title: A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts.
abstract: The electrical potential gradient across the mitochondrial membrane (delta psi m) in perfused rat hearts was estimated by calculating the equilibrium distribution of the lipophilic cation tetraphenylphosphonium (TPP+), using measured kinetic constants of uptake and release of TPP+. First-order rate constants of TPP+ uptake were measured during 30-min perfusions of intact rat hearts with tracer amounts (5.0 nM) of tritium-labeled TPP+ ([3H]TPP+) in the perfusate. This was followed by a 30-min washout, during which the first-order rate constant of efflux was estimated. Values of [3H]TPP+ outside the heart and total [3H]TPP+ inside the heart at equilibrium were calculated. From this information and separately estimated time-averaged plasma membrane potentials (delta psi c) it was possible to calculate free cytosolic [3H]TPP+ at equilibrium. It was also possible to calculate free intramitochondrial [3H]TPP+ at equilibrium as the difference between total tissue [3H]TPP+ minus free cytosolic TPP+ and the sum of all the bound [3H]TPP+. Bound [3H]TPP+ was determined from [3H]TPP+ binding constants measured in separate experiments, using both isolated mitochondria and isolated cardiac myocytes under conditions where both delta psi m and delta psi c were zero. Delta psi m was calculated from the intramitochondrial and cytosolic free TPP+ concentrations using the Nernst equation. Values of delta psi m were 144.9 +/- 2.0 mV in hearts perfused with 5 mM pyruvate and 118.2 +/- 1.4 mV in hearts perfused with 11 mM glucose, in good agreement with delta psi m obtained from isolated rat heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
authors.forename: B
authors.initials: B
authors.lastname: Wan
authors.forename: C
authors.initials: C
authors.lastname: Doumen
authors.forename: J
authors.initials: J
authors.lastname: Duszynski
authors.forename: G
authors.initials: G
authors.lastname: Salama
authors.forename: K F
authors.initials: KF
authors.lastname: LaNoue
mesh_terms: D000818:Animals; D004594:Electrophysiology / Q000379:methods*; D066298:In Vitro Techniques; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D008297:Male; D008566:Membranes / Q000502:physiology; D008929:Mitochondria, Heart / Q000378:metabolism / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism; D009861:Onium Compounds / Q000493:pharmacokinetics; D009943:Organophosphorus Compounds / Q000493:pharmacokinetics; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D007202:Indicators and Reagents; D009861:Onium Compounds; D009943:Organophosphorus Compounds; C013289:tetraphenylphosphonium