against zinc toxicity. We then noted with LC-MS/MS assay that exposure to pyruvate, and to a lesser degree oxaloacetate, increased levels of citrate and isocitrate, which are known zinc chelators. While pyruvate added only during zinc exposure did not reduce zinc toxicity, citrate and isocitrate added only during zinc exposure, as did extracellular zinc chelator CaEDTA, completely blocked it. Furthermore, addition of pyruvate after zinc exposure substantially reduced intracellular zinc levels. Our results suggest that the remarkable protective effect of pyruvate against zinc cytotoxicity may be mediated indirectly by the accumulation of intracellular citrate and isocitrate, which act as intracellular zinc chelators.
Asunto(s)
Quelantes/farmacología , Ácido Cítrico/farmacología , Isocitratos/farmacología , Fármacos Neuroprotectores/farmacología , Ácido Pirúvico/farmacología , Zinc/toxicidad , Animales , Células Cultivadas , Técnicas de Cocultivo , Ratones , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculate mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with gx values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11beta-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spinal signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spinal parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and a high spin ferric gxy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas/metabolismo , Mitocondrias/metabolismo , Aminoglutetimida/farmacología , Animales , Calcio/farmacología , Desoxicorticosterona/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Concentración de Iones de Hidrógeno , Isocitratos/farmacología , Metirapona/farmacología , Pregnenolona/farmacología , Ratas , Succinatos/farmacologíaRESUMEN
Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.
Asunto(s)
Cuerpo Lúteo/enzimología , Isocitrato Deshidrogenasa/aislamiento & purificación , NADP/farmacología , Animales , Western Blotting , Cationes Bivalentes , Fenómenos Químicos , Química Física , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Glicerol/farmacología , Concentración de Iones de Hidrógeno , Isocitrato Deshidrogenasa/metabolismo , Isocitratos/farmacología , Cinética , Cloruro de Magnesio/farmacología , Peso Molecular , Desnaturalización Proteica , PorcinosRESUMEN
Insulin secretion from ß cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing ß cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues ß cell function in T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/fisiopatología , Endopeptidasas/fisiología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Isocitratos/metabolismo , Animales , Dominio Catalítico , Membrana Celular/metabolismo , Cisteína Endopeptidasas , Diabetes Mellitus Tipo 2/patología , Endopeptidasas/biosíntesis , Endopeptidasas/deficiencia , Endopeptidasas/genética , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Técnicas de Inactivación de Genes , Glucosa/metabolismo , Glucosa/farmacología , Glutatión/farmacología , Células HEK293 , Homeostasis , Humanos , Insulina/farmacología , Secreción de Insulina , Islotes Pancreáticos/fisiopatología , Isocitrato Deshidrogenasa/fisiología , Isocitratos/farmacología , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , NADP/metabolismo , Especificidad de Órganos , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Secretoras/metabolismo , Transducción de Señal , SumoilaciónRESUMEN
Previous investigations have established that spironolactone (SL) administration to guinea pigs decreases adrenal mitochondrial and microsomal cytochrome P-450 content, and that the latter requires microsomal activation of the drug. Studies were carried out to determine if adrenal mitochondrial metabolism (activation) of SL was similarly involved in the effects of the drug on mitochondrial cytochrome P-450 destruction. Incubation of guinea pig adrenal mitochondria with SL in the absence of NADPH resulted in the formation of 7 alpha-thio-SL as the only metabolite. In the presence of an NADPH-generating system, an unknown polar metabolite was also produced. The mass spectrum of the unknown compound suggested that it was a hydroxylated derivative of SL. Incubation of mitochondrial preparations with 7 alpha-thio-SL also resulted in the formation of a polar metabolite, but the latter had a different HPLC retention time than that of the SL metabolite. Formation of the polar SL metabolite was prevented by metyrapone, an 11 beta-hydroxylase inhibitor, and was greatest in mitochondria from the adrenal zone having the highest 11 beta-hydroxylase activity. Steroid substrates for 11 beta-hydroxylation inhibited the production of the SL metabolite. Mitochondrial incubations with SL or with 7 alpha-thio-SL in the presence or absence of an NADPH-generating system did not affect cytochrome P-450 concentrations. The results indicate that, unlike the microsomal effects of SL, local activation of SL is not responsible for the destruction of adrenal mitochondrial cytochromes P-450. The major adrenal mitochondrial metabolites of SL appear to be 11 beta-hydroxy-SL and 7 alpha-thio-SL.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Mitocondrias/metabolismo , Espironolactona/metabolismo , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Cobayas , Técnicas In Vitro , Isocitratos/farmacología , Masculino , Espectrometría de MasasRESUMEN
Rat adrenal mitochondria contain approximately equal levels of P450scc and P45011 beta, each reduced by NADPH through adrenodoxin reductase (ADX-reductase) and adrenodoxin (ADX). Constitutive cholesterol side-chain cleavage (SCC) can be increased over 20-fold through a combination of hormonal activation and inhibition of cholesterol metabolism in vivo prior to isolation of the mitochondria. This stimulation, which results from accumulated reactive cholesterol, does not significantly affect either the dependence of activities on the concentration of isocitrate (IC) and succinate (SU) or the ratio of maximum activities [3:1] supported by these reductants. Thus, the rate of cholesterol SCC is determined independently by electron transfer and the amount of reactive cholesterol. Hydroxylation of deoxycorticosterone (11 beta and 18 positions) required much higher levels of each reductant, indicating less effective reductant transfer to P45011 beta. Reactions at P450scc and P45011 beta, mediated by IC, are enhanced by low concentrations of various dicarboxylates anions (fumarate, SU). The actions of SU dehydrogenase inhibitors and the activity of fumarate, a poor direct reductant, suggest that higher production of NADPH results from malate-enhanced uptake of isocitrate. Only synergistic combinations of reductants are sufficient to sustain maximum rates of 11-deoxycorticosterone (DOC) metabolism, whereas IC is fully effective for P450scc. Increased reaction at P450scc (cholesterol loading or addition of 20 alpha-hydroxycholesterol) decreased simultaneous DOC metabolism at P45011 beta in inverse proportion to the estimated intramitochondrial generation of NADPH (1 mM or 10 mM SU > 1 mM IC > 10 mM IC). These decreases were reversed by inhibition of P450scc. Crossover inhibition caused by maximum DOC metabolism was less pronounced. EGTA/albumin treatment, which enhanced activities at both P450scc and P45011 beta, presumably via increased NADPH, diminished this cross-competition. The differential dependence on reductants and the characteristics of crossover competition are consistent with a roughly three-fold more favorable partitioning of electron transfer to P450scc, possibly caused by preferential interaction of reduced adrenodoxin with P450scc.
Asunto(s)
Corteza Suprarrenal/enzimología , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Transporte de Electrón , Isoenzimas/metabolismo , Mitocondrias/enzimología , Esteroide 11-beta-Hidroxilasa/metabolismo , Corteza Suprarrenal/ultraestructura , Adrenodoxina/metabolismo , Aminoglutetimida/análogos & derivados , Aminoglutetimida/farmacología , Animales , Colesterol/metabolismo , Cosintropina/farmacología , Desoxicorticosterona/metabolismo , Femenino , Ferredoxina-NADP Reductasa/metabolismo , Isocitratos/farmacología , Metirapona/farmacología , NADP/metabolismo , Oxidación-Reducción , Pregnenolona/biosíntesis , Ratas , Ratas Sprague-Dawley , Succinatos/farmacología , Ácido SuccínicoRESUMEN
BACKGROUND: Insulin inhibits contraction and migration of primary confluent, cultured canine vascular smooth muscle cells (VSMCs) with inducible nitric oxide synthase (iNOS) by stimulating cyclic GMP (cGMP) production. The present study was performed to determine how insulin stimulates guanylate cyclase activity in these cells. METHODS: Primary cultured VSMC were obtained from canine femoral arteries. Lactate and pyruvate levels were measured by enzymatic assays, cGMP production by radioimmunoassay, iNOS activity by conversion of arginine to citrulline, and cell contraction by photomicroscopy. RESULTS: Insulin (1 nmol/L) increased cGMP production fivefold in VSMC with iNOS while raising the lactate-to-pyruvate ratio (LPR) from 3.1 +/- 0.5 to 10.0 +/- 1.6 (P < .05), indicating a rise in the ratio of reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD+) redox state of the cell. Insulin's stimulation of cGMP production was blocked by 0.1 mmol/L NG-monomethyl-L-arginine (L-NMMA) indicating dependence on iNOS activity, but insulin did not affect iNOS activity. Blocking insulin's increase in LPR by pyruvate (0.5 mmol/L) or oxaloacetate (0.5 mmol/L) completely inhibited the insulin-stimulated component of cGMP production. Pyruvate also blocked insulin's inhibition of serotonin-induced contraction in nonproliferated cells. In the absence of insulin, 5 mmol/L lactate or isocitrate increased the LPR by 420% +/- 47% and 167% +/- 20%, respectively (both P < .05), and stimulated cGMP production by 1,045% +/- 272% and 278% +/- 33%, respectively (both P < .05) by an L-NMMA-inhibitable mechanism. Although cGMP production in cells with iNOS was increased by insulin, the stimulation of cGMP production in cells without iNOS by 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) was not affected by insulin, suggesting that insulin does not stimulate guanylate cyclase activity directly. CONCLUSION: We conclude that insulin increases cGMP production in VSMC with iNOS by raising the cell NADH/NAD+ redox state, which may increase the availability of iNOS-derived NO.
Asunto(s)
Guanilato Ciclasa/metabolismo , Insulina/farmacología , Músculo Liso Vascular/enzimología , NAD/metabolismo , Animales , Células Cultivadas , GMP Cíclico/biosíntesis , Perros , Indazoles/farmacología , Isocitratos/farmacología , Ácido Láctico/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Ácido Oxaloacético/farmacología , Oxidación-Reducción , Ácido Pirúvico/farmacología , Estimulación QuímicaRESUMEN
Rat liver rhodanese [EC 2.8.1.1] purified by ammonium sulfate fractionation, CM-cellulose and Sephadex G-200 chromatography yielded two active fractions (I & II). Their molecular weights were estimated to be 1.75 X 10(4) (I) and 1.26 X 10(4) (II) by the gel filtration method. Kinetic studies revealed that Fraction I rat liver rhodanese catalyzes thiocyanate formation from thiosulfate and cyanide by a double displacement mechanism. Carboxylic acids such as DL-isocitric, citric malic, pyruvic, and oxaloacetic acid were competitive inhibitors with respect to thiosulfate, whereas fumaric, succinic, and alpha-ketoglutaric acids were noncompetitive inhibitors with respect ot thiosulfate. Incubation of mitochondria with sulfate and alpha-ketoglutaric acid caused a significant decrease in rhodanese activity.
Asunto(s)
Ácidos Dicarboxílicos/farmacología , Ácidos Cetoglutáricos/farmacología , Hígado/enzimología , Sulfurtransferasas/antagonistas & inhibidores , Tiosulfato Azufretransferasa/antagonistas & inhibidores , Ácidos Tricarboxílicos/farmacología , Animales , Unión Competitiva , Cianuros/metabolismo , Isocitratos/farmacología , Cinética , Mitocondrias Hepáticas/enzimología , Peso Molecular , Ratas , Succinatos/farmacología , Sulfatos/farmacología , Tiosulfato Azufretransferasa/aislamiento & purificación , Tiosulfatos/metabolismoRESUMEN
AIM: To investigate further the intracellular ammonium metabolism of Helicobacter pylori and the mechanism of its urease mediated destruction. METHODS: The mechanism of the in vitro destruction of H pylori was investigated by incubating it in buffer solutions, at pH 6.0, containing isocitrate or alpha ketoglutarate in addition to urea concentrations which had previously been shown to destroy H pylori. RESULTS: The median (range) 5 minute survival of H pylori in 0.2 mol/l citrate buffer (pH 6.0) in the absence of urea was 88% (18-184%) and was similar to its survival in 0.2 mol/l isocitrate buffer in the absence of urea, median 88% (15-274%). In the presence of 50 mmol/l urea the survival of H pylori in the citrate buffer was reduced, 9.9% (0-146%), compared with its survival in isocitrate buffer with the same concentration of urea 37% (0-274%) (p < 0.01). A 72 hour preincubation of the organism with 10 mmol/l alpha ketoglutarate also increased the 5 minute survival of the organism in 0.2 mol/l citrate buffer containing 50 mmol/l urea to 36% (9-145%) compared with its survival in the same buffer but without preincubation with alpha ketoglutarate 0% (0-62%). CONCLUSION: The protection of H pylori from rapid destruction by the supply of compounds used in the intracellular metabolism of the ammonium shows that the urease mediated destruction of H pylori can be explained by intracellular depletion of alpha ketoglutarate as a result of over production of ammonium by uncontrolled urease activity.
Asunto(s)
Amoníaco/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Isocitratos/farmacología , Ácidos Cetoglutáricos/farmacología , Ureasa/fisiología , Citratos/farmacología , Ácido Cítrico , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Helicobacter pylori/fisiología , Humanos , Urea/farmacologíaRESUMEN
1. 2-Oxoglutarate, succinate, fumarate, malate and citrate, cis-aconitate and isocitrate stimulate conversion of cholesterol to progesterone in human placental mitochondria. 2. The stimulatory effect of dicarboxylic and tricarboxylic acids depends on the activity of malate dehydrogenase (decarboxylating) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), respectively.
Asunto(s)
Ciclo del Ácido Cítrico , Mitocondrias/metabolismo , Placenta/metabolismo , Progesterona/biosíntesis , Ácido Aconítico/farmacología , Citratos/farmacología , Sinergismo Farmacológico , Femenino , Fumaratos/farmacología , Humanos , Isocitrato Deshidrogenasa/metabolismo , Isocitratos/farmacología , Ácidos Cetoglutáricos/farmacología , Malato Deshidrogenasa/metabolismo , Malatos/farmacología , Malonatos/farmacología , Manganeso/farmacología , Mitocondrias/efectos de los fármacos , Embarazo , Succinatos/farmacologíaRESUMEN
Although there is evidence that astrocytes support neuronal function, the contribution of astrocytes to seizure onset and termination is not known. To determine whether there are changes in seizure susceptibility or neuronal damage when the ability of astrocytes to generate ATP is reduced, 0.5 nmol of fluorocitrate (FC) was injected into the right ventricle. Injection of FC alone did not produce electrographic or behavioral seizures and did not stress or injure neurons or astrocytes, as measured with silver stain and immunohistochemistry for HSP32 or HSP72. However, in animals pretreated with FC, administration of kainic acid, at a dose that does not initiate seizures in control animals (7 mg/kg), caused wet dog shakes and neuronal damage in the hilus. Wet dog shakes did not cause any neuronal damage in control animals. If the dose of FC was increased to 0.75 nmol, then subsequent administration of the same dose of kainic acid (7 mg/kg) caused stage 3-5 seizures. Injection of FC also reduced the dose of pilocarpine needed to produce seizures. Given simultaneously with FC, isocitrate, which bypasses the biochemical inhibition of aconitase, blocked the effects of FC in both kainic acid and pilocarpine treated animals. The data demonstrate that inhibition of aconitase in astrocytes lowers the doses of both kainic acid and pilocarpine that will cause behavioral seizures and may increase neuronal vulnerability to seizures.
Asunto(s)
Aconitato Hidratasa/antagonistas & inhibidores , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Citratos/farmacología , Convulsivantes/farmacología , Inhibidores Enzimáticos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Citratos/administración & dosificación , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo-Oxigenasa 1 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Isocitratos/farmacología , Ácido Kaínico/farmacología , Masculino , Neuronas/efectos de los fármacos , Neuronas/patología , Pilocarpina/farmacología , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Vimentina/biosíntesisRESUMEN
Polymorphonuclear leukocytes (PMN) are important in corneal disease because of their role as effector cells in inflammation and ulceration. The favorable effect of citrate on corneal ulceration appears to result from inhibition of the PMN. Citrate does not enter the cells but chelates Ca2+ in the extracellular fluid and may promote a loss of some intracellular Ca2+. Isocitrate is the only tricarboxylic acid cycle intermediate that inhibits PMN, also by Ca2+ chelation. When isobutylcyanoacrylate is polymerized, a substance, probably formaldehyde, inhibitory to PMN, continuously leeches from the plastic. Although acetylcysteine has been reported to inhibit collagenase in vitro it has a direct effect of enhancing the respiratory burst and possibly degranulation of PMN stimulated by opsonized zymosan. Dexamethasone had no effect on PMN stimulation while prednisolone was partially inhibitory at high concentrations. Indomethacin exerts an inhibitory effect on all parameters of PMN stimulation. These studies clarify the site and mechanism of citrate action as well as show the importance of knowing the effect of drugs on the PMN.
Asunto(s)
Isocitratos/farmacología , Neutrófilos/efectos de los fármacos , Acetilcisteína/farmacología , Antiinflamatorios/farmacología , Antiinflamatorios no Esteroideos/farmacología , Ciclo del Ácido Cítrico , Humanos , Técnicas In Vitro , Piruvatos/farmacología , Esteroides , Adhesivos Tisulares/farmacologíaRESUMEN
Adriamycin is known to produce treatment limiting cardiotoxicity. We studied the effect of niacin, isocitrate, and N-acetyl-cysteine on cumulative adriamycin-induced cardiotoxicity in NMRI mice. Pathologic grading of the hearts indicated a significant reduction in myocardial injury. In addition, according to median survival and weight changes there was a significant decrease in toxicity. As demonstrated in Ehrlich ascites tumor, coadministration of N-acetyl-cysteine may interfere with the antitumor activity of adriamycin. In contrast, niacin and isocitrate did not show significant inhibition of antineoplastic activity. Our experiments suggest a promising role of niacin and isocitrate as potential cardioprotectors in the course of chemotherapy with adriamycin.