Gruwel M L, Kuzio B, Deslauriers R, Kupriyanov V V
Institute for Biodiagnostics, National Research Council, Winnipeg, Manitoba R3B 1Y6, Canada.
Am J Physiol. 1999 Jan;276(1):C193-200. doi: 10.1152/ajpcell.1999.276.1.C193.
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.
研究了在20℃下用Langendorff方法灌注的低温大鼠心脏的铷外流情况。在此温度下，87Rb-NMR外流实验表明存在两个87Rb池：细胞质池和线粒体池。大鼠心脏线粒体显示出线粒体Rb+与细胞质K+的交换非常缓慢。在洗脱出细胞质中的Rb+后，线粒体的Rb+水平在30多分钟内保持稳定。在模拟细胞内条件的灌注液存在下，用0.1 mM 2,4-二硝基苯酚（DNP）、通过肌膜通透化以及同时用皂素（0.01 mg/ml，处理4分钟）使细胞能量耗竭，或用ATP敏感性钾（KATP）通道开放剂刺激线粒体Rb+外流。DNP是一种线粒体解偶联剂，可引发线粒体Rb+交换；然而，洗脱并不完全（对照中为80%对56%）。使肌膜通透化的皂素导致的能量剥夺，会引起快速且完全的Rb+外流。在存在KATP通道抑制剂格列本脲（5 microM；k = 0.204±0.065 min-1；n = 8）时，或在皂素实验中存在ATP加磷酸肌酸（分别为1.0和5.0 mM；k = 0.134±0.021 min-1；n = 4）时，线粒体Rb+外流速率常数（k）降低（仅皂素处理时；k = 0.321±0.079 min-1；n = 3），这表明线粒体KATP通道受到抑制。因此，低温结合87Rb-NMR能够在不分离线粒体的情况下，对完整心脏中的线粒体K+池进行探测。
