RESUMO
Mevalonic aciduria (MVA) is caused by severe deficiency of mevalonic kinase activity leading to tissue accumulation and high urinary excretion of mevalonic acid (MA) and mevalonolactone (ML). Patients usually present severe neurologic symptoms whose pathophysiology is poorly known. Here, we tested the hypothesis that the major accumulating metabolites are toxic by investigating the in vitro effects of MA and ML on important mitochondrial functions in rat brain and liver mitochondria. ML, but not MA, markedly decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and the capacity to retain Ca2+ in the brain, besides inducing mitochondrial swelling. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (MPT) cyclosporine A and ADP, as well as by ruthenium red in Ca2+-loaded mitochondria, indicating the involvement of MPT and an important role for mitochondrial Ca2+ in these effects. ML also induced lipid peroxidation and markedly inhibited aconitase activity, an enzyme that is highly susceptible to free radical attack, in brain mitochondrial fractions, indicating that lipid and protein oxidative damage may underlie some of ML-induced deleterious effects including MTP induction. In contrast, ML and MA did not compromise oxidative phosphorylation in the brain and all mitochondrial functions evaluated in the liver, evidencing a selective toxicity of ML towards the central nervous system. Our present study provides for the first time evidence that ML impairs essential brain mitochondrial functions with the involvement of MPT pore opening. It is therefore presumed that disturbance of brain mitochondrial homeostasis possibly contributes to the neurologic symptoms in MVA.
Assuntos
Potencial da Membrana Mitocondrial/fisiologia , Deficiência de Mevalonato Quinase/induzido quimicamente , Deficiência de Mevalonato Quinase/metabolismo , Ácido Mevalônico/análogos & derivados , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ácido Mevalônico/toxicidade , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/agonistas , Poro de Transição de Permeabilidade Mitocondrial , Ratos , Ratos WistarRESUMO
Oxidative stress (OS) is a common event in most hepatopathies, leading to mitochondrial permeability transition pore (MPTP) formation and further exacerbation of both OS from mitochondrial origin and cell death. Intracellular Ca²âº increase plays a permissive role in these events, but the underlying mechanisms are poorly known. We examined in primary cultured rat hepatocytes whether the Ca²âº/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling pathway is involved in this process, by using tert-butyl hydroperoxide (tBOOH) as a pro-oxidant, model compound. tBOOH (500 µM, 15 min) induced MPTP formation, as assessed by measuring mitochondrial membrane depolarization as a surrogate marker, and increased lipid peroxidation in a cyclosporin A (CsA)-sensitive manner, revealing the involvement of MPTPs in tBOOH-induced radical oxygen species (ROS) formation. Intracellular Ca²âº sequestration with BAPTA/AM, CaM blockage with W7 or trifluoperazine, and CaMKII inhibition with KN-62 all fully prevented tBOOH-induced MPTP opening and reduced tBOOH-induced lipid peroxidation to a similar extent to CsA, suggesting that Ca²âº/CaM/CaMKII signaling pathway fully mediates MPTP-mediated mitochondrial ROS generation. tBOOH-induced apoptosis, as shown by flow cytometry of annexin V/propidium iodide, mitochondrial release of cytochrome c, activation of caspase-3 and increase in the Bax-to-Bcl-xL ratio, and the Ca²âº/CaM/CaMKII signaling antagonists fully prevented these effects. Intramitochondrial CaM and CaMKII were partially involved in tBOOH-induced MPTP formation, since W7 and KN-62 both attenuated the tBOOH-induced, MPTP-mediated swelling of isolated mitochondria. We concluded that Ca²âº/CaM/CaMKII signaling pathway is a key mediator of OS-induced MPTP formation and the subsequent exacerbation of OS from mitochondrial origin and apoptotic cell death.