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Differential multidrug resistance-associated protein 1 through 6 isoform expression and function in human intestinal epithelial Caco-2 cells

Lookup NU author(s): Dr Hannah Prime-Chapman, Richard Fearn, Emeritus Professor Barry Hirst

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Abstract

Multidrug resistance-associated protein (MRP) isoforms 1 through 6 mRNA are expressed in the human intestine and Caco-2 cells. In Caco-2 cells, the rank order for mRNA expression was MRP2 ≥ MRP6 > MRP4 ≥ MRP3 > MRP1 = MRP5. The functional expression of MRP-like activity was quantified as the efflux of the fluorescent probe calcein from confluent, polarized monolayers of Caco-2 cells. Calcein efflux was sensitive to temperature, energy depletion, and the MRP antagonist MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2- dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]. Calcein efflux across the apical membrane of Caco-2 cells exceeded that across the basolateral by approximately 2-fold, correlating with the apical localization of MRP2 visualized by immunocytochemical staining. T84 cells do not express MRP2 and show a predominance of basolateral calcein efflux over apical efflux. MRP3 was localized by immunocytochemical staining to the basolateral membrane. MRP1 staining was not localized to either membrane domain and MRP5 staining was not detected. Thus, basolateral calcein efflux may reflect a function of MRP3 or MRP4 and 6 inferred by their basolateral localization in other tissues. Basolateral, but not apical, calcein efflux was sensitive to glutathione depletion with buthionine-sulfoximine, indicating that whereas MRP2-mediated apical efflux is independent of glutathione, basolateral efflux is glutathione-dependent. Benzbromarone, probenecid, pravastatin, and diclofenac were able to inhibit both apical and basolateral calcein efflux. The apical calcein efflux in Caco-2 cells was selectively sensitive to indomethacin and propranolol, but not verapamil or erythromycin, whereas the converse was observed for basal efflux. The differential pharmacological sensitivity of apical (MRP2) and basolateral calcein efflux provides tools for dissecting MRP isoform functional roles.

Publication metadata

Author(s): Prime-Chapman HM, Fearn RA, Cooper AE, Moore V, Hirst BH

Publication type: Article

Publication status: Published

Journal: Journal of Pharmacology and Experimental Therapeutics

Year: 2004

Volume: 311

Issue: 2

Pages: 476-484

ISSN (print): 0022-3565

ISSN (electronic): 1521-0103

Publisher: American Society for Pharmacology and Experimental Therapeutics

URL: http://dx.doi.org/10.1124/jpet.104.068775

DOI: 10.1124/jpet.104.068775

PubMed id: 15210835

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