Efflux transporters expressed in human placental brush border membranes have an important role in protecting the fetus from xenobiotics and drugs. Accordingly, the extent of fetal exposure to a therapeutic administered to the pregnant woman could, in part, be dependent on the activity of efflux transporters. Therefore, we developed a method for determining the activity of P-glycoprotein, expressed in term human placental brush border membranes, for efflux of its prototypic substrate Taxol.
Brush border membrane vesicles (BBMVs) were prepared from human term placental trophoblast tissue. An affinity chromatography column was used to enrich the vesicle preparation with those oriented inside-out. The kinetics of the ATP-dependent P-gp-mediated uptake of [3H] Taxol by the inside-out placental BBMVs were determined.
The vesicle preparation exhibited a 12 ± 4 fold enrichment of its brush border membranes over syncytiotrophoblast lysate by the activity of the marker enzyme alkaline phosphatase. Inside-out vesicle preparations isolated by affinity chromatography were approximately 75% oriented inside out. The ATP-dependent uptake of [3H] Taxol by placental BBMVs was 2.5 ± 1.4 fold greater in enriched inside-out preparations over the crude vesicles i.e. before affinity chromatography. The ATP-dependent uptake of [3H] Taxol by P-gp of the inside-out vesicles exhibited classical saturation kinetics with an apparent Vmax of 13 ± 6 pmol/mg protein.min and an apparent Kt of 58 ± 19 nM.
Our data indicate that a method has been established to determine the activity of the efflux transporter P-gp, of placental brush border membranes, in the transfer of its prototypic substrate Taxol. This method is currently used to investigate the activity of human placental P-gp-mediated transport of a variety of therapeutics used during pregnancy. Supported by a grant from NIDA to (MSA) and NICHD to (G.D.H.)
© 2008 Mosby, Inc. Published by Elsevier Inc. All rights reserved.