A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts

• Published in: The American journal of physiology Vol. 265; no. 2 Pt 2; pp. H445 - H452
• Main Authors: Wan, B, Doumen, C, Duszynski, J, Salama, G, LaNoue, K F
• Format: Journal Article
• Language: English
• Published: United States 01-08-1993
• Subjects: Animals; Electrophysiology - methods; In Vitro Techniques; Indicators and Reagents - pharmacokinetics; Male; Membranes - physiology; Mitochondria, Heart - metabolism; Mitochondria, Heart - physiology; Myocardium - metabolism; Onium Compounds - pharmacokinetics; Organophosphorus Compounds - pharmacokinetics; Perfusion; Rats; Rats, Sprague-Dawley
• ISSN: 0002-9513
• DOI: 10.1152/ajpheart.1993.265.2.H445

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.