title: In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP.
abstract: Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.
authors.affiliation: Laboratory of Bioenergetics, U.S.S.R. Cardiology Research Center, Moscow.
authors.forename: V A
authors.initials: VA
authors.lastname: Saks
authors.forename: Y O
authors.initials: YO
authors.lastname: Belikova
authors.forename: A V
authors.initials: AV
authors.lastname: Kuznetsov
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D002469:Cell Separation; D003402:Creatine Kinase / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008855:Microscopy, Electron, Scanning; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption; D051381:Rats; D011919:Rats, Inbred Strains; D012503:Saponins / Q000494:pharmacology
chemical_list: D012503:Saponins; D000244:Adenosine Diphosphate; D007770:L-Lactate Dehydrogenase; D003402:Creatine Kinase