RESUMO
The use of potentiometry to measure plasma antioxidant capacity to contribute to oxidative stress evaluation is presented. In this assay, plasma (n=60) diluted (0.3 to 1 ml) in phosphate buffer, pH 7.4, NaCl 9%, was submitted to potentiometry. A platinum wire was the working electrode and saturated calomel the reference. The results are presented as the difference between sample and buffer potential (ΔE). ΔE presented a good inverse correlation with added increasing concentrations of ascorbate (2.5-75 µmol/L; R=-0.99), urate (9.0-150 µmol/L; R=-0.99), and bilirubin (0.78-13 µmol/L; R=-0.99). Increase in the antioxidant capacity decreased ΔE. Depletion of the antioxidant capacity by tert-butylhydroperoxide (6.5-50 µmol/L) presented a direct correlation (0.97) with ΔE. Furthermore, ΔE presented an inverse correlation (R=-0.99) with increased antioxidant capacity of plasma (FRAP) induced by the addition of ascorbate (2.5-75 µmol/L). The response of the potentiometric method proved be adequate for measuring the plasma antioxidant depletion induced by acute exhaustive exercise in rats (control, n=15; exercised, n=15). This exercise decreased the concentration of urate (p<0.05), decreased FRAP (p<0.5), increased TBARS (p<0.5), and decreased the potentiometer sensor response (p=6.5×10⻳). These results demonstrate the adequacy of potentiometry for evaluating the antioxidant capacity of blood plasma samples.
Assuntos
Antioxidantes/metabolismo , Plasma/metabolismo , Potenciometria/métodos , Adolescente , Animais , Antioxidantes/análise , Ácido Ascórbico/sangue , Ácido Ascórbico/metabolismo , Exercício Físico , Humanos , Masculino , Estresse Oxidativo , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ácido Úrico/sangue , Ácido Úrico/metabolismo , Adulto JovemRESUMO
Exercise can alter gene transcriptional and protein translational rates leading to changes in protein abundance toward adaptation to exercise. We investigated the alterations in protein abundance in skeletal muscle after one bout of an exhaustive exercise through proteomic analysis. Gastrocnemius muscles were sampled from non-exercised control rats and from rats exercised on a treadmill with incremental increases in speed until exhaustion (approximately 30 min). Rats were sacrificed 3 and 24 h after exercise cessation. Two-dimensional gel electrophoresis was performed and spots with a significant alteration in relative volume were identified by mass spectrometry. Six spots presented statistically significant altered abundances after exercise. The spots identified as the metabolic related proteins triosephosphate isomerase 1, glyceraldehyde-3-phosphate dehydrogenase, the ß subunit of pyruvate dehydrogenase E(1) and carnitine palmitoyltransferase 2 were all more abundant after exercise. One spot identified as heat shock cognate 70 was also more abundant after exercise. One spot demonstrated a decreased abundance after exercise and was identified as α-actin. These results suggest that a single session of exhaustive incremental exercise in untrained muscle can alter thin filaments synthesis/degradation rate and enhance cytosolic and mitochondrial proteins synthesis. The identified proteins may be important to a general preconditioning of skeletal muscle for subsequent exercise sessions.
Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Proteoma , Animais , Eletroforese em Gel Bidimensional , Masculino , Espectrometria de Massas , Proteômica , Ratos , Ratos WistarRESUMO
Anesthetics can affect the structure and biological function of tissues and systems differentially. The aim of the present study was to compare three injectable anesthetics generally used in experiments with animals in terms of the degree of hemolysis and glycogenolysis occurring after profound anesthesia. Twenty-four male Wistar rats (330-440 g) were divided into three groups (N = 8): chloral hydrate (CH), ketamine + xylazine (KX), Zoletil 50® (zolazepam and tiletamine) + xylazine (ZTX). After deep anesthesia, total blood was collected. The liver and white (WG) and red gastrocnemius (RG) muscles were also immediately removed. The degree of serum hemolysis was quantified on the basis of hemoglobin concentration (g/L). Hepatic and muscular glycogen concentrations (mmol/kg wet tissue) were quantified by the phenol-sulfuric method. The CH and KX groups exhibited serum hemolysis (4.0 ± 2.2 and 1.9 ± 0.9 g/L, respectively; P < 0.05) compared to the ZTX group, which presented none. Only KX induced elevated glycogenolysis (mmol/kg wet tissue) in the liver (86.9 ± 63.2) and in WG (18.7 ± 9.0) and RG (15.2 ± 7.2; P < 0.05). The CH and ZTX groups exhibited no glycogenolysis in the liver (164.4 ± 41.1 and 176.8 ± 54.4, respectively), WG (28.8 ± 4.4, 32.0 ± 6.5, respectively) or RG (29.0 ± 4.9; 25.3 ± 8.6, respectively). Our data indicate that ZTX seems to be an appropriate general anesthetic for studies that seek to simultaneously quantify the concentration of glycogen and serum biochemical markers without interferences. ZTX is reasonably priced, found easily at veterinary markets, quickly induces deep anesthesia, and presents a low mortality rate.
Assuntos
Animais , Masculino , Ratos , Anestésicos Gerais/farmacologia , Glicogenólise/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Glicogênio Hepático/metabolismo , Músculos/efeitos dos fármacos , Biomarcadores/análise , Combinação de Medicamentos , Ketamina/farmacologia , Músculos/enzimologia , Ratos Wistar , Tiletamina/farmacologia , Xilazina/farmacologia , Zolazepam/farmacologiaRESUMO
Anesthetics can affect the structure and biological function of tissues and systems differentially. The aim of the present study was to compare three injectable anesthetics generally used in experiments with animals in terms of the degree of hemolysis and glycogenolysis occurring after profound anesthesia. Twenty-four male Wistar rats (330-440 g) were divided into three groups (N = 8): chloral hydrate (CH), ketamine + xylazine (KX), Zoletil 50(R) (zolazepam and tiletamine) + xylazine (ZTX). After deep anesthesia, total blood was collected. The liver and white (WG) and red gastrocnemius (RG) muscles were also immediately removed. The degree of serum hemolysis was quantified on the basis of hemoglobin concentration (g/L). Hepatic and muscular glycogen concentrations (mmol/kg wet tissue) were quantified by the phenol-sulfuric method. The CH and KX groups exhibited serum hemolysis (4.0 +/- 2.2 and 1.9 +/- 0.9 g/L, respectively; P < 0.05) compared to the ZTX group, which presented none. Only KX induced elevated glycogenolysis (mmol/kg wet tissue) in the liver (86.9 +/- 63.2) and in WG (18.7 +/- 9.0) and RG (15.2 +/- 7.2; P < 0.05). The CH and ZTX groups exhibited no glycogenolysis in the liver (164.4 +/- 41.1 and 176.8 +/- 54.4, respectively), WG (28.8 +/- 4.4, 32.0 +/- 6.5, respectively) or RG (29.0 +/- 4.9; 25.3 +/- 8.6, respectively). Our data indicate that ZTX seems to be an appropriate general anesthetic for studies that seek to simultaneously quantify the concentration of glycogen and serum biochemical markers without interferences. ZTX is reasonably priced, found easily at veterinary markets, quickly induces deep anesthesia, and presents a low mortality rate.
Assuntos
Anestésicos Gerais/farmacologia , Glicogenólise/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Glicogênio Hepático/metabolismo , Músculos/efeitos dos fármacos , Animais , Biomarcadores/análise , Combinação de Medicamentos , Ketamina/farmacologia , Masculino , Músculos/enzimologia , Ratos , Ratos Wistar , Tiletamina/farmacologia , Xilazina/farmacologia , Zolazepam/farmacologiaRESUMO
Overreaching (OVR) is defined as the initial phase of overtraining syndrome and is known as a metabolic imbalance leading to short-term fatigue. Exercise increases reactive oxygen species production, which can oxidize intracellular structures impairing cell function and thus leads to OVR process. The aim of this work is to study the behavior of oxidative stress markers in subjects submitted to an OVR protocol. Thirty rats were divided in exercise and control group, and submitted to an 8-week-endurance training (ET) and a 3-week-OVR protocol. Thiobarbituric acid reactive substances (TBARs), reactive carbonylated derivatives (RCD), glutathione reductase (GR), catalase (CAT) and citrate synthase (CS) activities and stress protein HSP72 were measured in soleus (SO), extensor digital longus (EDL) and semitendinuous (ST) muscles. ET induced significant enhancement (P<0.05) in CS, GR, CAT, TBARs, RCD and HSP72 in SO, EDL and ST. OVR induced higher levels (P<0.05) of TBARs, RCD and HSP72 compared with ET only in SO, while in EDL and ST all measured parameters ranged at same levels reached during ET. We concluded that stress-induced OVR protocol is fiber type dependent, the SO muscle fiber type I being the most affected by this treatment.
Assuntos
Antioxidantes/metabolismo , Tolerância ao Exercício , Proteínas de Choque Térmico/metabolismo , Fadiga Muscular/fisiologia , Músculo Esquelético/metabolismo , Estresse Oxidativo/fisiologia , Esforço Físico , Animais , Antioxidantes/fisiologia , Catalase , Citrato (si)-Sintase , Glutationa Redutase , Masculino , Músculo Esquelético/fisiologia , Ratos , Ratos Wistar , Tiobarbitúricos , Fatores de TempoRESUMO
A novel method to measure oxidative stress resulting from exhaustive exercise in rats is presented. In this new procedure we evaluated the erythrocyte antioxidant enzymes, catalase (CAT) and glutathione reductase (GR), the plasma oxidative attack markers, reactive carbonyl derivatives (RCD) and thiobarbituric reactive substances (TBARS). Muscular tissue damage was evaluated by monitoring plasma creatine kinase (CK) and plasma taurine (Tau) concentrations. Also, we monitored total sulphydryl groups (TSG) and uric acid (UA), and the level of the 70 kDa heat shock protein (HSP70) in leukocytes as a marker of oxidative stress. In the study we found a correspondence between erythrocyte CAT and GR activities and leukocyte HSP70 levels, principally 3 h after the acute exercise, and this suggested an integrated mechanism of antioxidant defense. The increase in levels of plasma Tau was coincident with the increasing plasma levels of CK and TBARS, principally after two hours of exercise. Thus tissue damage occurred before the expression of any anti-oxidant system markers and the monitoring of Tau, CK or TBARS may be important for the estimation of oxidative stress during exhaustive exercise. Furthermore, the integrated analyses could be of value in a clinical setting to quantify the extent of oxidative stress risk and reduce the need to perform muscle biopsies as a tool of clinical evaluation.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Leucócitos/metabolismo , Estresse Oxidativo , Condicionamento Físico Animal , Animais , Biomarcadores/análise , Masculino , Ratos , Ratos WistarRESUMO
Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 +/- 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 +/- 0.65, P < 0.05) and soleus (9.8 +/- 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Mitocôndrias Musculares/enzimologia , Músculo Esquelético/enzimologia , Fosforilação Oxidativa , Condicionamento Físico Animal/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Coloração e Rotulagem/métodos , Animais , Calorimetria , Masculino , Ratos , Ratos WistarRESUMO
Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.
Assuntos
Animais , Masculino , Ratos , Eletroforese em Gel de Poliacrilamida , Condicionamento Físico AnimalRESUMO
The development of an amperometric sensor for glutathione reductase (GR) activity in erythrocyte hemolysate to contribute to oxidative stress evaluation is presented. In this assay, the reduced form of glutathione, the product of the GR reaction, reacts with 5,5(')-dithiobis(2-nitrobenzoic acid), producing GSTNB, which is easily reduced in the electrode surface. The current was recorded during 180 s after the sample addition, applying a potential of -300 mV. The sensor presented a suitable sensitivity, a good operational range, and precision. The effects of pH variations and specific uncompetitive inhibitor (safranin-O) in the enzyme activity were also evaluated. The GR activity determination in human erythrocyte hemolysate using this method has provided results that are statistically equal to those obtained by the classical spectrophotometric method, with 95% of confidence. The advantages of this method are the saved time, reagents, and samples and the possibility of its use in the field.
Assuntos
Eritrócitos/enzimologia , Glutationa Redutase/análise , Glutationa Redutase/metabolismo , Extratos Celulares , Eletroquímica , Eletrodos , Glutationa/análise , Glutationa/química , Humanos , Nitrobenzoatos/química , Oxirredução , Reprodutibilidade dos TestesRESUMO
Protein malnutrition leads to functional impairment in several organs, which is not fully restored with nutritional recovery. Little is known about the role of oxidative stress in the genesis of these alterations. This study was designed to assess the sensitivity of blood oxidative stress biomarkers to a dietary protein restriction. Male Wistar rats were divided into two groups, according to the diet fed from weaning (21 days) to 60 day old: normal protein (17% protein) and low protein (6% protein). Serum protein, albumin, free fatty acid and liver glycogen and lipids were evaluated to assess the nutritional status. Blood glutathione reductase (GR) and catalase (CAT) activities, plasma total sulfhydryl groups concentration (TSG) as well as plasma thiobarbituric acid reactive substances (TBARs) and reactive carbonyl derivatives (RCD) were measured as biomarkers of the antioxidant system and oxidative damage, respectively. The glucose metabolism in soleus muscle was also evaluated as an index of stress severity imposed to muscular mass by protein malnutrition. No difference was observed in muscle glucose metabolism or plasma RCD concentration between both groups. However, our results showed that the low protein group had higher plasma TBARs (62%) concentration and lower TSG (44%) concentration than control group, indicating increased reactive oxygen species production in low protein group. The enhancement of erythrocyte GR (29%) and CAT (28%) activities in this group also suggest an adaptation to the stress generated by the protein deficiency. Taken together, the results presented here show that the biomarkers used were able to reflect the oxidative stress level induced by this specific protein deficient diet.
Assuntos
Glucose/metabolismo , Estresse Oxidativo/fisiologia , Deficiência de Proteína/sangue , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Biomarcadores , Catalase/sangue , Glutationa Redutase/sangue , Masculino , Estado Nutricional , Deficiência de Proteína/enzimologia , Ratos , Ratos Wistar , Compostos de Sulfidrila/sangueRESUMO
Given the potential of reactive oxygen species to damage intracellular proteins during subsequent bouts of muscle contractions, it was suggested that, when this production exceeds the antioxidant capacity, the preexisting antioxidant pathways may be complemented by the synthesis of the defense mechanism represented by heat shock proteins (HSPs), stress proteins with the function of repair and maintaining protein folding. To test this hypothesis, we analyzed reactive carbonyl derivatives in plasma and the expression of HSP72 and activities of enzymes from the oxidative and antioxidant defense systems in the soleus muscle of sedentary rats and rats trained by two protocols: continuous and intermittent. We analyzed all three groups at rest and 2 h after acute exercise. After 8 wk of training, the animals from both groups clearly demonstrated higher resistance to exercise. Both trained groups showed significantly higher citrate synthase, catalase, and glutathione reductase activities than the control group (P < 0.01). After acute exercise, catalase and glutathione reductase activities significantly decreased (P < 0.01) and plasma reactive carbonyl derivatives significantly increased (P < 0.05) in the sedentary group, suggesting an oxidative-stress condition as responsible for exhaustion in this group. Finally, after acute exercise, the induction of HSP72 expression occurred only in the sedentary group, suggesting that HSP72 acts as a complementary protective mechanism in exercise-induced oxidative stress.
Assuntos
Proteínas de Choque Térmico/fisiologia , Músculo Esquelético/fisiologia , Estresse Oxidativo , Esforço Físico/fisiologia , Animais , Catalase/metabolismo , Citrato (si)-Sintase/metabolismo , Glutationa Redutase/metabolismo , Proteínas de Choque Térmico HSP72 , Masculino , Contração Muscular , Ratos , Ratos WistarRESUMO
Pedagogic education of graduate students, when and where it exists, is restricted to theoretical courses or to the participation of the students as teachers' assistants. This model is essentially reproductive and offers few opportunities for any significant curriculum innovation. To open an opportunity for novelty we have introduced a new approach in "Biochemistry Teaching", a course included in the Biochemistry Graduate Program of the Biochemistry Department (Universidade Estadual de Campinas and Universidade de São Paulo). The content of the course consists of a) choosing the theme, b) selecting and organizing the topics, c) preparing written material, d) establishing the methodological strategies, e) planning the evaluation tools and, finally, f) as teachers, conducting the course as an optional summer course for undergraduate students. During the first semester the graduate students establish general and specific educational objectives, select and organize contents, decide on the instructional strategies and plan evaluation tools. The contents are explored using a wide range of strategies, which include computer-aided instruction, laboratory classes, small group teaching, a few lectures and round table discussions. The graduate students also organize printed class notes to be used by the undergraduate students. Finally, as a group, they teach the summer course. In the three versions already developed, the themes chosen were Biochemistry of Exercise (UNICAMP), Biochemistry of Nutrition (UNICAMP) and Molecular Biology of Plants (USP). In all cases the number of registrations greatly exceeded the number of places and a selection had to be made. The evaluation of the experience by both graduate and undergraduate students was very positive. Graduate students considered this experience to be unique and recommended it to their schoolmates; the undergraduate students benefited from a more flexible curriculum (more options) and gave very high scores to both the courses and the teachers.
Assuntos
Bioquímica/educação , Educação de Pós-Graduação , Ensino , Currículo , HumanosRESUMO
Pedagogic education of graduate students, when and where it exists, is restricted to theoretical courses or to the participation of the students as teachers' assistants. This model is essentially reproductive and offers few opportunities for any significant curriculum innovation. To open an opportunity for novelty we have introduced a new approach in "Biochemistry Teaching", a course included in the Biochemistry Graduate Program of the Biochemistry Department (Universidade Estadual de Campinas and Universidade de São Paulo). The content of the course consists of a) choosing the theme, b) selecting and organizing the topics, c) preparing written material, d) establishing the methodological strategies, e) planning the evaluation tools and, finally, f) as teachers, conducting the course as an optional summer course for undergraduate students. During the first semester the graduate students establish general and specific educational objectives, select and organize contents, decide on the instructional strategies and plan evaluation tools. The contents are explored using a wide range of strategies, which include computer-aided instruction, laboratory classes, small group teaching, a few lectures and round table discussions. The graduate students also organize printed class notes to be used by the undergraduate students. Finally, as a group, they teach the summer course. In the three versions already developed, the themes chosen were Biochemistry of Exercise (UNICAMP), Biochemistry of Nutrition (UNICAMP) and Molecular Biology of Plants (USP). In all cases the number of registrations greatly exceeded the number of places and a selection had to be made. The evaluation of the experience by both graduate and undergraduate students was very positive. Graduate students considered this experience to be unique and recommended it to their schoolmates; the undergraduate students benefited from a more flexible curriculum (more options) and gave very high scores to both the courses and the teachers.
Assuntos
Humanos , Bioquímica , Educação , Docentes , Educação de Pós-GraduaçãoRESUMO
The non-covalent interaction between two molecular entities namely, phospholipase A2 and crotapotin, results in the main toxin, crotoxin, present in the venom of the South American rattlesnake Crotalus durissus terrificus. High performance liquid chromatography has enabled us the isolation of three phospholipase A2 isoforms (F1, F2 and F3), characterized through denaturing and non-denaturing polyacrylamide gel electrophoresis and also through the N-terminal amino acid sequence analysis. The effect of each purified phospholipase A2 isoform on isolated rat liver mitochondria was determined through mitochondrial swelling and O2 consumption during respiratory state 4. F1 showed a dose-dependent stimulation of O2 consumption while F2 and F3 caused stimulation only at low doses and inhibition at high amounts. These effects were completely suppressed by the presence of 0.1% bovine serum albumin or 0.5 mM EGTA in the incubation medium. Taking the mitochondrial swelling as an activity parameter, all of them presented the same behaviour at different intensities, leading to permeabilization of the mitochondrial membrane. In this case, addition of EGTA prevented it whereas bovine serum albumin was ineffective, indicating that the lipid microenvironment was affected. These results suggest that free fatty acids are directly responsible for the observed effects induced by phospholipase A2 isoforms on oxygen consumption experiments. The protection conferred by cyclosporin-A on swelling induced by the isoforms, when present in low concentrations, may suggest that cyclosporin-A binds to a mitochondrial membrane site protecting the membrane against the phospholipase A2 attack.
Assuntos
Venenos de Crotalídeos/enzimologia , Crotalus , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosfolipases A/toxicidade , Sequência de Aminoácidos , Animais , Ácido Egtázico/farmacologia , Feminino , Isoenzimas/isolamento & purificação , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Dilatação Mitocondrial , Dados de Sequência Molecular , Consumo de Oxigênio/efeitos dos fármacos , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Soroalbumina Bovina/farmacologiaRESUMO
The effect of different agents on inner-mitochondrial-membrane permeabilization and lipoperoxidation induced by Ca2+ and the pyridine-nucleotide oxidant t-butylhydroperoxide or inorganic phosphate was investigated. Comparing the protection conferred by ADP, a substrate of the ADP/ATP carrier, dithiothreitol, a disulfide reductant and butylhydroxytoluene, a radical scavenger, it was found that ADP was always the most effective against mitochondrial damage, when present in the incubation medium from the beginning. Moreover, carboxyatractyloside, a specific inhibitor of the ADP/ATP carrier, abolished completely the protective effect of ADP on both the lipoperoxidation and mitochondrial swelling processes. Experiments where deenergized mitochondria were previously incubated with Ca2+ showed a decrease in the content of active ADP/ATP carrier, indicating a direct involvement of this protein in the formation of a non-specific Ca(2+)-dependent pore. Our results also eliminate the possibility of an attack of oxygen radicals on lipids or proteins of the mitochondrial membrane as the primary event triggering the permeability transition of the inner mitochondrial membrane.
Assuntos
Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Atractilosídeo/análogos & derivados , Atractilosídeo/farmacologia , Hidroxitolueno Butilado/farmacologia , Cálcio/metabolismo , Ditiotreitol/farmacologia , Feminino , Membranas Intracelulares/efeitos dos fármacos , Peroxidação de Lipídeos , Dilatação Mitocondrial/efeitos dos fármacos , Oxidantes/farmacologia , Permeabilidade , Peróxidos/farmacologia , Fosfatos/farmacologia , Ratos , Ratos Wistar , terc-Butil HidroperóxidoRESUMO
Lipoperoxidation was investigated as a step for membrane protein thiol oxidation of rat liver mitochondria incubated in the presence of Ca2+ and t-butylhydroperoxide, by the determination of thiobarbituric acid reactive substances. Lipoperoxidation occurred only when the incubation medium contained 125 microM t-butylhydroperoxide (t-buOOH) in the presence of Ca2+ and phosphate (Pi). No lipoperoxidation was observed when acetate replaced Pi as permeant anion, or when the oxidant was omitted, even at high Ca2+ and Pi concentrations (up to 120 microM Ca2+ and 5 mM Pi), conditions under which the mitochondria are fully permeabilized. In both cases, ADP protected efficiently against permeabilization, indicating the possible involvement of the ADP/ATP carrier in the earlier stages of the process.
Assuntos
Cálcio/farmacologia , Peroxidação de Lipídeos , Mitocôndrias Hepáticas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Permeabilidade da Membrana Celular , Masculino , Fósforo/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Lipoperoxidation was investigated as a step for membrane protein thiol oxidation of rat liver mitochondria incubated in the presence of Ca2 and t-butylhydroproxide, by the determination of thiobarbituric acid reactive substances. Lipoperoxidation occured only when the incubation medium contained 125 µM t-butylhdroperoxide (t-buOOH) in the presence of Ca2+ and phosphate (Pi). No lipoperoxidation was observed when acetate replaced Pi as permeant anion, or when the oxidant was omitted, even at high Ca2 and Pi concentrations (up to 120 µMCa2+ and 5mMPi), conditions under which the mitochondria are fully permeabilized. In both cases, ADP protected efficiently against permeabilization, indicating the possible involvement of the ADP/ATP carrier in the earlier stages of the process
Assuntos
Animais , Masculino , Ratos , Cálcio/farmacocinética , Mitocôndrias Hepáticas/metabolismo , Peroxidação de Lipídeos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Permeabilidade da Membrana Celular , Ratos EndogâmicosRESUMO
The use of digitonin to permeabilize Leishmania mexicana mexicana, Leishmania agamae, and Crithidia fasciculata plasma membranes enabled us to study Ca2+ transport in situ. The present results show that the mitochondria of these trypanosomatids are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by inorganic phosphate. Large amounts of Ca2+ were retained by C. fasciculata mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butylhydroperoxide and diamide. In contrast, Ca2+ was not retained in the matrix of Leishmania sp. mitochondria for long periods of time. In addition to the mitochondrial Ca2+ uptake, a vanadate-sensitive Ca2(+)-transporting system was also detectable in these trypanosomatids.
Assuntos
Cálcio/metabolismo , Digitonina/farmacologia , Trypanosoma/metabolismo , Animais , Transporte Biológico , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/metabolismo , Potenciais da Membrana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Trypanosoma/efeitos dos fármacosRESUMO
Deenergized rat liver or AS-30D hepatoma mitochondria underwent extensive swelling when incubated in the presence of Ca2+ and an oxidant of mitochondrial pyridine nucleotides. This swelling was stimulated by phosphate and inhibited by ruthenium red, Mg2+ plus ATP and dibucaine. The degree of inhibition by dibucaine was shown to vary inversely with the Ca2+ concentration. The inhibition caused by ruthenium red was completely removed by the Ca2+ ionophore ionomycin indicating that the effect of the cation is mediated by internal binding sites. Since mitochondria were totally deenergized the data definitely rule out the requirement for Ca2+ cycling across the membrane in this mechanism.