Uptake Studies for Evaluating Activity of Efflux Transporters in a Cell Line Representative of the Blood‐Brain Barrier

Peter S. Silverstein1, D. Nedra Karunaratne1, Kenneth L. Audus1

1 University of Kansas, Lawrence, Kansas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.7
DOI:  10.1002/0471141755.ph0707s23
Online Posting Date:  February, 2004
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Abstract

In this unit, protocols are presented for performing uptake studies with bovine brain microvessel endothelial cells (BBMECs) using either radiolabeled compounds, or rhodamine 123 as a marker for a P‐glycoprotein substrate. The protocols can easily be modified for the investigation of substrate uptake in other cell lines.

Keywords: efflux transporter; P‐glycoprotein; uptake assay; rhodamine 123; BBMEC

     
 
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Table of Contents

  • Basic Protocol 1: Uptake of a Fluorescent P‐Glycoprotein Substrate in BBMECs
  • Alternate Protocol 1: Uptake of Radiolabeled Taxol by BBMECs
  • Support Protocol 1: Plating and Growth of Bovine Brain Microvessel Endothelial Cells (BBMECs)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Uptake of a Fluorescent P‐Glycoprotein Substrate in BBMECs

  Materials
  • BBMECs grown to at least 75% confluence in 12‐well or 24‐well culture plates (see protocol 3)
  • PBSA prewarmed to 37°C (see recipe)
  • Test compound
  • Cyclosporin A (see recipe)
  • 10 µM rhodamine 123 (see recipe)
  • PBS, room temperature and ice‐cold (see recipe)
  • Lysis buffer (see recipe)
  • BCA protein assay kit (Pierce)
  • Microplate incubator/shaker (e.g., Boekel Jitterbug)
  • 96‐well black‐walled plate
  • Fluorescent plate reader (e.g., Bio‐Tek FL‐600, Bio‐Tek Instruments)
  • Incubator suitable for mammalian tissue culture (37°C, 5% CO 2)
  • EIA plate reader

Alternate Protocol 1: Uptake of Radiolabeled Taxol by BBMECs

  Materials
  • 10 mM cyclosporin A (see recipe)
  • PBSA (see recipe)
  • 3H taxol (14.7 Ci/mmol; Moravek Biochemicals)
  • BBMECs grown in 12‐ or 24‐well culture plates (see protocol 3)
  • PBS (see recipe), ice cold
  • Lysis buffer (see recipe)
  • Scintillation cocktail (e.g., Scintiverse, Fisher)
  • 7‐ml scintillation vials and caps (Fisher)
  • Microplate incubator/shaker (e.g., Boekel Jitterbug)
  • Scintillation counter
  • Additional reagents and equipment for measuring protein content (see protocol 1)

Support Protocol 1: Plating and Growth of Bovine Brain Microvessel Endothelial Cells (BBMECs)

  Materials
  • 4 mg/ml rat‐tail collagen (Collaborative Biomedical Products)
  • 50 µg/ml fibronectin (see recipe)
  • BBMEC (Applied Cell Biology Research Institute)
  • Plating medium (see recipe)
  • 50 mg/ml heparin (Sigma)
  • Growth medium (see recipe)
  • 12‐ or 24‐well tissue culture plates
  • Ammonia hood
  • 15‐ or 50‐ml centrifuge tubes
  • 18‐G needle attached to a sterile 10‐ml syringe
  • 37°C, 5% CO 2 incubator
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Figures

Videos

Literature Cited

Literature Cited
   Allen, J.D., Brinkhuis, R.F., Wijnholds, J., and Schinkel, A.H. 1999. The mouse Bcrp1/Mxr/Abcp gene: Amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res. 59:4237–4241.
   Ambudkar, S.V., Dey, S., Hrycyna, C.A., Ramachandra, M., Pastan, I., and Gottesman, M.M. 1999. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol. 39:361–398.
   Audus, K.L., Ng, L., Wang, W., and Borchardt, R.T. 1996. Brain microvessel endothelial cell culture systems. In Models for Assessing Drug Absorption and Metabolism (R.T. Borchardt, P.L. Smith, and, G. Wilson, eds.) pp. 239–258. Plenum Press, New York.
   Borst, P. and Elferink, R.O. 2002. Mammalian ABC transporters in health and disease. Annu. Rev. Biochem. 71:537–592.
   Daoud, R. Kast, C., Gros, P., and Georges, E. 2000. Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1). Biochemistry 39:15344–15352.
   Feller, N., Broxterman, H.J., Wahrer, D.C., and Pinedo, H.M. 1995. ATP‐dependent efflux of calcein by the multidrug resistance protein (MRP): No inhibition by intracellular glutathione depletion. FEBS Lett. 368:385–388.
   Fontaine, M., Elmquist, W.F., and Miller, D.W. 1996. Use of rhodamine 123 to examine the functional activity of P‐glycoproteinin primary cultured brain microvessel endothelial cell monolayers. Life Sci. 59:1521–1531.
   Gumbleton, M. and Audus, K.L. 2001. Progress and limitations in the use of in vitro cell culture to serve as a permeability screen for the blood‐brain barrier. J. Pharm. Sci. 90:1681–1698.
   Hamilton, K.O., Yazdanian, M.A., and Audus, K.L. 2001. Modulation of P‐glycoprotein activity in Calu‐3 cells using steroids and β‐ligands. Int. J. Pharm. 228:171–179.
   Legrand, O., Simonin, G., Perrot, J.Y., Zittoun, R., and Marie, J.P. 1998. Pgp and MRP activities using calcein‐AM are prognostic factors in adult acute myeloid leukemia patients. Blood 91:4480–4488.
   Litman, T., Druley, T.E., Stein, W.D., and Bates, S.E. 2001. From MDR to MXR: New understanding of multidrug resistance systems, their properties and clinical significance. Cell. Mol. Life Sci. 58:931–959.
   Regina, A., Romero, I.A., Greenwood, J., Adamson, P., Bourre, J.M., Couraud, P.O., and Roux, F. 1999. Dexamethasone regulation of P‐glycoprotein activity in an immortalized rat brain endothelial cell line, GPNT. J. Neurochem. 73:1954–1963.
   Rose, J.M. and Audus, K.L. 1999. AT1 receptors mediate angiotensin II uptake and transport by bovine brain microvessel endothelial cells in primary culture. J. Cardiovasc. Pharmacol. 33:30–35.
   Shapiro, A.B. and Ling, V. 1998. Stoichiometry of coupling of rhodamine 123 transport to ATP hydrolysis by P‐glycoprotein. Eur. J. Biochem. 254:189–193.
   Shi, F., Bailey, C., Malick, A.W., and Audus, K.L. 1993. Biotin uptake and transport across bovine brain microvessel endothelial cell monolayers. Pharm. Res. 10:282–288.
   Tan, B., Piwnica‐Worms, D., and Ratner, L. 2000. Multidrug resistance transporters and modulation. Curr. Opin. Oncol. 12:450–458.
   Utoguchi, N., Chandorkar, G.A., Avery, M., and Audus, K.L. 2000. Functional expression of P‐glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells. Reprod. Toxicol. 14:217–234.
   Zaman, G.J.R., Flens, M.J., Vanleusden, M.R., Dehaas, M., Mulder, H.S., Lankelma, J., Pinedo, H.M., Scheper, R.J., Baas, F., Broxterman, H.J., and Borst, P. 1994. The human multidrug resistance‐associated protein MRP is a plasma membrane drug‐efflux pump. Proc. Natl. Acad. Sci. U.S.A. 91:8822–8826.
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