Köhnke D, Schramm M, Daut J
Institut für Normale und Pathologische Physiologie, Universität Marburg, Germany.
Mol Cell Biochem. 1997 Sep;174(1-2):101-13.
A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100-500 micrograms were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37 degrees C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 microM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 microM atractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 microM alpha-cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H+ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 +/- 3.5 mW cm-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells.
开发了一种新型流动量热技术来研究心肌线粒体的能量转换。从豚鼠心脏中分离出重量为100 - 500微克的圆柱形心肌束（小梁），并将其安装在管状记录室中，该记录室在37℃下用生理盐溶液持续灌注。以高分辨率测量记录室上下游之间的温差，该温差与小梁的产热速率成正比。通过这种方式，可以连续记录分离的心肌的能量消耗速率数小时。当用含有5 mM丙酮酸和2 mM苹果酸作为底物的“细胞内”溶液对制剂进行灌流时，用25 microM洋地黄皂苷使肌膜通透化会在存在2 mM ADP的情况下导致测量的热速率显著增加。大部分对ADP敏感的产热（83%）可被400 microM阿托伐他汀（一种腺嘌呤核苷酸转位酶抑制剂）和600 microM α - 氰基 - 4 - 羟基肉桂酸（一种单羧酸/H⁺共转运抑制剂）阻断。在缺氧溶液中，阿托伐他汀敏感的产热被消除。这些结果表明，阿托伐他汀敏感的产热（21.8±3.5 mW/cm³组织）归因于氧化磷酸化。用洋地黄皂苷处理后线粒体显然保持完整，因为应用解偶联剂2,4 - 二硝基苯酚（DNP）会使热速率大幅增加。在通透化的心肌制剂中，ADP诱导的热速率的一小部分（17%）对阿托伐他汀不敏感。在应用洋地黄皂苷之前也观察到了这一成分，它可能与外切核苷酸酶有关。总之，我们的量热技术允许“原位”研究心肌线粒体的能量代谢，即不破坏心肌细胞的微观结构。
