Transport and accumulation of lipophilic dye cations at the mitochondria of HeLa cells in situ.

The vital staining of mitochondrial in HeLa cells was investigated with various cationic styrylindolenines and indocarbocyanines. These dyes differed greatly in their lipophilic properties which were characterized by the partition coefficient Po/w between octanol (o) and water (w). The microspectra of the stained cells were measured in absorption and fluorescence and indicated that the dyes were not metabolized within the cells. In addition, the spectra suggested that the dye molecules were accumulated in strongly lipophilic areas of the mitochondria. Investigations of the mitochondrial ultrastructure as well as the respiratory activity and the rate of cell division indicated that the extremely lipophilic enzymes of the oxidative phosphorylation in the inner mitochondrial membrane were the favoured binding partners. The kinetics of dye accumulation was investigated with the concentration jump method. The flow Jo at the start of dye incubation at time t = 0 and the maximum fluorescence intensity Imax at t = infinity were measured. The influence of respiratory inhibitors, uncouplers, and ionophores on Jo and Imax were also investigated. The flow Jo at t = 0 describes the transfer of the dye through the cell membrane. Jo strongly depended on the lipophilicity of the dye molecules. With growing Po/w Jo first linearly increased and later leveled off. The same effect was observed in kinetic studies of the dye transfer in the model system octanol/water. The maximum concentration of bound dye molecules is given by Imax. 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 influence of TMP and Po/w on the dye accumulation are discussed in detail. Both trans-membrane potential and hydrophobic interactions are involved in strong dye binding at the mitochondria.