Abstract
Purpose. The effect of drugs on P-glycoprotein (P-gp) is normally studied in transfected or overexpressing cell lines derived from tumor cells or animal tissue. We wanted to develop an assay using normal healthy human tissue to study and characterize the drug-transporter interaction.
Methods. Lymphocytes were isolated from healthy human blood. The effect of inhibitors of P-gp (cyclosporine, tacrolimus, verapamil, quinidine, vinblastine) and of other transporters (indomethacin, probenecid, sulfinpyrazone) on intracellular accumulation of rhodamine 123 was evaluated by flow cytometry.
Results. The efflux of rhodamine 123 was inhibited by P-gp inhibitors in a saturable, concentration-dependent manner. The potency of inhibition of P-gp was cyclosporine > tacrolimus > quinidine > verapamil > vinblastine. Vinblastine inhibited P-gp at lower concentrations, whereas at high concentrations, there was an activation of rhodamine 123 efflux from lymphocytes. The multidrug resistance associated protein (MRP) inhibitors, sulfinpyrazone and probenecid, did not have any significant effect on intracellular accumulation of rhodamine 123, but indomethacin caused a concentration-dependent increase in retention of rhodamine 123, indicating the involvement of other uncharacterized transporters.
Conclusions. Lymphocytes can serve as a model tissue for studying modulation of P-gp activity by drugs. Both inhibitors and inducers of P-gp activity can be evaluated.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
REFERENCES
C. F. Higgins. ABC transporters: from microorganisms to multidrug efflux systems. Microbiol. Rev. 60:575-608 (1992).
S. P. C. Cole, G. Bhardwaj, J. H. Gerlach, J. E. Mackie, C. E. Grant, K. C. Almquist, A. J. Stewart, E. U. Kurz, A. M. V. Duncan, and R. G. Deeley. Overexpression of a transporter gene in a multidrug-resistnat human lung cancer cell line. Science 258:1650-1654 (1992).
M. M. Gottesman and I. Pastan. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62:385-427 (1993).
J. P. O'Brien and C. Cordon-Cardo. P-glycoprotein expression in normal human tissues. In S. Gupta and T. Tsuruo (eds.), Multidrug Resistance in Cancer Cells, John Wiley, New York, 1996, pp. 285-291.
H. W. van Veen and W. N. Konigs. Multidrug transporters from bacteria to man: similarities in structure and function. Sem. Cancer Bio. 8:183-191 (1997).
J. M. Zamora, H. L. Pearce, and W. T. Beck. Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic ells. Mol. Pharmacol. 33:454-462 (1988).
V. J. Wacher, C-Y. Wu, and L. Z. Benet. Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: Implications for drug delivery and activity in cancer chemotherapy. Mol. Carcinogen. 13:129-134 (1995).
K. Ueda, Y. Taguchi, and M. Morishima. How does P-glycoprotein recognize its substrates? Sem. Cancer Biol. 8:151-159 (1997).
A. Sparreboom, J. van Asperen, U. Mayer, A. H. Schinkel, J. W. Smit, D. K. F. Meijer, P. Borst, W. J. Nooijen, J. H. Beijnen, and O. van Tellingen. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc. Natl. Acad. Sci. USA 94:2031-2035 (1997).
Z. Hollo, L. Homolya, T. Hegedus, and B. Sarkadi. Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells. FEBS Lett. 383:99-104 (1996).
F. Tiberghien and F. Loor. Ranking of P-glycoprotein substrates and inhibitors by a calcein-AM fluorometry screening assay. Anti-Cancer Drugs 7:568-578 (1996).
S. D. Flanagan and L. Z. Benet. Net secretion of furosemide is subject to indomethacin inhibition, as observed in Caco-2 monolayers and excised rat jejunum. Pharm. Res. 16:221-224 (1999).
A. H. Schinkel, J. J. M. Smit, O. van Tellingen, J. H. Beijnen, E. Wagenaar, L. van Deemter, C. A. A. M. Mol, M. A. van der Valk, E. C. Robanus-Maandag, H. P. J. te Riele, A. J. M. Berns, and P. Borst. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77:491-502 (1994).
M. M. Hoffman and P. D. Roepe. Analysis of ion transport perturbations caused by hu MDR 1 protein overexpression. Biochemistry 36:11153-11168 (1997).
S. Aggarwal, T. Tsuruo, and S. Gupta. Altered expression and function of P-glycoprotein (170 kDa), encoded by the MDR 1 gene, in T cell subsets from aging humans. J. Clin. Immunol. 17:448-454 (1997).
A. B. Shapiro and V. Ling. The mechanism of ATP-dependent multidrug transport by P-glycoprotein. Acta Physiol. Scand. Suppl. 643:227-234 (1998).
A. Andreana, S. Aggarwal, S. Gollapudi, D. Wien, T. Tsuruo, and S. Gupta. Abnormal expression of a 170-kilodalton P-glycoprotein encoded by MDR1 gene, a metabolically active efflux pump, in CD4+ and CD8+ T cells from patients with human immunodeficiency virus type1 infection. AIDS Res. Hum. Retrovir. 12:1457-1462 (1996).
J. Drach, A. Gsur, G. Hamilton, S. Zhao, J. Angerier, M. Fiegi, N. Zojer, M. Raderer, I. Haberl, M. Andreef, and H. Huber. Involvement of P-glycoprotein in the transmembrane transport of interleukin-2 (IL-2), IL-4, and interferon-γ in normal human T lymphocytes. Blood 88:1747-1754 (1996).
R. Robey, S. Bakke, W. Stein, B. Meadows, T. Litman, S. Patil, T. Smith, T. Fojo, and S. Bates. Efflux of rhodamine from CD56+ cells as a surrogate marker for reversal of P-glycoprotein-mediated drug efflux by PSC 833. Blood 93:306-314 (1999).
M. P. Draper, R. L. Martell, and S. B. Levy. Indomethacin-mediated reversal of multidrug resistance and drug efflux in human and murine cell lines overexpressing MRP, but not P-glycoprotein. Br. J. Cancer 75:810-815 (1997).
Z. Hollo, L. Homolya, C. W. Davis, and B. Sarkadi. Calcein accumulation as a flurometric functional assay of the multidrug transporter. Biochim. Biophys. Acta 1191:384-388 (1994).
A. Soldner, U. Christians, M. Susanto, V. J. Wacher, J. A. Silverman, and L. Z. Benet. Grapefruit juice activates P-glycoprotein-mediated drug transport. Pharm. Res. 16:478-485 (1999).
S. Hatse, E. De Clercq, and J. Balzarini. Enhanced 9-(2-phosphonylmethoxyethyl) adeninyl) adenine-esistant human erythroleukemia K562 cell line. Mol. Pharmacol. 54:907-917 (1998).
P. Annaert, J. Van Gelder, L. Naesens, E. De Clercq, G. Van den Mooter, R. Kinget, and P. Augustijns. Carrier mechanisms involved in the transepithelial transport of bis(POM)-PMEA and its metabolites across Caco-2 monolayers. Pharm. Res. 15:1168-1173 (1998).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Parasrampuria, D.A., Lantz, M.V. & Benet, L.Z. A Human Lymphocyte Based Ex Vivo Assay to Study the Effect of Drugs on P-glycoprotein (p-Gp) Function1. Pharm Res 18, 39–44 (2001). https://doi.org/10.1023/A:1011070509191
Issue Date:
DOI: https://doi.org/10.1023/A:1011070509191