A METHOD OF DETERMINING ELECTRICAL POTENTIAL GRADIENT ACROSS MITOCHONDRIAL-MEMBRANE IN PERFUSED RAT HEARTS

The electrical potential gradient across the mitochondrial membrane (D ELTApsi(m)) in perfused rat hearts was estimated by calculating the eq uilibrium 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-m in perfusions of intact rat hearts with tracer amounts (5.0 nM) of tri tium-labeled TPP+ ([H-3]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 [H-3]TPP+ outside the heart and total [H-3]TP P+ inside the heart at equilibrium were calculated. From this informat ion and separately estimated time-averaged plasma membrane potentials (DELTApsi(c)) it was possible to calculate free cytosolic [H-3]TPP+ at equilibrium. It was also possible to calculate free intramitochondria l [H-3]TPP+ at equilibrium as the difference between total tissue [H-3 ]TPP+ minus free cytosolic TPP+ and the sum of all the bound [H-3]TPP. Bound [H-3]TPP+ was determined from [H-3]TPP+ binding constants meas ured in separate experiments, using both isolated mitochondria and iso lated cardiac myocytes under conditions where both DELTApsi(m) and DEL TApsi(c) were zero. DELTApsi(m) was calculated from the intramitochond rial and cytosolic free TPP+ concentrations using the Nernst equation. Values of DELTApsi(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 glucos e, in good agreement with DELTApsi(m) obtained from isolated rat heart mitochondria. The higher value of DELTApsi(m) obtained from pyruvate- perfused hearts compared with glucose-perfused hearts correlated well with higher cytosolic phosphorylation potentials obtained in pyruvate- perfused hearts. The results indicate that the method developed in the present study provides an adequate means to evaluate DELTApsi(m) in t he perfused heart.