Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.

• MeSH
• Cell Line MeSH
• Citric Acid Cycle drug effects   MeSH
• Citrate (si)-Synthase drug effects   metabolism   MeSH
• Glutamate Dehydrogenase drug effects   metabolism   MeSH
• Isocitrate Dehydrogenase drug effects   metabolism   MeSH
• Ketoglutarate Dehydrogenase Complex antagonists & inhibitors   metabolism   MeSH
• Muscle, Skeletal enzymology   MeSH
• Rats MeSH
• Malate Dehydrogenase drug effects   metabolism   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Onium Compounds pharmacology   MeSH
• Rats, Wistar MeSH
• Pyruvate Dehydrogenase Complex drug effects   metabolism   MeSH
• Mitochondria, Muscle drug effects   enzymology   MeSH
• Triphenylmethyl Compounds pharmacology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Citrate (si)-Synthase MeSH
• Glutamate Dehydrogenase MeSH
• Isocitrate Dehydrogenase MeSH
• Ketoglutarate Dehydrogenase Complex MeSH
• Malate Dehydrogenase MeSH
• Onium Compounds MeSH
• Pyruvate Dehydrogenase Complex MeSH
• triphenylmethylphosphonium MeSH Browser
• Triphenylmethyl Compounds MeSH

BACKGROUND: The lipophilic positively charged moiety of triphenylphosphonium (TPP+) has been used to target a range of biologically active compounds including antioxidants, spin-traps and other probes into mitochondria. The moiety itself, while often considered biologically inert, appears to influence mitochondrial metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We used the Seahorse XF flux analyzer to measure the effect of a range of alkylTPP+ on cellular respiration and further analyzed their effect on mitochondrial membrane potential and the activity of respiratory complexes. We found that the ability of alkylTPP+ to inhibit the respiratory chain and decrease the mitochondrial membrane potential increases with the length of the alkyl chain suggesting that hydrophobicity is an important determinant of toxicity. CONCLUSIONS/SIGNIFICANCE: More hydrophobic TPP+ derivatives can be expected to have a negative impact on mitochondrial membrane potential and respiratory chain activity in addition to the effect of the biologically active moiety attached to them. Using shorter linker chains or adding hydrophilic functional groups may provide a means to decrease this negative effect.

• MeSH
• Cell Line MeSH
• Electron Transport Chain Complex Proteins metabolism   MeSH
• Heterocyclic Compounds pharmacology   MeSH
• Rats MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Organophosphorus Compounds pharmacology   MeSH
• Rats, Wistar MeSH
• Oxygen Consumption drug effects   MeSH
• Mitochondria, Muscle metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Electron Transport Chain Complex Proteins MeSH
• Heterocyclic Compounds MeSH
• Organophosphorus Compounds MeSH
• tris(o-phenylenedioxy)cyclotriphosphazene MeSH Browser

BACKGROUND: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. METHODS: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses. RESULTS: Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p 50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations. CONCLUSION: Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes. GENERAL SIGNIFICANCE: The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.

• Keywords
• AA, antimycin A, BNE, Blue Native PAGE, COX, cytochrome c oxidase, Diagnostics, FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, GP, glycerol-3-phosphate, GPDH, mitochondrial FAD-dependent glycerophosphate dehydrogenase, Lymphocytes, Mitochondrial diseases, OXPHOS, oxidative phosphorylation, Oxidative phosphorylation, PAGE, polyacrylamide gel electrophoresis, Respirometry, TMPD, tetramethylphenylenediamine, TMRM, tetramethylrhodamine methyl ester, cI–cV, respiratory chain complexes I–V, s3, state 3-ADP, s3u, state 3-uncoupled, s4o, state 4-oligomycin, ΔΨm, mitochondrial membrane potential,
• Publication type
• Journal Article MeSH

Dysfunction of mitochondrial ATPase (F1F(o)-ATP synthase) due to missense mutations in ATP6 [mtDNA (mitochondrial DNA)-encoded subunit a] is a frequent cause of severe mitochondrial encephalomyopathies. We have investigated a rare mtDNA mutation, i.e. a 2 bp deletion of TA at positions 9205 and 9206 (9205DeltaTA), which affects the STOP codon of the ATP6 gene and the cleavage site between the RNAs for ATP6 and COX3 (cytochrome c oxidase 3). The mutation was present at increasing load in a three-generation family (in blood: 16%/82%/>98%). In the affected boy with severe encephalopathy, a homoplasmic mutation was present in blood, fibroblasts and muscle. The fibroblasts from the patient showed normal aurovertin-sensitive ATPase hydrolytic activity, a 70% decrease in ATP synthesis and an 85% decrease in COX activity. ADP-stimulated respiration and the ADP-induced decrease in the mitochondrial membrane potential at state 4 were decreased by 50%. The content of subunit a was decreased 10-fold compared with other ATPase subunits, and [35S]-methionine labelling showed a 9-fold decrease in subunit a biosynthesis. The content of COX subunits 1, 4 and 6c was decreased by 30-60%. Northern Blot and quantitative real-time reverse transcription-PCR analysis further demonstrated that the primary ATP6--COX3 transcript is cleaved to the ATP6 and COX3 mRNAs 2-3-fold less efficiently. Structural studies by Blue-Native and two-dimensional electrophoresis revealed an altered pattern of COX assembly and instability of the ATPase complex, which dissociated into subcomplexes. The results indicate that the 9205DeltaTA mutation prevents the synthesis of ATPase subunit a, and causes the formation of incomplete ATPase complexes that are capable of ATP hydrolysis but not ATP synthesis. The mutation also affects the biogenesis of COX, which is present in a decreased amount in cells from affected individuals.

• MeSH
• Electrophoresis, Gel, Two-Dimensional methods   MeSH
• Adenine metabolism   MeSH
• Adenosine Triphosphate biosynthesis   MeSH
• Adenosine Triphosphatases chemistry   physiology   MeSH
• Fibroblasts chemistry   enzymology   metabolism   pathology   MeSH
• Intracellular Membranes chemistry   enzymology   MeSH
• Cells, Cultured MeSH
• Skin pathology   MeSH
• Humans MeSH
• Membrane Potentials genetics   MeSH
• RNA, Messenger biosynthesis   MeSH
• DNA, Mitochondrial biosynthesis   genetics   MeSH
• Mitochondrial Proton-Translocating ATPases biosynthesis   MeSH
• Mitochondria chemistry   enzymology   MeSH
• Mutation genetics   MeSH
• Child, Preschool MeSH
• Electron Transport Complex IV biosynthesis   chemistry   metabolism   physiology   MeSH
• Sequence Deletion genetics   MeSH
• Oxygen Consumption genetics   physiology   MeSH
• Thymidine metabolism   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine MeSH
• Adenosine Triphosphate MeSH
• Adenosine Triphosphatases MeSH
• RNA, Messenger MeSH
• DNA, Mitochondrial MeSH
• Mitochondrial Proton-Translocating ATPases MeSH
• MT-ATP6 protein, human MeSH Browser
• Electron Transport Complex IV MeSH
• Thymidine MeSH