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

The rubidium efflux from hypothermic rat hearts perfused by theLangendorff method at 20°C was studied. At thistemperature ⁸⁷Rb-NMR efflux experiments showed theexistence of two ⁸⁷Rb pools: cytoplasmic and mitochondrial.Rat heart mitochondria showed a very slow exchange of mitochondrialRb⁺ for cytoplasmic K⁺. After washout ofcytosolic Rb⁺, mitochondria kept a stable Rb⁺level for >30 min. Rb⁺ efflux from mitochondria wasstimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmalpermeabilization and concomitant cellular energy depletion by saponin(0.01 mg/ml for 4 min) in the presence of a perfusate mimickingintracellular conditions, or by ATP-sensitive K (K_ATP)channel openers. DNP, a mitochondrial uncoupler, caused the onset ofmitochondrial Rb⁺ exchange; however, the washout was notcomplete (80 vs. 56% in control). Energy deprivation by saponin, whichpermeabilizes the sarcolemma, resulted in a rapid and completeRb⁺ efflux. The mitochondrial Rb⁺ efflux rateconstant (k) decreased in the presence of glibenclamide, aK_ATP channel inhibitor (5 µM;k = 0.204 ± 0.065 min¹; n = 8),or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM,respectively; k = 0.134 ± 0.021 min¹;n = 4) in the saponin experiments (saponin only;k = 0.321 ± 0.079 min¹; n = 3),indicating the inhibition of mitochondrial K_ATP channels. Thus hypothermia in combination with ⁸⁷Rb-NMR allowed theprobing of the mitochondrial K⁺ pool in whole heartswithout mitochondrial isolation.