Komlodi 2014 Abstract MiP2014

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Methylenblue, an enhancer of mitochondrial substrate-level phosphorylation.
Link:
Komlodi T
Mitochondr Physiol Network 19.13 - MiP2014

Komlodi T, Horvath G, Adam V, Tretter L (2014)

Event: MiP2014

Neurodegenerative disorders are associated with mitochondrial disfunction. In Alzheimer’s and Parkinson’s disease defects of respiratory system components have been described. Methylenblue (MB), a potential neuroprotective agent, is efficient in various Alzheimer and Parkinson models. The mitochondrial effects of MB are explained by the so-called alternative electron transfer model.

In the present study the effect of MB on mitochondrial substrate-level phosphorylation (SLP) was investigated. SLP in mitochondria is attributed to succinyl-CoA ligase catalyzing the formation of ATP and succinate from succinyl-CoA, ADP and Pi, in the citric acid cycle. This reaction can be considered an alternative way of ATP synthesis, because it is partially independent from the electron transfer-pathway (ET-pathway) and from the mitochondrial protonmotive force. Therefore, it has a great significance in hypoxia or when the ET-pathway is impaired. Besides that, SLP plays an important role during thermogenesis, when the mitochondria are uncoupled by the activation of thermogenin. MB can transfer electrons from Complex I to cytochrome c, bypassing the intermediate components of the ET-pathway. In case of Complex I inhibition it avoids NADH accumulation and thus inhibition of mitochondrial dehydrogenases, particulary α-ketoglutarate dehydrogenase (α-KGDH). Therefore, in the presence of MB the α-KGDH reaction can proceed and provide succinyl-CoA for matrix SLP. Our measurements were carried out on isolated mitochondria prepared from guinea-pig brain cortex. In mitochondria, ATP can be generated by oxidative phosphorylation, substrate-level phosphorylation and the adenylate kinase reaction catalyzing the formation of AMP and ATP from 2 mol ADP. The latter reaction was inhibited by AP5 during our measurements. ATP synthesis was measured spectrophotometrically with a coupled enzyme assay. The mitochondrial membrane potential was detected by safranine fluorescence and oxygen consumption was measured by high-resolution respirometry (Oroboros Oxygraph-2k).

In the presence of ET-pathway inhibitors, mitochondrial ATP synthesis was inhibited. In the presence of oligomycin, OXPHOS was inhibited but a low level of SLP could be detected. In the presence of ET-pathway inhibitors, addition of MB partially restores ΔΨmt and electron flow, resulting in ATP synthesis from OXPHOS and SLP. In the presence of oligomycin, addition of MB resulted in increase of SLP, which could be further stimulated by uncouplers. In the simultaneous presence of ET-pathway inhibitors and oligomycin administration of MB stimulates SLP and the formed ATP can contribute to the alleviation of energetic insufficiency. The effects of MB on SLP, however, are substrate dependent. α-ketoglutarate supports MB’s effect on SLP, but addition of succinate does not stimulate SLP.

We conclude that MB mediated stimulation of SLP can be an important factor to maintain energetic competence of mitochondria.

Supported by OTKA (NK 81983), TÁMOP (4.2.2./B-09/1), MTA (MTA TKI 2013), National Brain Research Program ( KTIA_13_NAP-A-III/6).


O2k-Network Lab: HU Budapest Tretter L


Labels: MiParea: Respiration  Pathology: Alzheimer's, Parkinson's  Stress:Ischemia-reperfusion  Organism: Guinea pig  Tissue;cell: Nervous system  Preparation: Isolated mitochondria 

Regulation: ATP production, mt-Membrane potential  Coupling state: OXPHOS, ET  Pathway: N, S  HRR: Oxygraph-2k, O2k-Fluorometer  Event: B1, Poster  MiP2014 

Affiliation

Dep Medical Biochemistry, Semmelweis Univ, Budapest. - komlodi.timea@med.semmelweis-univ.hu