The Effect of Albumin on MRP2 and BCRP in the Vesicular Transport Assay

Abstract

The ABC transporters multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) are of interest in drug development, since they affect the pharmacokinetics of several drugs. Membrane vesicle transport assays are widely used to study interactions with these proteins. Since albumin has been found to affect the kinetics of metabolic enzymes in similar membrane preparations, we investigated whether albumin affects the kinetic parameters of efflux transport. We found that albumin increased the Vmax of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) and estradiol-17-β-D-glucuronide uptake into MRP2 vesicles in the presence of 0.1% bovine serum albumin (BSA) by 2 and 1.5-fold, respectively, while BSA increased Lucifer yellow uptake by 30% in BCRP vesicles. Km values increased slightly, but the change was not statistically significant. The effect of BSA on substrate uptake was dependent on the vesicle amount, while increasing BSA concentration did not significantly improve substrate uptake. These results indicate a minor effect of albumin on MRP2 and BCRP, but it should be considered if albumin is added to transporter assays for example as a solubilizer, since the effect may be substrate or transporter specific.

Fig 1. The effect of 0.1% BSA on uptake kinetics.

Uptake of (A) 5(6)-carboxy-2’,7’-dichlorofluorescein (CDCF) in MRP2 vesicles, (B) Estradiol-17-β-glucuronide (E₂17βG) in MRP2 vesicles and (C) Lucifer Yellow (LY) in BCRP vesicles in the absence (control, open circles) and presence of 0.1% BSA (closed circles). All assays were performed in triplicate and each point represents the mean (± SD) of 2–4 separate experiments normalized to the calculated V_max of the control. Curves represent the results from model fitting (see Materials and Methods).

Fig 2. The effect of 0.1% bovine serum albumin (BSA) in BCRP vesicles not loaded with cholesterol.

Uptake of Lucifer Yellow (LY) into BCRP vesicles not loaded with cholesterol is shown in the absence (control, open circles) and presence (closed circles) of 0.1% BSA. The V_max of control was set to 100% and the results normalized to this. Each point represents the mean ± SD, n = 3. Black solid curves represent the fitting of the data for vesicles without cholesterol loading and grey dashed curves represent the corresponding fitting from cholesterol loaded vesicles.

Fig 3. Relationship between the albumin effect and amount of vesicles used in the assay.

Transport activity with varying amount of vesicles (measured as total protein in μg per well) were examined in the presence and absence of 0.1% BSA. Substrate concentrations in the assay were (left panel) 50 μM for CDCF (MRP2 vesicles) and (right panel) 200 μM for Lucifer Yellow (BCRP vesicles). Data is expressed as relative transport, normalized to the ATP-dependent uptake in the absence of BSA. Each bar represents the mean (± SD), n = 3–6.

Fig 4. The comparison of 0.1% and 1.0% BSA effect on uptake kinetics.

(Left panel) CDCF uptake into MRP2 vesicles, (right panel) Lucifer Yellow (LY) uptake into BCRP vesicles. Each bar represents the mean (± SD) of 2–3 separate experiments. The uptake rate of the control (0% BSA) was set to 100% and uptake rates in the presence of 0.1% and 1.0% BSA were normalized to control. * p < 0.05 and ** p < 0.01 compared to the uptake in the absence of BSA.

Fig 5. Inhibition of 5 μM CDCF uptake into MRP2 vesicles and 50 μM LY uptake into BCRP vesicles by oleic acid.

Data is presented as a relative transport activity normalized to uptake in the absence of oleic acid. Small, closed circles show results from inhibition studies and larger black/white circles represent the retention of CDCF or LY in vesicles in studies on unspecific membrane effects of oleic acid (for details see Materials and Methods). All data points were assayed in triplicate and curve fitting was performed using the four parameter logistic equation. Graphs show the results from a representative experiment and data is represented as mean ± SD. IC₅₀ (95% confidence interval) for BCRP was 28.2 μM (21.7–37.7). In the case of MRP2, the IC₅₀ was not applicable.