RESUMEN
Cells degrade excess and effete organelles by the process of autophagy. Autophagic stimulation of rat hepatocytes by serum deprivation and glucagon (1 M) caused a fivefold increase of spontaneously depolarizing mitochondria to about 1.5% of total mitochondria after 90 min. Cyclosporin A (CsA, 5 M), an immunosuppressant that blocks the mitochondrial permeability transition (MPT), prevented this depolarization. Depolarized mitochondria moved into acidic vacuoles labeled by LysoTracker Red. These autophagosomes also increased several-fold after autophagic stimulation. CsA blocked autophagosomal proliferation, whereas tacrolimus, an immunosuppressant that does not block the MPT, did not. In conclusion, the MPT initiates mitochondrial depolarization after autophagic stimulation and the subsequent sequestration of mitochondria into autophagosomes.
Asunto(s)
Autofagia , Hepatocitos/fisiología , Mitocondrias/metabolismo , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero , Ciclosporina/farmacología , Glucagón/farmacología , Hepatocitos/efectos de los fármacos , Inmunosupresores/farmacología , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Potenciales de la Membrana , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Permeabilidad , Fagosomas/metabolismo , RatasRESUMEN
Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye's syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye's syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.
Asunto(s)
Apoptosis , Autofagia , Mitocondrias/fisiología , Necrosis , Animales , Calcimicina/farmacología , Calcio/metabolismo , Células Cultivadas/efectos de los fármacos , Ciclosporina/farmacología , Fluoresceínas , Concentración de Iones de Hidrógeno , Microscopía Confocal , Mitocondrias/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo , Permeabilidad , Peróxidos , Especies Reactivas de Oxígeno/metabolismo , Rodaminas , Superóxidos/metabolismo , terc-ButilhidroperóxidoRESUMEN
Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.
Asunto(s)
Apoptosis , Autofagia , Permeabilidad de la Membrana Celular , Microscopía Confocal , Mitocondrias/metabolismo , Animales , Calcio/metabolismo , Humanos , Mitocondrias/ultraestructura , Necrosis , Síndrome de Reye/etiologíaRESUMEN
Intravenous infusion of Vibrio vulnificus lipopolysaccharide (LPS) (1 mg/kg body wt) in rats caused a dramatic drop in mean arterial pressure within 10 min and a further decline in mean arterial pressure and heart rate which lead to death between 25 and 70 min. Rats treated with LPS followed 10 min later by the intravenous infusion of NG-monomethyl-L-arginine (L-NMMA, 20 mg/kg body wt) showed an initial drop in mean arterial pressure owing to the LPS infusion, followed by a transient rise in mean arterial pressure which lasted for approximately 40 min after the infusion of L-NMMA. The pressure values then remained level for at least 150 min post-LPS infusion. Control rats treated with equivalent volumes of saline infusion showed stable values of mean arterial pressure and heart rate. Additional control rats receiving L-NMMA alone showed the transient rise in mean arterial pressure, followed by a return to the baseline values. The results indicate that the symptoms of endotoxic shock resulting from V. vulnificus LPS may result in part from the stimulation of the activity of nitric oxide synthase. Inhibition of nitric oxide synthase by L-NMMA is a possible treatment for toxic shock induced by V. vulnificus.