Efficiency of P-glycoprotein-mediated exclusion of rhodamine dyes from multidrug-resistant cells is determined by their passive transmembrane movement rate.

The aim of the present study was to examine the relationship between the rate of the passive transmembrane movement of multidrug resistance (MDR)-type substrates and the ability of P-glycoprotein to extrude them from MDR cells. For this purpose, seven rhodamine dyes were examined for their P-glycoprotein-mediated exclusion from MDR cells, their localization in wild-type drug-sensitive cells, their capacity to stimulate the ATPase activity of P-glycoprotein reconstituted in proteoliposomes, and their transmembrane movement rate in artificial liposomes. All these rhodamine dyes were accumulated in wild-type drug-sensitive cells and were localized mainly in the mitochondria. All the dyes tested were substrates of reconstituted P-glycoprotein and cellular P-glycoprotein and were excluded to a variable degree from MDR cells. The transmembrane movement rate proved the major factor determining the efficacy of the P-glycoprotein-mediated exclusion of rhodamine dyes from MDR cells. Thus, rhodamine B, the poorest cellular P-glycoprotein substrate, exhibited a high affinity toward reconstituted P-glycoprotein, but was the fastest membrane-traversing dye. In contrast, tetramethylrosamine, the best cellular MDR probe, exhibited high affinity toward reconstituted P-glycoprotein and slow transmembrane movement rate. Therefore, an anticancer drug with a fast transmembrane movement rate is expected to overcome the MDR phenomenon. Furthermore, the widely used MDR marker, rhodamine 123, was a poor cellular MDR substrate compared with other rhodamine dyes, especially tetramethylrosamine, which was a superior cellular MDR substrate for functional dye-exclusion studies.