Expression and Localization of Multidrug Resistant Protein mrp 2 in Rat Small Intestine 1

The expression of multidrug resistance-associated protein isoform 2 (mrp2), the ATP-dependent export pump that mediates the transport of glucuronic acid-, glutathione-, and sulfateconjugated derivatives, was studied in rat small intestine. The small intestine was divided into nine equal segments, and mrp2 content was analyzed in homogenate and brush border membrane preparations by Western analysis. mrp2 protein was present mainly in brush border membrane of the proximal segments and gradually decreased from jejunum to the distal ileum. We also analyzed the content of mrp2 in three different populations of proximal enterocytes obtained from the upper and lower villus and the crypt regions. The export pump was mainly expressed in the villus cells and to a lesser degree in the crypt cells of the epithelium. Immunohistochemical analysis performed in duodenum, jejunum, and ileum confirmed in situ the Western blot findings. Analysis of mRNA encoding mrp2 in proximal and distal segments revealed a similar content in both regions, whereas distribution along the villus-crypt axis was similar to the protein gradient. Because conjugating enzymes are distributed similarly to mrp2, we conclude that they may act coordinately to contribute to first-pass metabolism of drugs and other xenobiotics in the proximal small intestine. Although the primary function of the small intestine is to absorb food and water, it also serves as a major portal of entry for many chemicals, including drugs and toxic compounds in the environment. It therefore has one of the greatest exposures to xenobiotics in the body. The epithelial cells of the small intestine, the enterocytes, are also able to catalyze numerous biotransformation reactions and provide the first site for metabolism of orally ingested xenobiotics. Numerous enzymes catalyzing phase I reactions, e.g., cytochrome P-450, and phase II reactions, e.g., UDP-glucuronosyltransferases, glutathione S-transferases, and sulfotransferases, have been localized to enterocytes (reviewed in Laitinen and Watkins, 1986; Lin et al., 1999). Conjugation with glucuronic acid, glutathione, and sulfate represent the major phase II pathways identified in small intestine. The relevance of the intestine in conjugating reactions is not restricted to xenobiotics because several endogenous compounds are also efficiently metabolized by this tissue. Bilirubin and steroid hormones are the most common endogenous substances that undergo intestinal conjugation (Peters et al., 1989; Radominska-Pandya et al., 1998). Because xenobiotics and endogenous substrates may enter the enterocyte by different routes, namely blood, bile, and after oral ingestion, the contribution of the different regions of the intestine to specific phase II reactions may differentially affect biotransformation and thereby, bioavailability of substrates. The distribution of activities of conjugating enzymes along the small intestine depends on the species, enzyme, and substrate studied. In humans, glucuronidation of bilirubin, glutathione conjugation with 1-chloro-2,4-dinitrobenzene, and sulfotransferase activities toward 2-naphthol and terbutaline decrease, whereas glucuronidation of planar phenols and androgens increases from proximal to distal intestine; in contrast, there is no distinct pattern for glucuronidation of bulky phenols (Peters et al., 1989; RadominskaPandya et al., 1998; Lin et al., 1999). In the rat, glucuronic acid, glutathione, and sulfate conjugation of the most common endogenous and exogenous substrates share the same pattern of distribution, with the highest activities present in the proximal portion with a decrease observed further down the intestinal tract (Clifton and Kaplowitz, 1977; Pinkus et al., 1977; Schwarz and Schwenk, 1984; Koster et al., 1985). A gradient between the villus and the crypt of the intestinal mucosa has also been described for these enzymes in the rat, suggesting a major role for the villus tip cells in glucuronic acidand glutathione-mediated biotransformation of exogeReceived for publication December 20, 1999. 1 This work was supported by Public Health Service Grants GM55343 and NS31220. 2 Permanent address: Instituto de Fisiologı́a Experimental, CONICETUniversidad Nacional de Rosario, Facultad de Ciencias Bioquı́micas y Farmacéuticas, Suipacha 570, (2000) Rosario, Argentina. ABBREVIATIONS: mrp, multidrug resistance-associated protein; ABC, ATP-binding cassette; BBM, brush border membrane; EHBR, Eisai hyperbilirubinemic rats; MDR1, multidrug resistance 1. 0022-3565/00/2933-0717$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 293:717–723, 2000 /2403/826723 717 at A PE T Jornals on O cber 1, 2017 jpet.asjournals.org D ow nladed from nous compounds (Pinkus et al., 1977; Chowdhury et al., 1985). Transport of substrates into the intestinal cells and/or release of their conjugated metabolites rather than the biotransformation enzyme activity have been postulated to be the rate-limiting steps in overall intestinal metabolism (Koster and Noordhoek, 1983; Wollenberg and Rummel, 1985). The transport of glucuronide and glutathione conjugates into the extracellular space has been characterized as a primaryactive, ATP-dependent transport and is mediated by members of the ATP-binding cassette (ABC) transporters known as multidrug resistance-associated protein (MRP) 1 and 2 (reviewed in Keppler et al., 1997). One of these isoforms, mrp2 or canalicular multispecific organic anion transporter (cMOAT), mediates the transport of conjugated compounds across apical membrane domains. The expression and function of this export pump are highly significant in liver, although other tissues, such as the proximal tubular epithelium of the kidney, also express mrp2 (Schaub et al., 1997). The expression of mRNA encoding mrp2 was reported in small intestine from laboratory animals and humans (Paulusma et al., 1996; Kool et al., 1997; Gotoh et al., 2000). However, expression of the transport protein per se has not been described in intestine so that it is not known whether it has a similar distribution as that previously seen for phase II biotransformation enzymes. Conjugate formation of xenobiotics precedes their mrp2-mediated transport across apical membranes; thus, the effectiveness of the intestinal secretory process is dependent on a coordinate action between conjugating enzymes and the export pump. In these studies, we examined the expression and localization of mrp2 protein in rat small intestine using Western blot and immunohistochemistry techniques. The pattern of distribution of mrp2 mRNA was studied as well. We report a significant expression of the mrp2 protein in the proximal region of the small intestine, following a similar pattern of distribution as the conjugation enzymes, not only along the intestinal tract but also along the villus. Materials and Methods Chemicals. Leupeptin, phenylmethylsulfonyl fluoride, and pepstatin A were obtained from Sigma Chemical Co. (St. Louis, MO). The specific antibody against the C terminus of rat mrp2 (Liu et al., 1999) and mrp2 cDNA (Madon et al., 1997) were generous gifts from Dr. Peter Meier (University Hospital, Zurich, Switzerland). The horseradish peroxidase-linked secondary antibody used in Western blot studies was from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ), whereas a biotinylated secondary antibody (Vector Laboratories Inc., Burlingame, CA) was used for immunohistochemical studies. All other chemicals were of analytical grade purity and were