INHIBITORS OF P-GLYCOPROTEIN-MEDIATED DAUNOMYCIN TRANSPORT IN RAT-LIVER CANALICULAR MEMBRANE-VESICLES

P-glycoprotein (P-gp), the multidrug resistance (MDR) gene product, is exclusively located on the canalicular membrane of hepatocytes. Recen t studies using isolated rat canalicular liver plasma membrane (cLPM) vesicles indicate that daunomycin (DNM) is a substrate for the ATP-dep endent P-gp efflux system in the rat liver. The isoforms of P-gp prese nt in cLPM and in cancer cell lines differ in that the major form pres ent in the liver represents the gene product of mdr2 in mice (MDR3 in humans; class III) while the isoform of P-gp in cancer cells is the ge ne product of mdr1 in mice (MDR1 in humans, class I). The objective of this study was to examine the inhibitory effects of various organic c ompounds, most of which have been studied previously in MDR cancer cel ls, on P-gp-mediated [H-3]DNM uptake into cLPM. Also, the stereospecif icity of P-gp for its substrates was investigated by comparing the inh ibitory effects of the enantiomers and the racemic mixtures of verapam il and propranolol. DNM exhibited ATP-dependent active transport into rat liver cLPM with a K-m of 26.8 +/- 13.4 mu M and a V-max of 4.9 +/- 0.8 nmol/45 s/mg of protein (n = 4). ADP, AMP, and a nonhydrolyzable ATP analogue did not increase DNM transport over the control value. Th irty-one potential inhibitors were examined; only acridine orange, dox orubicin, verapamil, propranolol, phosphatidylcholine, beta-estradiol glucuronide, and DNM itself showed statistically significant inhibitio n of [H-3]DNM uptake into cLPM. These results suggest that only a limi ted number of substrates bind to or are transported across the hepatic canalicular membrane via P-gp. Phosphatidylcholine, a substrate for t he gene product of the class III P-gp gene, produced significant inhib ition of [H-3]DNM transport (30.6% at a 10-fold-higher substrate conce ntration), suggesting that transport may be mediated, at least in part , by this P-gp gene product. There were no statistically significant d ifferences in the inhibitory effects of the enantiomers and racemate o f verapamil on [H-3]DNM transport into cLPM, but the enantiomers of pr opranolol exhibited stereospecific inhibition of DNM transport. (R)-()-Propranolol produced a statistically significant inhibition of [H-3] DNM transport similar to that observed with the racemic mixture, while (S)(-)-propranolol showed no inhibition. These findings suggest that bile canalicular P-gp may exhibit stereospecificity of binding or tran sport for its substrates.