Skip to main content
Log in

Kinetic Analysis of P-Glycoprotein-Mediated Transport by Using Normal Human Placental Brush-Border Membrane Vesicles

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose. 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 (K t 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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. R. L. Juliano and V. Ling. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta 455:152-162 (1976).

    Google Scholar 

  2. P. Borst, A. H. Schinkel, J. J. Smit, E. Wagenaar, L. Van Deemter, A. J. Smith, E. W. Eijdems, F. Baas, and G. J. Zaman. Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals. Pharmacol. Ther. 60:289-299 (1993).

    Google Scholar 

  3. I. Sugawara. Expression and functions of P-glycoprotein (mdr1 gene product) in normal and malignant tissues. Acta Pathol. Jpn. 40:545-553 (1990).

    Google Scholar 

  4. Y. Tanigawara, N. Okamura, M. Hirai, M. Yasuhara, K. Ueda, N. Kioka, T. Komano, and R. Hori. Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1). J. Pharmacol. Exp. Ther. 263:840-845 (1992).

    Google Scholar 

  5. F. Thiebaut, T Tsuruo, H. Hamada, M. M. Gottesman, I. Pastan, and M. C. Willingham. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc. Natl. Acad. Sci. USA 84:7735-7738 (1987).

    Google Scholar 

  6. A. Tsuji, T. Terasaki, Y. Takabatake, Y. Terada, I. Tamai, T. Yamashita, S. Moritani, T. Tsuruo, and J. Yamashita. P-Glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells. Life Sci. 51:1427-1437 (1992).

    Google Scholar 

  7. F. Ushigome, H. Takanaga, H. Matsuo, S. Yanai, K. Tsukimori, H. Nakano, T. Uchiumi, T. Nakamura, M. Kuwano, H. Ohtani, and Y. Sawada. Human placental transport of vinblastine, vincristine, digoxin and progesterone: contribution of P-glycoprotein. Eur. J. Pharmacol. 408:1-10 (2000).

    Google Scholar 

  8. T. Litman, T. Zeuthen, T. Skovsgaard, and W. D. Stein. Competitive, non-competitive and cooperative interactions between substrates of P-glycoprotein as measured by its ATPase activity. Biochim. Biophys. Acta 1361:169-176 (1997a).

    Google Scholar 

  9. T. Litman, T. Zeuthen, T. Skovsgaard, and W. D. Stein. Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity. Biochim. Biophys. Acta 1361:159-168 (1997b).

    Google Scholar 

  10. J. Hunter, M. A. Jepson, T. Tsuruo, N. L. Simmons, and B. H. Hirstm. Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators. J. Biol. Chem. 268:14991-14997 (1993).

    Google Scholar 

  11. R. H. Stephens, C. A. O'Neill, A. Warhurst, G. L. Carlson, M. Rowland, and G. Warhurst. Kinetic profiling of P-glycoprotein-mediated drug efflux in rat and human intestinal epithelia. J. Pharmacol. Exp. Ther. 296:584-591 (2001).

    Google Scholar 

  12. H. Nakamura, F. Ushigome, N. Koyabu, S. Satoh, K. Tsukimori, H. Nakano, H. Ohtani, and Y. Sawada. Proton gradient-dependent transport of valproic acid in human placental brushborder membrane vesicles. Pharm. Res. 19:154-161 (2002).

    Google Scholar 

  13. C. H. Smith, D. M. Nelson, B. F. King, T. M. Donohue, S. M. Ruzycki, and L. K. Kelly. Characterization of a microvillous membrane preparation from human placental syncytiotrophoblast: A morphologic, biochemical and physiologic study. Am. J. Obstet. Gynecol. 128:190-196 (1977).

    Google Scholar 

  14. L. K. Kelley, C. H. Smith, and B. F. King. Isolation and partial characterization of the basal cell membrane of human placental trophoblasts. Biochim. Biophys. Acta 734:91-98 (1983).

    Google Scholar 

  15. U. K. Laemmli. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 (1970).

    Google Scholar 

  16. C. E. Hulstaert, J. L. Torringa, J. Koudstaal, M. J. Hardonk, and I. Molennaar. The characteristic distribution of alkaline phosphatase in full-term human placenta. Gynecol. Invest. 4:23-30 (1973).

    Google Scholar 

  17. M. Bohme, M. Muller, I. Leier, G. Jedlitscky, and D. Keppler. Cholestasis caused by inhibition of the adenosine triphosphate-dependent bile salt transport in rat liver. Gastroenterology 107:255-265 (1994).

    Google Scholar 

  18. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275 (1951).

    Google Scholar 

  19. T. Ikegawa, F. Ushigome, N. Koyabu, S. Morimoto, Y. Shoyama, M. Naito, T. Tsuruo, H. Ohtani, and Y. Sawada. Inhibition of P-glycoprotein by orange juice components, polymethoxyflavones in adriamycin-resistant human myelogeneous leukemia (K562/ADM) cells. Cancer Lett. 160:21-28 (2000).

    Google Scholar 

  20. F. G. M. Russel, P. E. M. van der Linden, W. G. Vermeulen, M. Heijn, C. H. van Os, and C.A.M. van Ginneken. Na+ and H+ gradient-dependent transport of p-aminohippurate in membrane vesicles from dog kidney cortex. Biochem. Pharmacol. 37:2639-2649 (1988).

    Google Scholar 

  21. M. Horio, M. M. Gottesman, and I. Pastan. ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc. Natl. Acad. Sci. USA 85:3580-3584 (1988).

    Google Scholar 

  22. H. Kusuhara, H. Suzuki, M. Naito, T. Tsuruo, and Y. Sugiyama. Characterization of efflux transport of organic anions in a mouse brain capillary endothelial cell line. J. Pharmacol. Exp. Ther. 285:1260-1265 (1998).

    Google Scholar 

  23. K. Yamaoka, Y. Tanigawara, T. Nakagawa, and T. Uno. A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharm-Dyn. 4:879-885 (1981).

    Google Scholar 

  24. Y. Nakamura, S. Ikeda, T. Furukawa, T. Sumizawa, A. Tani, S. Akiyama, and Y. Nagata. Function of P-glycoprotein expressed in placenta and mole. Biochem. Biophys. Res. Commun. 235:849-853 (1997).

    Google Scholar 

  25. M. V. St-Pierre, M. A. Serrano, R. I. Macias, U. Dubs, M. Hoechli, U. Lauper, P. J. Meier, and J. J. Marin. Expression of members of the multidrug resistance protein family in human term placenta. Am. J. Physiol. 279:R1495-R1503 (2000).

    Google Scholar 

  26. M. Tanabe, I. Ieiri, N. Nagata, K. Inoue, S. Ito, Y. Kanamori, M. Takahashi, Y. Kurata, J. Kigawa, S. Higuchi, N. Terakawa, and K. Otsubo. Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene. J. Pharmacol. Exp. Ther. 297:1137-1143 (2001).

    Google Scholar 

  27. S. Hoffmeyer, O. Burk, O. von Richter, H. P. Arnold, J. Brockmoller, A. Johne, I. Cascorbi, T. Gerloff, I. Roots, M. Eichelbaum, and U. Brinkmann. Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc. Natl. Acad. Sci. USA 97:3473-3478 (2000).

    Google Scholar 

  28. Y. Aalto, S. Teglund, U. Andersson, G. Blanco, S. Hammarström, and R. Henriksson. Intra-and inter-individual heterogeneity in exon 2 of the MDR1 gene in primary breast carcinoma and healthy individuals. Int. J. Oncol. 17:1077-1086 (1997).

    Google Scholar 

  29. I. Cascorbi, T. Gerloff, A. Johne, C. Meisel, S. Hoffmeyer, M. Schwab, E. Schaeffeler, M. Eichelbaum, U. Brinkmann, and I. Roots. Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin. Pharmacol. Ther. 69:169-174 (2001).

    Google Scholar 

  30. X. Declèves, S. Chevillard, C. Charpentier, P. Vielh, and J. L. Laplanche. A new polymorphism (N21D) in the exon 2 of the human MDR1 gene encoding the P-glycoprotein. Hum. Mutat. 15:486(2000).

    Google Scholar 

  31. S. Ito, I. Ieiri, M. Tanabe, A. Suzuki, S. Higuchi, and K. Otsubo. Polymorphism of the ABC transporter genes, MDR1, MRP1 and MRP2/cMOAT, in healthy Japanese subjects. Pharmacogenetics 11:175-184 (2001).

    Google Scholar 

  32. L. A. Mickley, J. S. Lee, Z. Weng, Z. Zhan, M. Alvarez, W. Wilson, S. E. Bates, and T. Fojo. Genetic polymorphism in MDR-1: a tool for examining allelic expression in normal cells, unselected and drug-selected cell lines, and human tumors. Blood 91:1749-1756 (1998).

    Google Scholar 

  33. D. Rund, I. Azar, and O. Shperling. A mutation in the promoter of the multidrug resistance gene (MDR1) in human hematological malignancies may contribute to the pathogenesis of resistant disease. Adv. Exp. Med. Biol. 457:71-75 (1999).

    Google Scholar 

  34. R. B. Kim, B. F. Leake, E. F. Choo, G. K. Dresser, S. V. Kubba, U. I. Schwarz, A. Taylor, H. G. Xie, J. McKinsey, S. Zhou, L. B. Lan, J. D. Schuetz, E. G. Schuetz, and G. R. Wilkinson. Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin. Pharmacol. Ther. 70:189-199 (2001).

    Google Scholar 

  35. M. Hitzl, S. Drescher, H. van der Kuip, E. Schaffeler, J. Fischer, M. Schwab, M. Eichelbaum, and M. F. Fromm. The C3435T mutation in the human MDR1 gene is associated with altered efflux of the P-glycoprotein substrate rhodamine 123 from CD56+ natural killer cells. Pharmacogenetics 11:293-298 (2001).

    Google Scholar 

  36. N. Kartner, D. Evernden-Porelle, G. Bradley, and V. Ling. Detection of P-glycoprotein in multi-drug resistant cell line by monoclonal antibodies. Nature 316:820-823 (1985).

    Google Scholar 

  37. N. Sawabu, M. Nakagen, T. Wakabayashi, K. Ozaki, D. Toya, N. Hattori, and M. Ishii. Gamma-glutamyltranspeptidase as a tumor marker. Nippon Rinsyo-Jpn. J. Clin. Med. 38:4606-4613 (1980).

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yasufumi Sawada.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ushigome, F., Koyabu, N., Satoh, S. et al. Kinetic Analysis of P-Glycoprotein-Mediated Transport by Using Normal Human Placental Brush-Border Membrane Vesicles. Pharm Res 20, 38–44 (2003). https://doi.org/10.1023/A:1022290523347

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1022290523347

Navigation