Subcellular distribution and analysis of technetium-99m-MIBI in isolated perfused rat hearts.
Subcellular distribution and analysis of technetium-99m-MIBI in isolated perfused rat hearts.
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发表时间：
1992-08
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通讯作者：
P. A. Carvalho;Mary L. Chiu;J. Kronauge;M. Kawamura;Alun G. Jones;B. Holman;D. Piwnica-worms
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作者：
P. A. Carvalho;Mary L. Chiu;J. Kronauge;M. Kawamura;Alun G. Jones;B. Holman;D. Piwnica-worms
为了解决培养细胞和全心标本之间的明显差异，用标准的差示离心法分离了负载六(2-甲氧基异丁基异腈)氚(I)的朗宁多夫灌流大鼠心脏，并将其与线粒体标志物、苹果酸脱氢酶(MDH)和线粒体底物进行了相关分析。该方法测得的“胞浆”组分名义上含有99mTC-MIBI总量的89%+/-3%，但同时也含有总MDH活性的91%+/-1%。对“胞浆”部分活性的层析分析表明，95%以上的试剂以原始的游离阳离子络合物的形式存在；与小分子胞浆蛋白的结合不参与定位。将线粒体解偶联剂CCCP(5微米)添加到“线粒体”和“细胞碎片”微丸中，可释放高达84%+/-8%的99mTC-MIBI含量；在鱼藤酮(1微米)存在的情况下，添加线粒体底物琥珀酸酯(10微米)，可使99mTC-MIBI含量增加高达139%+/-52%。这些来自大鼠心脏的相关数据表明，体内约90%的99mTC-MIBI活性与线粒体以能量依赖的方式以自由阳离子络合物的形式结合，但在分离/离心过程中迁移。
To address the apparent discrepancy between cultured cells and whole heart preparations, Langendorff-perfused rat hearts loaded with hexakis (2-methoxyisobutyl isonitrile) technetium (I) (99mTc-MIBI) were fractionated by a standard differential centrifugation method and fractional contents of 99mTc-MIBI were correlated with the mitochondrial marker, malate dehydrogenase (MDH), and mitochondrial substrates. The "cytosolic" fraction nominally contained 89% +/- 3% of total 99mTc-MIBI, but also contained 91% +/- 1% of total MDH activity by this method. Chromatographic analysis of activity in the "cytosolic" fraction demonstrated greater than 95% of the agent was present as the original free cationic complex; binding to a small molecular weight cytosolic protein was not involved in localization. Addition of the mitochondrial uncoupler CCCP (5 microM) to both "mitochondrial" and "cell fragment" pellets released up to 84% +/- 8% of 99mTc-MIBI content and addition of the mitochondrial substrate succinate (10 microM) in the presence of rotenone (1 microM) enhanced 99mTc-MIBI content by up to 139% +/- 52% over the control. These correlative data from rat hearts indicate that approximately 90% of 99mTc-MIBI activity in vivo is associated with mitochondria in an energy-dependent manner as a free cationic complex, but migrates during fractionation/centrifugation.