M. L Kluppel

Effect of methotrexate (MTX) on NAD(P)+ dehydrogenases of HeLa cells: malic enzyme, 2-oxoglutarate and isocitrate dehydrogenases

The effects of methotrexate (MTX) on oxygen uptake by permeabilized HeLa cells were evaluated. MTX did not inhibit state III respiration when the oxidizable substrate was succinate, but when the substrates were 2‐oxoglutarate or isocitrate the respiration decreased about 50 per cent at 1·0 mM concentration of the drug. This effect was explained by inhibition of 2‐oxoglutarate and isocitrate dehydrogenases by MTX. No effect was observed on succinate dehydrogenase. An evaluation of the effects of MTX on malic enzyme activity as measured by pyruvate plus lactate production in intact cells supplied with malate showed a decrease of about 40 per cent in metabolite production using 0·4 mM MTX. HeLa cell malic enzyme, as observed for other tumour cells, is compartmentalized in mitochondria and cytosol, and is another example of a dehydrogenase inhibited by MTX.

Amiodarone effects on membrane organization evaluated by fluorescence polarization

The effects of amiodarone (0-100 microM) on the physical state of synthetic and native membranes were investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and of its anionic propionic acid derivative (DPH-PA), probing the outer regions of the bilayer. In the gel phase of dimyristoylphosphatidylcholine (DMPC) bilayers, amiodarone broadens the transition profile and shifts the phase transition midpoint to lower temperature values, as evaluated by both probes. On the other hand, the drug orders the fluid phase of the lipid either in hydrophobic core or in the outer regions of the bilayer, as detected by DPH and DPH-PA, respectively. The effects of amiodarone on the thermotropic behaviour of DPPC confirm and extend data in DMPC. Cholesterol concentration modulates to a great extent the effects of amiodarone in the fluid phase of DMPC. Thus, both probes, DPH and DPH-PA, detect either ordering effects of amiodarone for low cholesterol concentrations (< or = 20 mol%) or disordering amiodarone effects at higher cholesterol levels (> 20 mol%). In agreement with the results in models of synthetic lipids, the ordering effects of amiodarone in fluid native membranes of mitochondria and brain microsomes are depressed with the increase in intrinsic cholesterol. The ordering effects in mitochondria may induce bioenergetic dysfunctions and consequently disturbances in the electromechanic functioning of myocardium.

Possible mechanism of action of perhexiline maleate on heart mitochondria.

Abstract Oxidative phosphorylation and oxidation of NAD+-linked substrates by rat heart mitochondria were depressed by 6.75 × 10−5 M perhexiline maleate (PM), while the succinate oxidation was increased to 320 per cent activity. The drug had no effect on mitochondrial succinic and malate dehydrogenase, NADH-ferricyanide reductase and NADH-CoQ reductase (340 nm); therefore, NADH-oxidase, mitochondrial electron-transporting particles (EP1), NADH-CoQ reductase (550 nm) and aspartate aminotransferase were inhibited. It is suggested that PM would act. preventing the reoxidation of NADH+ + H+ through the respiratory chain.

Caracterizaçäo de miopatias mitocondriais através da avaliaçäo das atividades enzimáticas envolvidas no metabolismo energético

The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome c oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.Foi determinada a atividade das enzimas NADH desidiogenase, NADH citocromo e redutase, succinato desidiogenase, succinato citocromo e redutase, citocromo e oxidase e citrato sintase em mitocondrias de musculo esqueletico humano normal e doente (suspeito de miopatia mitocondrial). O grupo controle foi constituido de 13 individuos normais e que nao faziam uso continuo de farrnacos. O grupo doente era constituido de 10 pacientes cujo diagnostico anatomopatologico indicava suspeita de miopatia mitocondrial. Observou-se reducao na atividade das enzimas em todos os pacientes: 7 com anormalidades em todas as enzimas ensaiadas; 2 com deficiencias em todas as enzimas exceto na citocromo e oxidase; e 1 paciente com disfuncao apenas na atividade da succinato desidiogenase e succinato citocromo e redutase. Este perfil possibilitou caracterizar multiplas deficiencias ou deficiencia combinada da cadeia respiratoria, alem da disfuncao na citrato sintase em 9 pacientes. Um dos casos constituiu excecao, sendo a deficiencia enzimatica restrita ao complexo II. Foi possivel concluir que a metodologia usada e adequada e facilmente aplicavel aos objetivos clinicos. Os resultados obtidos possibilitam a caracterizacao dos complexos enzimaticos mitocondriais deficientes, mostrando que tais enfermidades sao originadas de disfuncao no metabolismo energetico.