Fumihiko Ushigome

Human placental transport of vinblastine, vincristine, digoxin and progesterone: contribution of P-glycoprotein.

To elucidate the role of P-glycoprotein in human placenta, we examined its expression in placenta, and the transcellular transport and uptake of P-glycoprotein substrates in cultured human placental choriocarcinoma epithelial cells (BeWo cells). The uptake of [(3)H]vinblastine and [(3)H]vincristine into BeWo cells was increased in the presence of a metabolic inhibitor, sodium azide. The basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was greater than the apical-to-basolateral transcellular transport. In the presence of cyclosporin A, the basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was significantly increased, and the apical-to-basolateral transcellular transport was decreased. The uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells was significantly enhanced in the presence of several inhibitors, such as verapamil or mouse monoclonal antibody anti-P-glycoprotein MX-MDR (MRK16) as well as cyclosporin A. Although progesterone significantly enhanced the uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells, the uptake of [(3)H]progesterone was not affected by these inhibitors. Immunoblot analysis revealed that P-glycoprotein with a molecular weight of 172 kDa was expressed in BeWo cells and isolated trophoblast cells. Furthermore, P-glycoprotein was detected in human placental brush-border membrane vesicles, but not in human placental basolateral membrane vesicles. In conclusion, these data suggest that P-glycoprotein is expressed on the brush-border membrane (maternal side) of human placental trophoblast cells. P-Glycoprotein is considered to regulate the transfer of several substances including vinblastine, vincristine and digoxin from mother to fetus, and to protect the fetus from toxic substances.

Effects of grapefruit juice and orange juice components on P‐glycoprotein‐ and MRP2‐mediated drug efflux

We investigated the effects of grapefruit juice (GFJ) and orange juice (OJ) on drug transport by MDR1 P‐glycoprotein (P‐gp) and multidrug resistance protein 2 (MRP2), which are efflux transporters expressed in human small intestine. We examined the transcellular transport and uptake of [3H]vinblastine (VBL) and [14C]saquinavir in a human colon carcinoma cell line (Caco‐2) and in porcine kidney epithelial cell lines transfected with human MDR1 cDNA and human MRP2 cDNA, LLC‐GA5‐COL150, and LLC‐MRP2, respectively. In Caco‐2 cells, the basal‐to‐apical transports of [3H]VBL and [14C]saquinavir were greater than those in the opposite direction. The ratio of basal‐to‐apical transport to apical‐to‐basal transport of [3H]VBL and [14C]saquinavir by Caco‐2 cells was reduced in the presence of MK571 (MRPs inhibitor), verapamil (P‐gp inhibitor), cyclosporin A (inhibitor of both), 50% ethyl acetate extracts of GFJ and OJ, or their components (6′,7′‐dihydroxybergamottin, bergamottin, tangeretin, hepatomethoxyflavone, and nobiletin). Studies of transport and uptake of [3H]VBL and [14C]saquinavir with MDR1 and MRP2 transfectants showed that VBL and saquinavir are transported by both P‐gp and MRP2. GFJ and OJ components inhibited the transport by MRP2 as well as P‐gp. However, their inhibitory potencies for P‐gp or MRP2 were substrate‐dependent. The present study has revealed that GFJ and OJ interact with not only P‐gp but also MRP2, both of which are expressed at apical membranes and limit the apical‐to‐basal transport of VBL and saquinavir in Caco‐2 cells.

Basal Membrane Localization of MRP1 in Human Placental Trophoblast

The placental trophoblast is considered to act as a barrier between mother and fetus, mediating the exchange of various materials across the placenta. ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug-resistance protein (MRP) are expressed in the placenta and function as efflux transport systems for xenobiotics. In the present study, we aimed to determine the localization of MRP1 in the human placenta in comparison with that of P-gp. Western blotting analysis with human placental membrane vesicles indicated that P-gp and MRP1 are localized on the brush-border membranes and basal membranes, respectively. Immunohistochemical analysis with human normal full-term placenta showed that anti-P-gp monoclonal antibody F4 stained the brush-border side of the trophoblast cells, whereas anti-MRP1 monoclonal antibody MRPr1 stained the basal side. These results confirm that P-gp and MRP1 are located on the brush-border membranes and basal membranes, respectively, of human full-term placental trophoblast. MRP1 was also detected on the abluminal side of blood vessels in the villi. Accordingly, MRP1 may play a role distinct from that of P-gp, which is considered to restrict the influx of xenobiotics into the fetus.

Kinetic analysis of P-glycoprotein-mediated transport by using normal human placental brush-border membrane vesicles

AbstractPurpose. P-Glycoprotein (Pgp) plays an important role in drug disposition and excretion in various tissues such as the brain, intestine, and kidney. Moreover, we have demonstrated that Pgp is expressed on the brush-border membranes of trophoblast cells in the placenta and restricts drug transfer from the maternal circulation to the fetus. However, the transport kinetics of physiologically expressed Pgp has scarcely been investigated. Methods. In this study, we assessed the functional kinetics of transport mediated by Pgp that is physiologically expressed in normal tissue by using human placental brush-border membrane vesicles (BBMVs). Digoxin and vinblastine were used as typical substrates of Pgp. Results. The uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly increased in the presence of an ATP-regenerating system. The ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs exhibited saturable kinetics. The Michaelis constants (Kt values) were 2.65 ± 1.80 μM and 21.9 ± 3.37 μM, respectively. In the presence of a Pgp inhibitor such as verapamil, cyclosporine A, or progesterone, the ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly reduced. Anti-Pgp monoclonal antibody C219 completely inhibited the uptake of [3H]digoxin. Conclusions. The transport kinetics of [3H]digoxin and [3H]vinblastine by physiologically expressed Pgp were successfully evaluated by using BBMVs prepared from normal human placenta. The present method enabled us to evaluate the function of physiologically expressed Pgp and is superior to the use of cultured transfectants in terms of the yield of vesicles. The present method may also be applicable to investigating the influence of various factors such as the genotype of the MDR1 gene or various pathophysiologic states of neonates on the function of Pgp.

Proton gradient-dependent transport of valproic acid in human placental brush-border membrane vesicles.

AbstractPurpose. To investigate the transport mechanism of valproic acid across the human placenta, we used human placental brush-border membrane vesicles and compared them with that of lactic acid. Methods. Transport of [3H]valproic acid and [14C]lactic acid was measured by using human placental brush-border membrane vesicles. Results. The uptakes of [3H]valproic acid and [14C]lactic acid into brush-border membrane vesicles were greatly stimulated at acidic extravesicular pH. The uptakes of [3H]valproic acid and [14C]lactic acid were inhibited by various fatty acids, p-chloromercuribenzene sulfonate, α-cyano-4-hydroxycinnamate, and FCCP. A kinetic analysis showed that it was saturable, with Michaelis constants (Kt) of 1.04 ± 0.41 mM and 1.71 ± 0.33 mM for [3H]valproic acid and [14C]lactic acid, respectively. Furthermore, lactic acid competitively inhibited [3H]valproic acid uptake and vice versa.Conclusion. These results suggest that the transport of valproic acid across the microvillous membrane of human placenta is mediated by a proton-linked transport system that also transports lactic acid. However, some inhibitors differentially inhibited the uptakes of [3H]valproic acid and [14C]lactic acid, suggesting that other transport systems may also contribute to the elevated fetal blood concentration of valproic acid in gravida.

Inhibitory effects of angiotensin II receptor antagonists and leukotriene receptor antagonists on the transport of human organic anion transporter 4.

Human organic anion transporter 4 (OAT4) is the only member of the OAT family that is expressed in the placenta and also expressed in kidney. Although OAT4 has been shown to transport certain organic anions as well as other members of the OAT family, fewer numbers of substrates have been identified for OAT4 compared with OAT1 and OAT3, suggesting that the substrate specificity of OAT4 is greater than other OAT members. However, the substrate specificity of OAT4 remains to be investigated in detail. The aim of this study was to examine the effects of various drugs on the OAT4‐mediated transport of estrone‐3‐sulfate, a typical substrate of OAT4, by using human embryonic kidney cells stably transfected with OAT4 (HEK‐OAT4). HEK‐OAT4 cells exhibited concentration‐dependent uptake of estrone‐3‐sulfate, with a Km value of 20.9 ± 3.53 μM. Dehydroepiandrosterone sulfate and probenecid potently inhibited estrone‐3‐sulfate uptake. We also searched for the potential inhibitors of OAT4 and identified candesartan, candesartan cilexetil, losartan, losartan carboxyl (EXP3174) and valsartan as inhibitors of OAT4, with Ki values of 88.9, 135.2, 24.8, 13.8 and 19.6 μM, respectively. The above angiotensin II receptor antagonists and leukotriene receptor antagonists share a common structural feature, that is the tetrazole group. Although pranlukast is devoid of anionic motifs other than the tetrazole group, it potently inhibited the OAT4‐mediated uptake of estrone‐3‐sulfate, indicating that a tetrazole group may be one important structural feature in substrate recognition by OAT4.