title: Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.
abstract: Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.
authors.affiliation: Division of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, MB, Canada R2H 2A6 srchowdhury@sbrc.ca.
authors.forename: Subir Roy
authors.initials: SR
authors.lastname: Chowdhury
authors.affiliation: Division of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, MB, Canada R2H 2A6.
authors.forename: Jelena
authors.initials: J
authors.lastname: Djordjevic
authors.affiliation: Division of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, MB, Canada R2H 2A6 Department of Pharmacology & Therapeutics, University of Manitoba, Winnipeg, MB, Canada R3E 0T6.
authors.forename: Benedict C
authors.initials: BC
authors.lastname: Albensi
authors.affiliation: Division of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, MB, Canada R2H 2A6 Department of Pharmacology & Therapeutics, University of Manitoba, Winnipeg, MB, Canada R3E 0T6.
authors.forename: Paul
authors.initials: P
authors.lastname: Fernyhough
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002540:Cerebral Cortex / Q000378:metabolism* / Q000473:pathology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010101:Oxygen Consumption / Q000187:drug effects* / Q000235:genetics; D010619:Phenazines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012235:Rhodamines / Q000494:pharmacology*
chemical_list: D010619:Phenazines; D012235:Rhodamines; C401833:tetramethylrhodamine methyl ester; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C009195:safranine T
keywords: Alzheimer's disease; TMRM; cortex; membrane potential; mitochondrial respiration; safranin O