RESUMEN
AIM: To determine the effect of pioglitazone treatment on in vivo and ex vivo muscle mitochondrial function in a rat model of diabetes. METHODS: Both the lean, healthy rats and the obese, diabetic rats are Zucker Diabetic Fatty (ZDF) rats. The homozygous fa/fa ZDF rats are obese and diabetic. The heterozygous fa/+ ZDF rats are lean and healthy. Diabetic Zucker Diabetic Fatty rats were treated with either pioglitazone (30 mg/kg/day) or water as a control (n = 6 per group), for 2 weeks. In vivo ¹H and ³¹P magnetic resonance spectroscopy was performed on skeletal muscle to assess intramyocellular lipid (IMCL) content and muscle oxidative capacity, respectively. Ex vivo muscle mitochondrial respiratory capacity was evaluated using high-resolution respirometry. In addition, several markers of mitochondrial content were determined. RESULTS: IMCL content was 14-fold higher and in vivo muscle oxidative capacity was 26% lower in diabetic rats compared with lean rats, which was, however, not caused by impairments of ex vivo mitochondrial respiratory capacity or a lower mitochondrial content. Pioglitazone treatment restored in vivo muscle oxidative capacity in diabetic rats to the level of lean controls. This amelioration was not accompanied by an increase in mitochondrial content or ex vivo mitochondrial respiratory capacity, but rather was paralleled by an improvement in lipid homeostasis, that is lowering of plasma triglycerides and muscle lipid and long-chain acylcarnitine content. CONCLUSION: Diminished in vivo muscle oxidative capacity in diabetic rats results from mitochondrial lipid overload and can be alleviated by redirecting the lipids from the muscle into adipose tissue using pioglitazone treatment.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Metabolismo de los Lípidos/efectos de los fármacos , Enfermedades Mitocondriales/prevención & control , Músculo Esquelético/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Tiazolidinedionas/uso terapéutico , Animales , Biomarcadores/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Hipertrigliceridemia/complicaciones , Hipertrigliceridemia/prevención & control , Hipoglucemiantes/efectos adversos , Hipolipemiantes/uso terapéutico , Masculino , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Enfermedades Mitocondriales/complicaciones , Recambio Mitocondrial/efectos de los fármacos , Músculo Esquelético/metabolismo , Obesidad/complicaciones , Fosforilación Oxidativa/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , Pioglitazona , Ratas Zucker , Tiazolidinedionas/efectos adversosRESUMEN
The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.
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Glucólisis/fisiología , Músculo Esquelético/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/análisis , Calcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Simulación por Computador , Concentración de Iones de Hidrógeno , Isquemia/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Masculino , Modelos Biológicos , Contracción Muscular/fisiología , Fosfocreatina/análisis , Fosfocreatina/metabolismo , Fosfofructoquinasa-1 Tipo Muscular/química , Fosfofructoquinasa-1 Tipo Muscular/metabolismo , Condicionamiento Físico Animal/fisiología , Ratas , Ratas WistarRESUMEN
AIMS/HYPOTHESIS: Insulin resistance and type 2 diabetes have been associated with ectopic lipid deposition. This study investigates the derangements in postprandial lipid handling in liver and skeletal muscle tissue at different stages during the pathogenesis of type 2 diabetes in a rat model. METHODS: Four groups (n = 6) of male Zucker diabetic fatty rats were used for this study: prediabetic fa/fa rats and healthy fa/+ littermates at the age of 6 weeks, and diabetic fa/fa rats and healthy fa/+ littermates at the age of 12 weeks. In vivo (1)H-[(13)C] magnetic resonance spectroscopy measurements were performed in liver and tibialis anterior muscle at baseline and 4, 24 and 48 h after oral administration of 1.5 g [U-(13)C]algal lipid mixture per kilogram body weight. Total and (13)C-labelled intracellular lipid contents were determined from the magnetic resonance spectra. RESULTS: In both prediabetic and diabetic rats, total lipid contents in muscle and liver were substantially higher than in healthy controls and this was accompanied by a 2.3-fold greater postprandial lipid uptake in the liver (p < 0.001). Interestingly, in prediabetic rats, skeletal muscle appeared to be protected from excess lipid uptake whereas after developing overt diabetes muscle lipid uptake was 3.4-fold higher than in controls (p < 0.05). Muscle lipid use was significantly lower in prediabetic and diabetic muscle, indicative of impairments in lipid oxidation. CONCLUSIONS/INTERPRETATION: In vivo postprandial lipid handling is disturbed in both liver and skeletal muscle tissue in prediabetic and diabetic rats, but the uptake of dietary lipids in muscle is only increased after the development of overt diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Hígado/metabolismo , Músculo Esquelético/metabolismo , Periodo Posprandial/fisiología , Estado Prediabético/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/fisiología , Espectroscopía de Resonancia Magnética , Masculino , Estado Prediabético/fisiopatología , Ratas , Ratas ZuckerRESUMEN
Muscle fiber conduction velocity (MFCV) has often been shown to decrease during standardized fatiguing isometric contractions. However, several studies have indicated that the MFCV may remain constant during fatiguing dynamic exercise. It was investigated if these observations can be related to the absence of a large decrease in pH and if MFCV can be considered as a good indicator of acidosis, also during dynamic bicycle exercise. High-density surface electromyography (HDsEMG) was combined with read-outs of muscle energetics recorded by in vivo (31)P magnetic resonance spectroscopy (MRS). Measurements were performed during serial exhausting bouts of bicycle exercise at three different workloads. The HDsEMG recordings revealed a small and incoherent variation of MFCV during all high-intensity exercise bouts. (31)P MRS spectra revealed a moderate decrease in pH at the end of exercise (~0.3 units down to 6.8) and a rapid ancillary drop to pH 6.5 during recovery 30 s post-exercise. This additional degree of acidification caused a significant decrease in MFCV during cycling immediately after the rest period. From the data a significant correlation between MFCV and [H(+)] ([H(+)] = 10(-pH)) was calculated (p < 0.001, Pearson's R = -0.87). Our results confirmed the previous observations of MFCV remaining constant during fatiguing dynamic exercise. A constant MFCV is in line with a low degree of acidification, considering the presence of a correlation between pH and MFCV after further increasing acidification.
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Acidosis/fisiopatología , Ciclismo/fisiología , Ejercicio Físico/fisiología , Contracción Isométrica/fisiología , Fatiga Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Conducción Nerviosa/fisiología , Adulto , Electromiografía , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Pressure ulcers are localized areas of soft tissue breakdown due to mechanical loading. Susceptible individuals are subjected to pressure relief strategies to prevent long loading periods. Therefore, ischemia-reperfusion injury may play an important role in the etiology of pressure ulcers. To investigate the inter-relation between postischemic perfusion and changes in skeletal muscle integrity, the hindlimbs of Brown Norway rats were subjected to 4-h ischemia followed by 2-h reperfusion. Dynamic contrast-enhanced MRI was used to examine perfusion, and changes in skeletal muscle integrity were monitored with T2-weighted MRI. The dynamic contrast-enhanced MRI data showed a heterogeneous postischemic profile in the hindlimb, consisting of areas with increased contrast enhancement (14-76% of the hindlimb) and regions with no-reflow (5-77%). For T2, a gradual increase in the complete leg was observed during the 4-h ischemic period (from 34 to 41 msec). During the reperfusion phase, a heterogeneous distribution of T2 was observed. Areas with increased contrast enhancement were associated with a decrease in T2 (to 38 msec) toward preischemic levels, whereas no-reflow areas exhibited a further increase in T2 (to 42 msec). These results show that reperfusion after prolonged ischemia may not be complete, thereby continuing the ischemic condition and aggravating tissue damage.
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Compuestos Heterocíclicos , Imagen por Resonancia Magnética/métodos , Músculo Esquelético/patología , Enfermedades Musculares/patología , Compuestos Organometálicos , Úlcera por Presión/patología , Daño por Reperfusión/patología , Animales , Medios de Contraste , Femenino , Gadolinio , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Mitochondria are thought to play a crucial role in the etiology of muscle insulin resistance (IR). The aim of this study was to gain more insight into the timing and nature of mitochondrial adaptations during the development of high-fat-diet (HFD)-induced IR. Adult Wistar rats were fed HFD or normal chow for 2.5 and 25 wk. Intramyocellular lipids (IMCLs) were quantified in vivo using (1)H magnetic resonance spectroscopy (MRS). Muscle oxidative capacity was assessed in vivo using (31)P MRS and in vitro by measuring mitochondrial DNA copy number and oxygen consumption in isolated mitochondria. MRS in tibialis anterior muscle revealed 3.3-fold higher IMCL content and 1.2-fold increased oxidative capacity after 2.5 wk of HFD feeding. The latter result could be fully accounted for by increased mitochondrial content. After 25 wk of HFD, maximal ADP-stimulated oxygen consumption in isolated mitochondria oxidizing pyruvate plus malate remained unaffected, while IMCL and mitochondrial content had further increased compared to controls (5.1-fold and 1.4-fold, respectively). Interestingly, in vivo oxidative capacity at this time point was identical to controls. These results show that skeletal muscle in HFD-induced IR accompanied by IMCL accumulation requires a progressively larger mitochondrial pool size to maintain normal oxidative capacity in vivo.
Asunto(s)
Grasas de la Dieta/metabolismo , Resistencia a la Insulina , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Animales , Carnitina/análogos & derivados , Carnitina/metabolismo , Dieta , Grasas de la Dieta/administración & dosificación , Masculino , Oxidación-Reducción , Consumo de Oxígeno , Ratas , Ratas WistarRESUMEN
(31)P magnetic resonance spectroscopy (MRS) has been used to assess skeletal muscle mitochondrial function in vivo by measuring 1) phosphocreatine (PCr) recovery after exercise or 2) resting ATP synthesis flux with saturation transfer (ST). In this study, we compared both parameters in a rat model of mitochondrial dysfunction with the aim of establishing the most appropriate method for the assessment of in vivo muscle mitochondrial function. Mitochondrial dysfunction was induced in adult Wistar rats by daily subcutaneous injections with the complex I inhibitor diphenyleneiodonium (DPI) for 2 wk. In vivo (31)P MRS measurements were supplemented by in vitro measurements of oxygen consumption in isolated mitochondria. Two weeks of DPI treatment induced mitochondrial dysfunction, as evidenced by a 20% lower maximal ADP-stimulated oxygen consumption rate in isolated mitochondria from DPI-treated rats oxidizing pyruvate plus malate. This was paralleled by a 46% decrease in in vivo oxidative capacity, determined from postexercise PCr recovery. Interestingly, no significant difference in resting, ST-based ATP synthesis flux was observed between DPI-treated rats and controls. These results show that PCr recovery after exercise has a more direct relationship with skeletal muscle mitochondrial function than the ATP synthesis flux measured with (31)P ST MRS in the resting state.
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Adenosina Trifosfato/biosíntesis , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Fosfocreatina/metabolismo , Condicionamiento Físico Animal/fisiología , Adenosina Difosfato/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Masculino , Mitocondrias Musculares/efectos de los fármacos , Músculo Esquelético/ultraestructura , Compuestos Onio/farmacología , Fosforilación Oxidativa , Consumo de Oxígeno , Ratas , Ratas WistarRESUMEN
Prolonged mechanical loading of soft tissues adjacent to bony prominences can lead to degeneration of muscle tissue, resulting in a condition termed pressure-related deep tissue injury. This type of deep pressure ulcers can develop into a severe wound, associated with problematic healing and a variable prognosis. Limited knowledge of the underlying damage pathways impedes effective preventive strategies and early detection. Traditionally, pressure-induced ischaemia has been thought to be the main aetiological factor for initiating damage. Recent research, however, proposes tissue deformation per se as another candidate for initiating pressure-induced deep tissue injury. In this study, different strain parameters were evaluated on their suitability as a generic predictive indicator for deep tissue injury. With a combined animal-experimental numerical approach, we show that there is a reproducible monotonic increase in damage with increasing maximum shear strain once a strain threshold has been exceeded. This relationship between maximum shear strain and damage seems to reflect an intrinsic muscle property, as it applied across a considerable number of the experiments. This finding confirms that tissue deformation per se is important in the aetiology of deep tissue injury. Using dedicated finite element modeling, a considerable reduction in the inherent biological variation was obtained, leading to the proposal that muscle deformation can prove a generic predictive indicator of damage.
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Modelos Animales de Enfermedad , Modelos Biológicos , Estimulación Física/efectos adversos , Úlcera por Presión/etiología , Úlcera por Presión/fisiopatología , Animales , Fuerza Compresiva , Simulación por Computador , Módulo de Elasticidad , Femenino , Presión , Ratas , Estrés MecánicoRESUMEN
The underlying mechanisms leading to deep tissue injury after sustained compressive loading are not well understood. It is hypothesized that initial damage to muscle fibers is induced mechanically by local excessive deformation. Therefore, in this study, an animal model was used to study early damage after compressive loading to elucidate on the damage mechanisms leading to deep pressure ulcers. The tibialis anterior of Brown-Norway rats was loaded for 2 h by means of an indenter. Experiments were performed in a magnetic resonance (MR)-compatible loading device. Muscle tissue was evaluated with transverse relaxation time (T2)-weighted MRI both during loading and up to 20 h after load removal. In addition, a detailed examination of the histopathology was performed at several time points (1, 4, and 20 h) after unloading. Results demonstrated that, immediately after unloading, T2-weighted MR images showed localized areas with increased signal intensity. Histological examination at 1 and 4 h after unloading showed large necrotic regions with complete disorganization of the internal structure of the muscle fibers. Hypercontraction zones were found bilateral to the necrotic zone. Twenty hours after unloading, an extensive inflammatory response was observed. The proposed relevance of large deformation was demonstrated by the location of damage indicated by T2-weighted MRI and the histological appearance of the compressed tissues. Differences in damage development distal and proximal to the indenter position suggested a contribution of perfusion status in the measured tissue changes that, however, appeared be to reversible.
Asunto(s)
Imagen por Resonancia Magnética , Úlcera por Presión/patología , Animales , Femenino , Inflamación/patología , Fibras Musculares Esqueléticas/patología , Necrosis/patología , Ratas , Ratas Endogámicas BN , Soporte de PesoRESUMEN
To study the aetiology of pressure ulcers an MR-compatible loading device was developed. Magnetic resonance imaging provides the possibility of non-invasive evaluation of muscle tissue after compressive loading. Pressure was applied to the tibialis anterior region of rats by means of an indenter. The developed MR-compatible loading device allowed high quality consecutive MR measurements for up to 6h. Tissue was evaluated both during and after loading. Two loading protocols were used; a large indentation of 4.5mm (mean pressure 150 kPa) was applied for 2h and a small indentation of 2.9 mm (mean pressure 50 kPa) was applied for 4h. T2-weighted MR images after the large indentation showed an immediate increase in signal intensity, associated with damage, following load removal. After 20 h the signal intensity remained higher in the affected regions. Afterwards the tissue was perfusion fixated for histological examination. Histological evaluation revealed an inflammatory response and severe muscle necrosis. No signal increase was observed after small indentation. With this new set-up, the different factors that may play a role in the onset of muscle damage can be studied, what we believe will lead to a better understanding of the contributing factors to pressure ulcer development.
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Imagen por Resonancia Magnética/métodos , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Estimulación Física/efectos adversos , Estimulación Física/métodos , Traumatismos de los Tejidos Blandos/diagnóstico , Soporte de Peso , Animales , Fuerza Compresiva , Diseño de Equipo , Análisis de Falla de Equipo , Femenino , Imagen por Resonancia Magnética/instrumentación , Presión , Úlcera por Presión/diagnóstico , Úlcera por Presión/etiología , RatasRESUMEN
Excitotoxicity is a paradigm used to explain the biochemical events in both acute neuronal damage and in slowly progressive, neurodegenerative diseases. Here, we show in a longitudinal magnetic resonance imaging study that Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the main active compound in marijuana, reduces neuronal injury in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain to elicit excitotoxicity. In the acute phase Delta(9)-THC reduced the volume of cytotoxic edema by 22%. After 7 d, 36% less neuronal damage was observed in treated rats compared with control animals. Coadministration of the CB(1) cannabinoid receptor antagonist SR141716 prevented the neuroprotective actions of Delta(9)-THC, indicating that Delta(9)-THC afforded protection to neurons via the CB(1) receptor. In Delta(9)-THC-treated rats the volume of astrogliotic tissue was 36% smaller. The CB(1) receptor antagonist did not block this effect. These results provide evidence that the cannabinoid system can serve to protect the brain against neurodegeneration.
Asunto(s)
Edema Encefálico/prevención & control , Cannabis , Dronabinol/farmacología , Fármacos Neuroprotectores/farmacología , Ouabaína/toxicidad , Enfermedad Aguda , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Edema Encefálico/inducido químicamente , Edema Encefálico/diagnóstico , Edema Encefálico/metabolismo , Enfermedad Crónica , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Estudios Longitudinales , Imagen por Resonancia Magnética , Microinyecciones , Ouabaína/administración & dosificación , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/antagonistas & inhibidores , Receptores de Droga/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Agua/metabolismoRESUMEN
The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 +/- 15% in a manner insensitive to the cannabinoid CB(1) receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 +/- 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.
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Ácidos Araquidónicos/farmacología , Lesiones Encefálicas/prevención & control , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Western Blotting , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Edema Encefálico/inducido químicamente , Edema Encefálico/patología , Edema Encefálico/prevención & control , Lesiones Encefálicas/inducido químicamente , Lesiones Encefálicas/patología , Moduladores de Receptores de Cannabinoides , Cannabinoides/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endocannabinoides , Inhibidores Enzimáticos , Glicéridos/metabolismo , Estudios Longitudinales , Imagen por Resonancia Magnética , Microinyecciones , Neuronas/metabolismo , Neuronas/patología , Ouabaína , Piperidinas/farmacología , Alcamidas Poliinsaturadas , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/antagonistas & inhibidores , RimonabantRESUMEN
Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.
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Ácidos Araquidónicos/fisiología , Cannabinoides/farmacología , Capsaicina/análogos & derivados , Capsaicina/metabolismo , Ácidos Grasos Insaturados/fisiología , Lipooxigenasa/fisiología , Degeneración Nerviosa/prevención & control , Fármacos Neuroprotectores/farmacología , Receptores de Droga/fisiología , Animales , Animales Recién Nacidos , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/enzimología , Células Sanguíneas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/metabolismo , Química Encefálica , Mapeo Encefálico , Moduladores de Receptores de Cannabinoides , Endocannabinoides , Etanolaminas/análisis , Etanolaminas/metabolismo , Lipooxigenasa/metabolismo , Masculino , Masoprocol/farmacología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/enzimología , Ouabaína/farmacología , Alcamidas Poliinsaturadas , Ratas , Ratas Wistar , Receptores de Droga/metabolismoRESUMEN
The inhibition of respiratory chain activities in rat liver, rat heart and bovine heart mitochondria by the anthracycline antibiotic adriamycin was measured in order to determine the adriamycin-sensitive sites. It appeared that complex III and IV are efficiently affected such that their activities were reduced to 50% of control values at 175 +/- 25 microM adriamycin. Complex I displayed a minor sensitivity to the drug. Of the complex-I-related activities tested, only duroquinone oxidation appeared sensitive (50% inhibition at approx. 450 microM adriamycin). Electron-transfer activities catalyzed by complex II remained essentially unaltered up to high drug concentrations. Of the activities measured for this complex, only duroquinone oxidation was significantly affected. However, the adriamycin concentration required to reduce this activity to 50% exceeded 1 mM. Mitochondria isolated from rat liver, rat heart and bovine heart behaved essentially identical in their response to adriamycin. These data support the conclusion that, in these three mitochondrial systems, the major drug-sensitive sites lie in complex III and IV. Cytochrome c oxidase and succinate oxidase activity in whole mitochondria exhibited a similar sensitivity towards adriamycin, as inner membrane ghosts, suggesting that the drug has direct access to its inner membrane target sites irrespective of the presence of the outer membrane. By measuring NADH and succinate oxidase activities in the presence of exogenously added cytochrome c, it appeared that adriamycin was less inhibitory under these conditions. This suggests that adriamycin competes with cytochrome c for binding to the same site on the inner membrane, presumably cardiolipin.
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Benzoquinonas , Doxorrubicina/farmacología , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Mitocondrias Cardíacas/enzimología , Mitocondrias Hepáticas/enzimología , Animales , Bovinos , Grupo Citocromo c/metabolismo , Masculino , Complejos Multienzimáticos/antagonistas & inhibidores , NADH Deshidrogenasa/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Oxidación-Reducción , Oxidorreductasas/antagonistas & inhibidores , Quinonas/metabolismo , Ratas , Succinato Deshidrogenasa/antagonistas & inhibidoresRESUMEN
Three functions have been suggested to be localized in contact sites between the inner and the outer membrane of mitochondria from mammalian cells: (i) transfer of energy from matrix to cytosol through the action of peripheral kinases; (ii) import of mitochondrial precursor proteins; and (iii) transfer of lipids between outer and inner membrane. In the contact site-related energy transfer a number of kinases localized in the periphery of the mitochondrion play a crucial role. Two examples of such kinases are relevant here: (i) hexokinase isoenzyme I which is capable of binding to the outer aspect of the outer membrane; and (ii) the mitochondrial isoenzyme of creatine kinase which is localized in the intermembrane space. Recently, evidence was presented that both hexokinase and creatine kinase are preferentially localized in contact sites (Adams, V. et al. (1989) Biochim. Biophys. Acta 981, 213-225). The aim of the present experiments was two-fold. First, to establish methods which enable the bioenergetic aspects of energy transfer mediated by kinases in contact sites to be measured. In these experiments emphasis was on hexokinase, while 31P-NMR was the major experimental technique. Second, we wanted to develop methods which can give insight into factors playing a role in the formation of contact sites involved in energy transfer. In the latter approach, mitochondrial creatine kinase was studied using monolayer techniques.
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Membranas Intracelulares/metabolismo , Mitocondrias Hepáticas/metabolismo , Animales , Sitios de Unión/fisiología , Creatina Quinasa/metabolismo , Citosol/metabolismo , Transferencia de Energía , Hexoquinasa/aislamiento & purificación , Hexoquinasa/metabolismo , Espectroscopía de Resonancia Magnética , Ratas , Saccharomyces cerevisiae/enzimologíaRESUMEN
Rat liver mitochondria were isolated by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation, as assessed by marker enzyme analysis, latency of cytochrome-c oxidase, respiratory control index and electron microscopy. Two different methods were compared for the separation of inner and outer membranes. In the swell-shrink-sonicate procedure glycerol was included resulting in the isolation of one outer membrane and two inner membrane fractions of high purity. Using digitonin a highly selective and gradual solubilization of the outer membrane could be accomplished. Analysis of the phospholipid composition of the intact mitochondria and all subfractions showed that the inner membrane was virtually devoid of phosphatidylinositol and -serine, while the outer membrane contained 23% of the total mitochondrial cardiolipin, which did not originate from inner membrane contamination and therefore is a true component of the outer membrane.
Asunto(s)
Membranas Intracelulares/ultraestructura , Lípidos de la Membrana/análisis , Mitocondrias Hepáticas/ultraestructura , Fosfolípidos/análisis , Partículas Submitocóndricas/ultraestructura , Animales , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad , Cromatografía en Capa Delgada , Digitonina , Membranas Intracelulares/análisis , Masculino , Lípidos de la Membrana/aislamiento & purificación , Microscopía Electrónica , Fosfolípidos/aislamiento & purificación , Povidona , Ratas , Ratas Endogámicas , Dióxido de Silicio , Partículas Submitocóndricas/análisisRESUMEN
The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.
Asunto(s)
Lípidos de la Membrana/metabolismo , Mitocondrias Hepáticas/enzimología , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Fosfolípidos/metabolismo , Animales , Calcio/farmacología , Hidrólisis , Cinética , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Micelas , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipasas A2 , RatasRESUMEN
The effects of the anti-tumor drug adriamycin on lipid polymorphism in cardiolipin-containing model membranes and in isolated inner mitochondrial membranes has been examined by 31P-NMR. Adriamycin binding does not affect the macroscopic structure or local order in the phosphate region of cardiolipin liposomes. In cardiolipin liposomes and in cardiolipin-phosphatidylcholine (1:1) liposomes, the drug inhibits the ability of Ca2+ to induce the hexagonal HII phase. Adriamycin interaction with both dioleoylphosphatidylethanolamine-cardiolipin (2:1) and dioleoylphosphatidylethanolamine-phosphatidylserine (1:1) liposomes results in structural phase separation into a liquid-crystalline hexagonal HII phase for the phosphatidylethanolamine and a liquid-crystalline lamellar phase for the negatively charged phospholipid. Combined high-resolution 31P-NMR, electron microscopy and light scattering studies reveal the prominent fusion capacity of adriamycin towards cardiolipin-phosphatidylcholine small unilamellar vesicles. Addition of Ca2+ to total rat liver inner mitochondrial membrane lipids, dispersed in excess buffer, results in hexagonal HII formation for part of the phospholipids. By contrast, the original bilayer structure is completely conserved when the above experiment is performed in the presence of adriamycin. 31P-NMR spectra of isolated inner mitochondrial membranes are indicative of a bilayer organization for the majority of the phospholipids. Approximately 15% of the signal intensity originates from phospholipids which experience isotropic motion. Adriamycin addition almost completely eliminates the latter spectral component. In the absence of adriamycin, Ca2+ addition greatly increases the percentage of the phospholipids giving rise to an isotropic signal possibly indicating the formation of non-lamellar lipid structures. Adriamycin which specifically binds to cardiolipin (K. Nicolay et al. (1984) Biochim. Biophys. Acta 778, 359-371) completely blocks the Ca2+-induced structural reorganization of the lipids in this membrane.
Asunto(s)
Cardiolipinas , Doxorrubicina , Lípidos de la Membrana , Calcio/metabolismo , Cardiolipinas/fisiología , Fenómenos Químicos , Química Física , Doxorrubicina/farmacología , Membranas Intracelulares/efectos de los fármacos , Membrana Dobles de Lípidos , Espectroscopía de Resonancia Magnética , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Modelos Biológicos , Fosforilación Oxidativa/efectos de los fármacos , Fosfatidilcolinas , Fosfatidiletanolaminas , FosfatidilserinasRESUMEN
In order to study the individual steps involved in the import of phosphatidylcholine (PC) into rat liver mitochondria, a number of PC analogues were introduced into the outer membrane of isolated mitochondria. Two fluorescent PC species, i.e. 1-palmitoyl-2-(16-bimanylthio)hexadecanoyl-PC (bimane-PC) and 1-palmitoyl-2-(10-pyrene)decanoyl-PC (pyrene-PC), and one radiolabeled PC species, i.e. 1-palmitoyl-2-[1-14C]oleoyl-PC (14C-POPC), were studied. The PC analogues were introduced from small unilamellar vesicles with the use of PC-specific transfer protein. The amount of PC imported was quantified by reisolation of the mitochondria. Import of the fluorescent PC species was monitored by on-line fluorescence spectroscopy. The distribution of the newly inserted PC between the outer and the inner membrane was assessed by separation of the two membranes using digitonin treatment. All analogues tested remained exclusively localized in the outer membrane thereby suggesting that additional (extramitochondrial) factors are required to initiate transfer of PC to the inner membrane.