Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

Figures & data

Figure 1.  Flow cytometric analysis of the homogeneity of the membrane vesicles preparations (top). (A) Forward scattering (FSC) histograms of E-IOVs indicating homogenous (bold solid line) and non-homogenous (solid line, filled curve) preparations. (B) Forward scattering (FSC) histograms of MRP1-Sf9-IOVs (bold solid line) and CTRL-Sf9-IOVs (solid line, filled curve). Determination of ATP-dependent BCPCF transport into membrane vesicles (bottom). Overlay of fluorescence intensity (FL-1) histograms for (C) E-IOVs incubated with 15 µM BCPCF for 60 min at 37°C and (D) MRP1-Sf9-IOVs incubated with 15 µM BCPCF for 30 min at 37°C. The ATP-dependent transport was calculated by subtraction of the MFI values of the histogram in the presence of ATP (bold solid line) and of the histogram in the presence of AMP (solid line). The plot of E-IOVs incubated in the presence of ATP (C) shows a typical profile that was similar in all preparations.

Figure 2.  Inhibition of ATP-dependent BCPCF transport into erythrocyte membrane inside-out vesicles by cyclosporin A. E-IOVs were incubated with 5 µM BCPCF for 60 min at 37°C in the absence (control) and in the presence of cyclosporin A (CsA) for flow cytometric analysis. Fluorescence (FL-1) histograms in the presence of ATP for control (bold solid line) and for CsA concentrations of 2 µM (solid line) and 10 µM (dotted line) and in the presence of AMP (dotted lines, filled curves) are shown as an overlay graph. This experiment is representative of three independent experiments.

Figure 3.  ATP-dependent transport of BCPCF into inside-out erythrocyte membrane vesicles. E-IOVs were incubated with BCPCF (5 µM) at 37°C. The ATP-dependent transport of BCPCF (⋄) was calculated as the difference of BCPCF accumulation measured by flow cytometry in the presence of ATP (▪) and in the presence of AMP (□). Median of the histogram fluorescence intensity was taken as a measure of the BCPCF accumulation of the sample. Values are the mean±SD of triplicate determinations in a single experiment. Two additional experiments for vesicles prepared from the blood of the other donors show similar results.

Figure 4.  Kinetics of ATP-dependent BCPCF transport into inside-out erythrocyte membrane vesicles. (A) The rate of BCPCF accumulation into E-IOVs was measured with varying BCPCF concentrations (1–15 µM) for 60 min at 37°C in the presence of ATP (▪) and in the presence of AMP (□). (B) The calculated rate of ATP-dependent transport of fluorophore into vesicles shown in Figure 4A (⋄), and data for E-IOVs prepared from the blood of another donor (▴), are plotted. Kinetic constants K_m and V_max for BCPCF transport were determined using nonlinear fitting to the hyperbolic Michaelis-Menten equation. Each value in Figure 4A and 4B is the mean±SD of three independent calibrated experiments made in duplicate.

Table I.  Kinetic constants K_m and V_max for ATP-dependent BCPCF transport into erythrocyte membrane inside-out vesicles.

donor	Km [µM]	Vmax [pmol mg^−1 h^−1]	calibration factor [pmol mg^−1 h^−1]
1	8.9±3.1	435±95	0.26±0.02
2	11.9±0.4	449±62	0.31±0.03
3	8.5±3.6	327±77	0.24±0.05
4	7.1±1.5	518±88	0.32±0.03
5	7.6±0.6	321±32	0.59±0.06

The ATP-dependent BCPCF transport into E-IOVs from five different donors was measured at various substrate concentrations (1–15 µM, 60 min, 37°C). K_m and V_max values were determined using nonlinear fitting to the hyperbolic Michaelis-Menten equation from calibrated flow cytometric measurements. The calibration factor was determined from linear regression analysis as a slope (normalised accumulation of BCPCF from spectrofluorimetric measurements versus the median fluorescence intensity from flow cytometric measurements). The presented data are the mean±SD from three experiments done in duplicate on IOVs from one preparation.

Figure 5.  Kinetics of ATP-dependent BCPCF transport into inside-out Sf9 cell membrane vesicles. (A) MRP1-Sf9-IOVs (▪, □) and CTRL-Sf9-IOVs (•, ○) were exposed for 30 min to various concentrations of BCPCF (1–25 µM) in the presence of either ATP (▪, •) or AMP (□, ○). Each data point is the mean value of duplicate determinations of the accumulation in a single experiment. Similar results were obtained in one additional independent experiment. (B) Kinetic constants K_m and V_max for BCPCF transport were determined using nonlinear fitting to the hyperbolic Michaelis-Menten equation. ATP-dependent transport values calculated (see legend to Figure 3) from data shown in Figure 5A for MRP1-Sf9-IOVs (▪) and CTRL-Sf9-IOVs (•) are plotted.

Figure 6.  Effect of the MRP1-specific antibodies QCRL-3 and QCRL-1 and of the MRP-specific inhibitor MK-571 on BCPCF transport into the lumen of inside-out membrane vesicles. The influence of modulators on ATP-dependent BCPCF (5 µM) transport into E-IOVs (60 min) and Sf9-IOVs (30 min) are shown in Figure 6A, 6B, 6C and in Figure 6D, respectively. Results are expressed as a percentage of control (in the absence of modulators). (A) Concentration dependent inhibition by the monoclonal MRP1-specific antibody QCRL-3 and (B) by the MRP-specific inhibitor MK-571. Bars represent the mean±SD of three experiments done in duplicate on preparations from different blood donors. (C) The effects of monoclonal MRP1-specific antibody QCRL-1 (4 µg/ml), sucrose (1 M), or Triton X-100 (0.02%). Bars represent the mean of two experiments done in duplicate. (D) The effects of QCRL-3 (4 µg/ml), QCRL-1 (4 µg/ml), sucrose (1 M), Triton X-100 (0.02%) and MK-571 (2 µM) on ATP-dependent transport into CTRL-Sf9-IOVs (grey bars) and MRP1-Sf9-IOVs (black bars). Bars represent the mean±SD (of three experiments done in duplicate) or the mean (of two experiments done in duplicate). Significant difference from control calculated with Student's t-test; *p < 0.05, **p < 0.01.

Figure 7.  The effect of various inhibitors on ATP-dependent BCPCF transport into human erythrocyte membrane inside-out vesicles. ATP-dependent transport of BCPCF (5 µM, 60 min, 37°C) in the presence of benzbromarone (BNB), verapamil (VER), and probenecid (PRO) is expressed as the percentage of the transport in the absence of inhibitors (control). The values of concentration-dependent inhibition are the mean±SD of duplicate determination in three experiments (each performed on E-IOVs preparation from the blood of a different donor). Significant difference from control calculated with Student's t-test; *p < 0.01, **p < 0.001.

Figure 8.  Inhibition of ATP-dependent BCPCF transport into erythrocyte membrane inside-out vesicles by benzbromarone and verapamil. ATP-dependent BCPCF transport was determined from uncalibrated flow cytometric measurements at various substrate concentrations (1–15 µM) for 60 min in the absence (▪) or in the presence of 5 µM benzbromarone (○) and in the presence of 10 µM verapamil (Δ). Data were plotted from a single experiment in duplicate and the kinetic constants K_m and V_maxrel were calculated using nonlinear fitting to the hyperbolic equation. Similar results were obtained in two additional experiments done with E-IOVs preparations from the blood of the other donors. Summarized results are shown in Table II.

Table II.  Effect of inhibitors on the kinetic constants K_m and V_max for BCPCF transport into erythrocyte membrane inside-out vesicles.

		Km [µM]		Vmax [pmol mg^−1 h^−1]
Inhibitor	concentration [µM]	control	in the presence of inhibitor	control	in the presence of inhibitor	Vmaxrel
MK-571	1	6.3±1.2	11.6±2.3*	200±29	131±19*	0.66±0.06*
Cyclosporine A	2	6.3±1.2	8.7±3.8	200±29	91±47*	0.45±0.21*
benzbromarone	5	8.3±3.2	12.0±5.2*	ND	ND	0.85±0.30
Verapamil	10	8.2±3.4	4.5±1.7**	ND	ND	0.43±0.06**

ATP-dependent BCPCF transport into E-IOVs was measured at various substrate concentrations (1–15 µM) for 60 min, at 37°C in the presence and in the absence of inhibitors. The values of the kinetic constants were determined using nonlinear fitting to the hyperbolic Michaelis-Menten equation for MK-571 and cyclosporin A from calibrated and for benzbromarone and verapamil from uncalibrated flow cytometric measurements. V_maxrel was calculated as the ratio of V_max determined in the presence of inhibitor to V_max of the control (in the absence of inhibitor). The presented data are the mean±SD from three experiments done on E-IOVs preparations from different donors. Significant difference from control calculated with Student's t-test; *p < 0.05; **p < 0.01; ND, not determined.

Figure 9.  Inhibition of ATP-dependent BCPCF transport into erythrocyte membrane inside-out vesicles by MK-571 (A) and cyclosporin A (B). The ATP-dependent BCPCF transport was determined from calibrated flow cytometric measurements at various substrate concentrations (1–15 µM) for 60 min at 37°C in the absence (▪, A and B) or in the presence of 1 µM MK-571 (○, A) and in the presence of 2 µM CsA (Δ, B). Data were plotted from a single experiment in duplicate and the kinetic constants K_m and V_max were calculated using nonlinear fitting to the hyperbolic equation. Similar results were obtained in two additional experiments done on E-IOVs from the blood of the other donors. Summarized results are shown in Table II. V_max determined for the controls were 176 pmol×mg⁻¹×h⁻¹ and 191 pmol×mg⁻¹×h⁻¹ in the Figure 9A and 9B, respectively.