Measurements of mitochondrial K+ fluxes in whole rat hearts using 87Rb-NMR.
The rubidium efflux from hypothermic rat hearts perfused by the Langendorff method at 20 degreesC was studied. At this temperature 87Rb-NMR efflux experiments showed the existence of two 87Rb pools: cytoplasmic and mitochondrial. 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 concomitant cellular energy depletion by saponin (0.01 mg/ml for 4 min) in the presence of a perfusate mimicking intracellular conditions, or by ATP-sensitive K (KATP) channel openers. DNP, a mitochondrial uncoupler, caused the onset of mitochondrial Rb+ exchange; however, the washout was not complete (80 vs. 56% in control). Energy deprivation by saponin, which permeabilizes the sarcolemma, resulted in a rapid and complete Rb+ efflux. The mitochondrial Rb+ efflux rate constant (k) decreased in the presence of glibenclamide, a KATP channel inhibitor (5 microM; k = 0.204 +/- 0.065 min-1; n = 8), or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM, respectively; k = 0.134 +/- 0.021 min-1; n = 4) in the saponin experiments (saponin only; k = 0.321 +/- 0.079 min-1; n = 3), indicating the inhibition of mitochondrial KATP channels. Thus hypothermia in combination with 87Rb-NMR allowed the probing of the mitochondrial K+ pool in whole hearts without mitochondrial isolation.
Gruwel
M L
ML
Institute for Biodiagnostics, National Research Council, Winnipeg, Manitoba R3B 1Y6, Canada. marco.gruwel@nrc.ca
Kuzio
B
B
Deslauriers
R
R
Kupriyanov
V V
VV
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D006321:Heart / Q000187:drug effects / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007035:Hypothermia / Q000378:metabolism; D066298:In Vitro Techniques; D007474:Ion Exchange; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium / Q000378:metabolism; D012414:Rubidium Radioisotopes; D012503:Saponins / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D010710:Phosphates; D012414:Rubidium Radioisotopes; D012503:Saponins; D014475:Uncoupling Agents; D012413:Rubidium; D019297:2,4-Dinitrophenol; D011188:Potassium