TRANSPORT AND ACCUMULATION OF LIPOPHILIC DYE CATIONS AT THE MITOCHONDRIA OF HELA-CELLS IN-SITU

The vital staining of mitochondria in HeLa cells was investigated with various cationic styrylindolenines and indocarbocyanines. These dyes differed greatly in their lipophilic properties which were characteriz ed by the partition coefficient P(o/w) between octanol (o) and water ( w). - The microspectra of the stained cells were measured in absorptio n and fluorescence and indicated that the dyes were not metabolized wi thin the cells. In addition, the spectra suggested that the dye molecu les were accumulated in strongly lipophilic areas of the mitochondria. Investigations of the mitochondrial ultrastructure as well as the res piratory activity and the rate of cell division indicated that the ext remely lipophilic enzymes of the oxidative phosphorylation in the inne r mitochondrial membrane were the favoured binding partners. The kinet ics of dye accumulation was investigated with the concentration jump m ethod. The flow J0 at the start of dye incubation at time t = 0 and th e maximum fluorescence intensity I(max) at t = infinity were measured. The influence of respiratory inhibitors, uncouplers, and ionophores o n J0 and I(max) were also investigated. The flow J0 at t = 0 describes the transfer of the dye through the cell membrane. J. strongly depend ed on the lipophilicity of the dye molecules. With growing P(o/w) J0 f irst linearly increased and later leveled off. The same effect was obs erved in kinetic studies of the dye transfer in the model system octan ol/water. The maximum concentration of bound dye molecules is given by I(max). It depended on the transmembrane potential (TMP) at the inner mitochondrial membrane as well as the hydrophobic interactions of the dye with the lipophilic substrates of the inner membrane. The influen ce of TMP and P(o/w) on the dye accumulation are discussed in detail. Both trans-membrane potential and hydrophobic interactions are involve d in strong dye binding at the mitochondria.