Determination of the osmotic active drug concentration in the cytoplasm of anthracycline-resistant and -sensitive K562 cells.

A fluorescence method was used to follow the interaction of 4'-o-tetrahydropyranyladriamycin (THP-ADR) with drug-resistant and -sensitive K562 cells. The amounts of drug bound to the nuclei at the steady state, Cn and at the equilibrium state, CN, once the membrane has been solubilized with Triton X-100, have been determined as a function of the pH outside the cells (pHe): Cn increased and CN decreased as pHe increased. At a given pH value outside the cells, CN is the same for both sensitive and resistant cells, whereas Cn is lower in resistant cells as compared to sensitive cells. Using the observation that the essential binding characteristics of THP-ADR in nuclei are the same for both types of cell, the osmotic active drug concentration, Ci, in the cytoplasm of the cells was determined at different values of pHe. Using fluorescent dye, the cytoplasmic pH was determined and found equal to 7.2 +/- 0.1 in both types of cell. In sensitive cells, the equilibrium transmembrane concentrations verified the relation [DH+]i/[DH+]e = [H +]i/[H+]e where [DH +]i and [DH +]e stand for the concentration of protonated form of the drug inside and outside the cells, respectively. This indicates that the uptake of the drug occurs through free permeation of the neutral form of the drug in response to delta pH gradient. Such a relation is not verified in the case of resistant cells.