Measurements of mitochondrial K+ fluxes in whole rat hearts using Rb-87-NMR

The rubidium efflux from hypothermic rat hearts perfused by the Langendorff method at 20 degrees C was studied. At this temperature Rb-87-NMR efflux e xperiments showed the existence of two Rb-87 pools: cytoplasmic and mitocho ndrial. Rat heart mitochondria showed a very slow exchange of mitochondrial Rb+ for cytoplasmic K+. After washout of cytosolic Rb+, mitochondria kept a stable Rb+ level for >30 min. Rb+ efflux from mitochondria was stimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmal permeabilization and c oncomitant cellular energy depletion by saponin (0.01 mg/ml for 4 min) in t he presence of a perfusate mimicking intracellular conditions, or by ATP-se nsitive K (K-ATP) channel openers. DNP, a mitochondrial uncoupler, caused t he onset of mitochondrial. Rbf exchange; however, the washout was not compl ete (80 vs. 56% in control). Energy deprivation by saponin, which permeabil izes the sarcolemma, resulted in a rapid and complete Rb+ efflux. The mitoc hondrial Rb+ efflux rate constant (K) decreased in the presence of glibencl amide, a KATP channel inhibitor (5 mu M; k = 0.204 +/- 0.065 min(-1) n = 8) , or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM, respectiv ely; k = 0.134 +/- 0.021 min(-1); n = 4) in the saponin experiments (saponi n only; K = 0.321 +/- 0.079 min(-1); n = 3), indicating the inhibition of m itochondrial K-ATP channels. Thus hypothermia in combination with Rb-87-NMR allowed the probing of the mitochondrial K+ pool in whole hearts without m itochondrial isolation.