RESUMO
The cell-toxic bile salt glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA) are responsible for hepatocyte demise in cholestatic liver diseases, while tauroursodeoxycholic acid (TUDCA) is regarded hepatoprotective. We demonstrate the direct mitochondrio-toxicity of bile salts which deplete the mitochondrial membrane potential and induce the mitochondrial permeability transition (MPT). The bile salt mediated mechanistic mode of destruction significantly differs from that of calcium, the prototype MPT inducer. Cell-toxic bile salts initially bind to the mitochondrial outer membrane. Subsequently, the structure of the inner boundary membrane disintegrates. And it is only thereafter that the MPT is induced. This progressive destruction occurs in a dose- and time-dependent way. We demonstrate that GCDCA and TCDCA, but not TUDCA, preferentially permeabilize liposomes containing the mitochondrial membrane protein ANT, a process resembling the MPT induction in whole mitochondria. This suggests that ANT is one decisive target for toxic bile salts. To our knowledge this is the first report unraveling the consecutive steps leading to mitochondrial destruction by cell-toxic bile salts.
Assuntos
Ácido Glicoquenodesoxicólico/toxicidade , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/agonistas , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Lipossomos/química , Fígado/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/química , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Translocases Mitocondriais de ADP e ATP/isolamento & purificação , Proteínas de Transporte da Membrana Mitocondrial/agonistas , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Miocárdio/química , Ratos , Ácido Tauroquenodesoxicólico/toxicidade , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/isolamento & purificaçãoRESUMO
Fluorometric measurements of 4-methylumbelliferone (4-MU) are generally used to screen lysosomal storage diseases (LSDs) using dried blood spots (DBSs). However, in DBS, it is difficult to measure lysosomal acid lipase (LAL) activity due to the influence of other lipases in whole blood. Recently, Hamilton used a fluorometric enzyme assay with 4-MU derivatives to measure the LAL activity in DBS. This method requires mercury chloride as stopping reagent, and the fluorescence intensity of 4-MU was measured at an acidic pH. We report a revised method to measure the LAL activity without using toxic mercury chloride and to measure the fluorescence intensity of 4-MU at a basic pH. For this measurement, we established a more practical method that does not require mercury chloride. The LAL activity in DBS was measured in 51 normal controls, seven obligate carriers and seven patients with CESD. The average LAL activities ± SD in the DBS from the normal, obligate carriers and CESD patients were 0.68 ± 0.2 (range: 0.3-1.08), 0.21 ± 0.1 (range: 0.11-0.41) and 0.02 ± 0.02 (range: 0-0.06) nmol/punch/h, respectively. There was a significant difference between the normal and the CESD. Our method does not require toxic mercury chloride and is an appropriate revised enzyme assay using DBS for screening patients with CESD.
Assuntos
Doença do Armazenamento de Colesterol Éster/sangue , Teste em Amostras de Sangue Seco/métodos , Fluorometria/métodos , Esterol Esterase/sangue , Doença de Wolman/sangue , Adulto , Biomarcadores/sangue , Carbamatos/química , Estudos de Casos e Controles , Doença do Armazenamento de Colesterol Éster/diagnóstico , Humanos , Concentração de Íons de Hidrogênio , Himecromona/química , Limite de Detecção , Esterol Esterase/antagonistas & inibidores , Tiadiazóis/química , Doença de Wolman/diagnósticoRESUMO
Acid phosphatases (APases) play a key role in phosphorus (P) acquisition and recycling in plants. White lupin (Lupinus albus L.) forms cluster roots (CRs) and produces large amounts of APases under P deficiency. However, the relationships between the activity of intracellular and extracellular APases (EC 3.1.3.2) and CR development are not fully understood. Here, comparative studies were conducted to examine the spatial variation pattern of APase activity during CR development using the enzyme-labelled fluorescence-97 (ELF-97) and the p-nitrophenyl phosphate methods. The activity of intracellular and extracellular APases was significantly enhanced under P deficiency in the non-CRs and CRs at different developmental stages. These two APases exhibited different spatial distribution patterns during CR development, and these distribution patterns were highly modified by P deficiency. The activity of extracellular APase increased steadily with CR development from meristematic, juvenile, mature to senescent stages under P deficiency. In comparison, P deficiency-induced increase in the activity of intracellular APase remained relatively constant during CR development. Increased activity of intracellular and extracellular APases was associated with enhanced expression of LaSAP1 encoding intracellular APase and LaSAP2 encoding extracellular APase. The expression levels of these two genes were significantly higher at transcriptional level in both mature and senescent CRs. Taken together, these findings demonstrate that both activity and gene expression of intracellular or extracellular APases exhibit a differential response pattern during CR development, depending on root types, CR developmental stages and P supply. Simultaneous in situ determination of intracellular and extracellular APase activity has proved to be an effective approach for studying spatial variation of APases during CR development.