Multichannel Liquid Chromatography–Tandem Mass Spectrometry Cocktail Method for Comprehensive Substrate Characterization of Multidrug Resistance-Associated Protein 4 Transporter

Abstract

The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.

Identification of Transporters Associated with Etoposide Sensitivity of Stomach Cancer Cell Lines and Methotrexate Sensitivity of Breast Cancer Cell Lines by Quantitative Targeted Absolute Proteomics

“…The protein amount of membrane vesicles was determined by the Lowry method using the DC protein assay reagent (Bio-Rad) with bovine serum albumin as a standard. Uptake of gemcitabine and MTX by membrane vesicles was determined by the rapid filtration method, as described previously (Uchida et al, 2007). Briefly, vesicular uptake was done in uptake medium (250 mM sucrose, 10 mM Tris-HCl, 10 mM MgCl 2 , 4 mM ATP, 10 mM phosphocreatine, and 100 mg/ml creatine phosphokinase [pH 7.4]) containing 100 mM gemcitabine or 10 mM MTX (a known substrate of MRP5).…”

Involvement of Multidrug Resistance-Associated Protein 4 in Efflux Transport of Prostaglandin E2 across Mouse Blood-Brain Barrier and Its Inhibition by Intravenous Administration of Cephalosporins

“…Control vesicles and MRP4-expressing membrane vesicles were derived from insect Sf9 cells. Uptake experiments were performed using the rapid filtration technique as described previously, with minor modifications (Reid et al, 2003;Uchida et al, 2007). In brief, experiments were performed in medium containing 5 g of membrane vesicles, 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), 10 mM MgCl 2 , 4 mM ATP, 10 mM phosphocreatine, 100 g/ml creatine phosphokinase, and 0.2 Ci [ 3 H]PGE 2 with or without inhibitor at the indicated concentration in a total volume of 55 l. The reactions were performed at 37°C and stopped by addition of 400 l of ice-cold stop solution [0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), and 100 mM NaCl].…”

“…3A). Cefmetazole is a selective substrate for human MRP4 with a K m value of 28.5 M and is not a substrate/inhibitor for other ATP-binding cassette transporters expressed at the BBB, such as P-glycoprotein (ABCB1) and rat breast cancer resistance protein (ABCG2) (Okumura et al, 2007;Uchida et al, 2007). The concentration-dependent inhibitory effect of cefmetazole on human MRP4-medi- ]PGE 2 (1 Ci) was injected into the S2 region of mouse brain.…”

Pharmacokinetics and Pharmacogenomics of β-Lactam-Induced Neutropenia

“…MRP4 is an efflux transporter present on both on the apical membrane of renal proximal tubule cells as well as the surface of CD34 + stem cells and other bone marrow cells [8]. It has been shown that various β-lactam antibiotics are substrates of MRP4 using tandem mass spectrometry and in vitro studies of membrane vesicles expressing human MRP4 [10,11]. MRP4 also was demonstrated to be involved in the luminal efflux of ceftizoxime and cefazolin in the renal tubule of a mouse model; MRP4 knockout mice demonstrated higher renal concentrations of the cephalosporin antibiotics [11].…”

“…MRP4 is also expressed on CD34 + stem cells and in other bone marrow cells [8]. β-lactam antibiotics are known substrates of both MRP4 and OAT, and mouse models lacking these MRP4 and OAT3 have demonstrated increased renal concentrations and increased plasma concentrations of β-lactam antibiotics, respectively [10][11][12][13] there is also evidence that MRP4 provides protection against hematopoietic toxicity secondary to thiopurine [14,15]. It is interesting to postulate that MRP4 and OATs protect against hematopoietic toxicity by controlling blood and renal levels of β-lactam antibiotics.…”