title: Fluorescence imaging of metabolic responses in single mitochondria.
abstract: The membrane potentials, rates of NAD(P)H formation, and rates of flavoprotein reduction have been measured for single mitochondria isolated from porcine hearts. These metabolic responses were elicited by the addition of malate and measured using fluorescence microscopy. For the measurements of mitochondrial membrane potential, mitochondria were stained with tetramethylrhodamine ethyl ester, and the membrane potentials of single mitochondria were determined. Individual mitochondria maintained the membrane potential at around -80 mV before addition of malate. Upon the addition of malate, each mitochondrion was rapidly polarized to around -100 approximately -140 mV and underwent repeated cycles of polarization and depolarization, which were probably caused by openings and closings of permeability transition pores. NAD(P)(+) and flavoprotein were reduced immediately after addition of malate and then slowly became reoxidized. Thus, single mitochondria can undergo rapid and repetitive changes in membrane potential, but not in the redox state of NAD(P)H and flavoprotein.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Tokyo University of Agriculture and Technology, Nakacho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakayama
authors.forename: Takanori
authors.initials: T
authors.lastname: Sakuyama
authors.forename: Shigeki
authors.initials: S
authors.lastname: Mitaku
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohta
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004305:Dose-Response Relationship, Drug; D005415:Flavins / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D008293:Malates / Q000494:pharmacology*; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure*; D008929:Mitochondria, Heart / Q000378:metabolism / Q000648:ultrastructure*; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D009942:Organometallic Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D013552:Swine; D013997:Time Factors
chemical_list: D005415:Flavins; D005420:Flavoproteins; D005456:Fluorescent Dyes; D008293:Malates; D009942:Organometallic Compounds; C110932:tetramethyl rhodamine ethyl ester; D009249:NADP; C030298:malic acid; D000255:Adenosine Triphosphate