FLUORESCENT CELLULAR INDICATORS ARE EXTRUDED BY THE MULTIDRUG-RESISTANCE PROTEIN

In this report we show that NIH-3T3 mouse fibroblasts stably expressin g the human multidrug transporter (MDR1 or P-glycoprotein), in contras t to the control NIH-3T3 cells, actively extrude the hydrophobic aceto xymethyl ester (AM) derivatives used for cellular loading of various f luorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporter, e.g . by verapamil, vincristine, sodium orthovanadate, oligomycin, and a m onoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the i ndicators are not exported by MDR1. We also demonstrate that in isolat ed cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescen t dyes whereas the free acid forms of the dyes are without effect. Sin ce (i) the AM derivatives of the fluorescent indicators rapidly permea te the cell membrane and are readily cleaved by high activity and larg e capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the A M compounds. These data also call attention to the possible interactio n of widely used hydrophobic fluorescent indicators with MDR1 and offe r an efficient detection of MDR1-expressing tumor cells as well as a s creening method for examining drug interactions with the multidrug tra nsporter.