Use of fluorescent probes to monitor the efflux transporters P-glycoprotein and MRP1 in BeWo cells

Abstract

Rhodamine, Fluorescein and the Acetoxy Methyl (AM) derivative (calcein AM) of the fluorescent indicator calcein were used to evaluate the functional role of the efflux transport system in BeWo cells. Uptake studies with P-glycoprotein or MRP1 inhibitors show significant enhancement of accumulation of calcein. Since both calcein and calcein AM are extruded by MRP1, whereas only Calcein AM is a substrate of P-glycoprotein, the effect of modulators on the transport of calcein AM is indicative of the functional activity of both P-glycoprotein and MRP1. However, studies with rhodamine (substrate of P-glycoprotein) did not reveal the expected functional activity in the presence of the same modulators. Uptake of Fluorescein (substrate of MRP1) increased in the presence of inhibitors of MRP1, but was unaffected by P-glycoprotein inhibitors. It was found that cyclosporinA, verapamil, indomethacin, probenecid, sodium orthovanadate and vinblastine affected the uptake, efflux and transport of calcein. All of the modulators except sodium orthovanadate and sodium azide increased the uptake of calcein. The transport was polarized with greater permeability from the apical to the basolateral direction. The inhibition of MRP1 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 calcein was decreased by all the compounds tested, indicating an inhibition of MRP1. Sodium orthovanadate also decreased the efflux but did not affect transport. Therefore, we believe that calcein AM can be used as an indicator of Pgp and MRP1 functional activity in BeWo cells. Fluorescein uptake was only affected by MRP1 inhibitors. P-glycoprotein inhibitors Verapamil and C219 had no effect on the uptake of fluorescein, suggesting that it is an effective indicator of MRP1 functionality. However the transport of fluorescein did not show polarization. This may be due to fluorescein showing a permeability (∼ 1×10-5 cms-1) ten times greater than that of calcein. Finally, rhodamine cannot 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.