SubcellularDistribution and Analysis of Technetium-99m-MIBIin Isolated PerfusedRat Hearts

ard dillerential centrifugation techniques, 84% was asso ciated with the cytosolic fraction (4). By use of similar techniques applied to newborn chick hearts, technetium complexes of the 2-carbomethoxy isopropyl isonitrile ii gand (99mTc@CPI), another modestly lipophilic cationic agent, were found predominantly in the cell membrane fraction (6 1%) and cytosolic fraction (25%) (5). As ex pected, other highly lipophilic cationic alkylisonitrile 99mTc To addressthe apparentdiscrepancybetweencultured cells and whole heart preparations, Langendorff-perfused rat heartsloadedwith hexakis(2-methoxyisobutylisonitrile)tech neticum (I) (@‘Tc-MlBl) were fractionated by a standard dif ferential centrifugation method and fractional contents of @‘Tc-MlBl were correlated with the mitochondrial marker, malatedehydrogenase (MDH),andmitochondrial substrates. The “cytosolic” fraction nominally contained 89% ±3% of total @“Tc-MIBl, butalsocontained91% ±I % of totalMDH activity by this method. Chromatographic analysis of activity in the “cytosolic” fractiondemonstrated>95% of the agent waspresent astheoriginal freecationic complex; binding to a small molecular weight cytosolic protein was not involved in localization.Addition of the mitochondnaluncouplerCCCP (5 pM)to both“mitochondnal” and “cell fragment” pellets releasedupto 84% ± 8% of @Tc-MlBI contentandaddition ofthemitochondnal substrate succinate (10 @@M) inthepres enceofrotenone (1 @M) enhanced @‘Tc-MIBl content byup to 139% ±52% over the control. These correlative data from rat hearts indicatethat approximately90% of @‘Tc-MlBI activity in vivo is associatedwith mitochondnain an energy dependentmanneras a free cationic complex, but migrates dunngfractionation/centrifugation.