Fluorescent compounds as functional indicators of the efflux transporters P-Glycoprotein and MRP1 in Bewo cells

Abstract

Rhodamine, fluorescein and the acetoxy methyl (AM) derivative (calceinAM) of the fluorescent indicator calcein were used to evaluate the functional role of the effiux transport system in BeWo cells. Uptake studies with P-glycoprotein or MRPI inhibitors show significant enhancement of accumulation of calcein. CyclosporinA, verapamil, indomethacin, probenecid, sodiuni orthovanadate and vinblastine affected the uptake, efflux and transport of calcein. The transport was polarized with greater permeability from the apical to the basolateral direction. The inhibition of MRPI and Pgp resulted in a decrease of apical to basolateral transport. However, none were able to decrease transport from basolateral to the apical side significantly. The efflux of caIcein was decreased by all the compounds tested, indicating an inhibition of MRPI. Since both calcein and calceinAM are extruded by MRPI, whereas only calceinAM is a substrate of P- glycoprotein, the effect of modulators on the transport of calceinAM is indicative of the functional activity of both P-glycoprotein and MRPI. Fluorescein uptake was only affected by MRPI inhibitors. P-glycoprotein inhibitors verapamil and C219 had no effect on the uptake of fluorescein, suggesting that it is an effective indicator of MRPI functionality. However the transport of fluorescein did not show polarization. This may be due to fluorescein showing permeability (- lxl0⁻⁵ cms⁻¹) ten times greater than that of caIcein. Studies with rhodamine (substrate of P-glycoprotein) did not reveal the expected functional activity in the presence of the same modulators. Therefore, rhodamine could not be used as a functional indicatior of P-glycoprotein activity in BeWo cells due to the presence of other transporters which interfere with rhodamine uptake and transport.