RESUMEN
BACKGROUND: The liver may be injured in situations where it is submitted to ischemia, such as partial hepatectomy and liver transplantation. In all cases, ischemia is followed by reperfusion and, although it is essential for the reestablishment of tissue function, reperfusion may cause greater damage than ischemia, an injury characterized as ischemia-reperfusion (I/R) damage. The aim of this work was to analyze the effect of ischemic preconditioning with the use of methylene blue (MB; 15 mg/kg) 5 or 15 minutes before I/R (IRMB5' and IRMB15', respectively) on the hepatic injury occurring after I/R. METHODS: Twenty-eight male Wistar rats were used, and liver samples submitted to partial ischemia (IR) or not (NI) were obtained from the same animal. The samples were divided into 7 groups. Data were analyzed statistically by means of the nonparametric Mann-Whitney test and Wilcoxon Matched test, with the level of significance set at 5% (P < .05). RESULTS: The rate of oxygen consumption by state 3 mitochondria was inhibited in all ischemic groups compared with the sham group (SH vs IR: P = .0052; SH vs IRMB5': P = .0006; SH vs IRMB15': P = .0048), which did not occur in the nonischemic contralateral portion of the same liver (SH vs NI: P = .7652; SH vs NIMB5': P = .059; SH vs NIMB15': P = .3153). The inhibition of the rate of oxygen consumption by state 3 mitochondria was maintained in the presence of MB (IR vs IRMB5': P = .4563; IR vs IRMB15': P = .9021). The respiratory control ratio was reduced in all ischemic groups compared with the sham group, owing to the inhibition of oxygen consumption in state 3 (SH vs IR: P = .0151; SH vs IRMB5': P = .005; SH vs IRMB15': P = .0007). CONCLUSIONS: Methylene blue had no effect on the mitochondrial respiratory parameters studied, but was able to reduce lipid peroxidation, preventing the production of reactive oxygen species (SH vs IRMB15': P = .0210).
Asunto(s)
Inhibidores Enzimáticos/administración & dosificación , Precondicionamiento Isquémico/métodos , Hígado/irrigación sanguínea , Azul de Metileno/administración & dosificación , Daño por Reperfusión/prevención & control , Animales , Isquemia/etiología , Isquemia/fisiopatología , Hígado/lesiones , Hígado/cirugía , Masculino , Mitocondrias/fisiología , Consumo de Oxígeno , Ratas , Ratas Wistar , Daño por Reperfusión/etiologíaRESUMEN
OBJECTIVES: We designed studies to test the hypotheses that hyperbaric oxygen (HBO) therapy should protect liver against subsequent ischemia/reperfusion (I/R) injury are scarce and controversial. The purpose of this study was to clarify some questions about the association of HBO with the processes of liver I/R. METHODS: We divided Wistar rats into 5 groups: (1) SHAM operation, (2) I/R, rats submitted to total pedicle ischemia for 30 minutes followed by 5 minutes of reperfusion; (3) HBO60I/R and (4) HBO120I/R, rats respectively submitted to 60 and 120 minutes of HBO therapy at 2 absolute atmospheres and immediately after submitted to the experimental protocol of I/R; (5) HBO120, rats submitted to 120 minutes of HBO therapy at 2 absolute atmospheres and then immediately after humanely killed. The experimental protocol included (1) serum levels of aspartate and alanine aminotransferase; (2) mitochondrial function; (3) tissue malondialdehyde (MDA); and (4) plasma nitrite/nitrate. Data were analyzed using the Mann-Whitney test and were considered significant P < 5%. RESULTS: The processes of liver ischemia/reperfusion caused tissue injury with hepatic mitochondrial functional impairment. A single exposure to 120 minutes of HBO caused an increase of tissue MDA. The time of HBO exposure as preconditioning before hepatic I/R is critical in the prevalence of beneficial or deleterious effects. Sixty minutes of hyperoxic preconditioning before liver I/R presents systemic benefits, but no significant tissue preservation. One hundred twenty minutes of hyperoxic preconditioning tissue liver benefits predominate compared with systemic benefits. CONCLUSIONS: The HBO preconditioning therapeutic benefits to liver I/R injury are time dependent, suggesting a therapeutic window that needs to be clearly defined in future studies.
Asunto(s)
Oxigenoterapia Hiperbárica/métodos , Precondicionamiento Isquémico/métodos , Oxígeno/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Daño por Reperfusión/fisiopatología , Alanina Transaminasa/sangre , Animales , Ácido Aspártico/sangre , Modelos Animales de Enfermedad , Radicales Libres , Hiperoxia , Hígado/fisiopatología , Masculino , Malondialdehído/química , Mitocondrias/metabolismo , Mitocondrias/patología , Nitratos/sangre , Nitritos/sangre , Consumo de Oxígeno , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The objective of the present study was to analyze hepatic mitochondrial function in patients with familial amyloidotic polyneuropathy (FAP) undergoing cadaveric donor orthotopic liver transplantation. From February 2005 to May 2007, eight patients with FAP, ranging in age from 34 to 41 years and with Model for End-Stage Liver Disease scores ranging from 24 to 29. Underwent orthotopic transplantation using a liver from a deceased donor by the piggyback method. Immediately before beginning the recipient hepatectomy in a patient with FAP, a biopsy was obtained for analysis of mitochondrial function (FAP group). The control group consisted of 15 patients undergoing hepatic surgery to treat small tumors of the liver. Mitochondrial respiration was determined on the basis of oxygen consumption by energized mitochondria using a polarographic method. The membrane potential of the mitochondria was determined spectrofluorometrically. Data were analyzed statistically by the Mann-Whitney test, with the level of significance set at 5%. State 3 and 4 values, respiratory control ratio, and membrane potential were 47 +/- 8 versus 28 +/- 10 natoms O/min/mg protein (P < .05); 14 +/- 3 vs 17 +/- 7 nat.O/min/mg.prot.mit. (P > .05); 3.6 +/- .5 vs 1.7 +/- 0.7 (P < .05); and 135 +/- 5.2 vs 135 +/- 6 mV (P > .05) for control versus FAP patients, respectively, demonstrating a decreased energy status of the liver in FAP.
Asunto(s)
Neuropatías Amiloides Familiares/metabolismo , Neuropatías Amiloides Familiares/cirugía , Trasplante de Hígado , Mitocondrias Hepáticas/metabolismo , Adulto , Femenino , Hepatectomía , Humanos , Masculino , Potenciales de la Membrana , Consumo de OxígenoRESUMEN
Some studies have indicated that mitochondria may be the target organelle of plants. We therefore decided to assess the effects of the aqueous extract of Hyptis pectinata leaves on liver mitochondrial respiratory function in vitro. Eight rat livers were subjected to isolation of mitochondria by differential centrifugation. In an adequate medium, the plant extract was added at different concentrations. The analyzed data were: state 3, state 4 and respiratory control ratio (RCR). H. pectinata extract caused a statistically significant decrease in state 3 (at 0.05, 0.1 and 0.2 mg/mg protein) and RCR (at 0.05, 0.1 and 0.2 mg/mg protein). Respiratory state 4 was not altered by the increasing concentrations. In conclusion, the aqueous extract of H. pectinata leaves may not injure the mitochondrial inner membrane but decreases significantly the oxidative phosphorylation.
Asunto(s)
Hyptis , Mitocondrias Hepáticas/efectos de los fármacos , Fitoterapia , Extractos Vegetales/farmacología , Animales , Respiración de la Célula/efectos de los fármacos , Masculino , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Hojas de la Planta , Ratas , Ratas WistarRESUMEN
To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3'-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (delta psi), osmotic swelling, Ca2+ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 microM Ca2+, 5 microM ruthenium red (RR) or 1 microM cyclosporin A (CsA) were used. The dose response-curves for both respiration release and delta psi dissipation were nearly linear, presenting an IC50 of approximately 10 microM and reaching saturation within 25-50 microM, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 microM Ca2+, followed by delta psi dissipation and Ca2+ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca2+. ADP, Mg2+, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H2O2 production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.
Asunto(s)
Ácido Flufenámico/farmacología , Mitocondrias Hepáticas/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Desacopladores/farmacología , Animales , Calcio/metabolismo , Cloruro de Calcio/farmacología , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Oxidación-Reducción , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Rotenona/farmacología , Rojo de Rutenio/farmacología , Ácido Succínico/farmacologíaRESUMEN
The ability of nonsteroidal anti-inflammatory drugs (NSAIDs) to induce Ca(2+)-mediated/cyclosporin A-sensitive mitochondrial membrane permeability transition (MMPT) was evaluated by monitoring swelling of isolated rat renal cortex mitochondria in the presence of 20 microM CaCl2. Dipyrone and paracetamol did not induce MMPT, while piroxicam and acetylsalicylic acid (and its metabolite salicylate) were poor inducers. In contrast, diclofenac sodium and mefenamic acid were potent triggering agents, inducing MMPT at 2 microM, a concentration below those previously shown to uncouple and/or inhibit oxidative phosphorylation. When compared to salicylate, a classical uncoupler and inducer of MMPT, the potency of diclofenac sodium and mefenamic acid was about 50-fold greater. Swelling was completely prevented by EGTA, cyclosporin A, or MgCl2, and only partially by ADP or dithiothreitol. Under the same experimental conditions as for the swelling assays, the drugs depressed the membrane potential of mitochondria, an effect prevented by cyclosporin A and restored by EGTA. Also, the drugs did not induce membrane lipid peroxidation or changes in GSSG levels, but led to a small decrease in protein thiol content, as well as to a substantial decrease in the NADPH levels of mitochondria. Hence, membrane depolarization and pyridine nucleotide oxidation seem to be involved in MMPT induction by these NSAIDs. The potency in eliciting the process, like the uncoupling activity, seems to be influenced by the lipophilic character of the molecules.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Diclofenaco/farmacología , Corteza Renal/efectos de los fármacos , Ácido Mefenámico/farmacología , Mitocondrias/efectos de los fármacos , Animales , Ciclosporina/farmacología , Corteza Renal/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Mitocondrias/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Oxidación-Reducción , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Salicilatos/farmacología , Ácido Salicílico , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 x 10(5) trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K0.5 values, and a decreased total Vmax. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100-solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels.
Asunto(s)
Cardiomiopatía Chagásica/enzimología , Miocardio/enzimología , ATPasas de Translocación de Protón/metabolismo , Enfermedad Aguda , Adenosina Trifosfato/metabolismo , Animales , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Masculino , Mitocondrias Cardíacas/enzimología , Ratas , Ratas Wistar , TemperaturaRESUMEN
The in vitro interference of some of most important nonsteroidal anti-inflammatory drugs (NSAIDs) with the respiration of rat kidney (renal cortex) mitochondria and ATP synthesis was evaluated. Acetylsalicylic acid, diclofenac sodium, mefenamic acid, and piroxicam both uncoupled and inhibited oxidative phosphorylation in mitochondria energized with glutamate plus malate or with succinate, while dipyrone only uncoupled and paracetamol only inhibited it. The drug concentrations affecting mitochondrial respiration were in the low to middle micromolar range for diclofenac, mefenamic acid, and piroxicam, and in the low millimolar range for acetylsalicylic acid, dipyrone, and paracetamol. The pattern of inhibition, except for the paracetamol, was similar to that expressed by the respiratory chain inhibitors. NSAIDs also inhibited the rate of ATP synthesis in mitochondria energized with glutamate plus malate, as well as the phosphorylation potential of mitochondria. The IC50 values for rate of ATP synthesis, using 2 mM ADP, were about 0.1 mM for diclofenac sodium and mefenamic acid, 0.7 mM for piroxicam, and in the range of 5-8 mM for acetylsalicylic acid, dipyrone, and paracetamol. The potential for renal energetic cytotoxicity of NSAIDs is discussed considering their ability to interact with the oxidative phosphorylation in rat renal cortex mitochondria. A comparison is made with the interference of salicylate, the main metabolite of acetylsalicylic acid, and a classical uncoupler of oxidative phosphorylation.