*2.4. P-gp Is Active and Can Be Chemically Inhibited in CHO-APP and SK-N-SH Cells*

Calcein-AM is a soluble hydrophobic non-fluorescent dye that rapidly crosses the plasma membrane and is actively exported by P-gp. When P-gp is active, calcein-AM is efficiently removed from the cell before it can undergo hydrolysis. If P-gp is inactive, intracellular esterases cleave calcein-AM to produce the free acid calcein, which cannot be transported by P-gp and thus remains trapped inside the cell where it produces an intense fluorescence [48]. Therefore, measuring the accumulation of fluorescent calcein is a rapid and sensitive method for studying P-gp activity.

β β β Experiments were firstly performed in the CHO-APP cells, which secrete relatively large quantities of Aβ peptides. P-gp protein was confirmed to be expressed in CHO-APP cells at a level consistent with that of the parental CHO-K1 cell line (Figure 3; right panel). Verapamil and nicardipine, both anti-hypertensive calcium channel blockers, were selected due to their well established and strong P-gp inhibitory activity [49,50]. Figure 4a,b show that addition of these P-gp inhibitors to CHO-APP cells increased fluorescence, indicating increased intracellular calcein accumulation, in a concentration-dependent manner. Experiments were subsequently replicated in SK-N-SH neuroblastoma cells, which have previously been established to secrete Aβ<sup>40</sup> and Aβ<sup>42</sup> peptides [51,52]. Data show a similar concentration-dependent effect of P-gp inhibition on intracellular fluorescence (Figure 4c,d). Together, these data indicate that P-gp is active and can be chemically inhibited by these two drugs in both cell lines. Furthermore, nicardipine was approximately three-fold more effective than verapamil at inhibiting P-gp-mediated export of calcein-AM (Figure 4), which corresponds with previously published findings [49,50].

Although calcein-AM is a substrate of both P-gp and the related multi-drug resistance transporter ABCC1/MRP1, verapamil and nicardipine do not directly affect ABCC1/MRP1 activity [53,54]. Furthermore, verapamil does not appear to affect the activity of other transporters including ABCG2/BCRP, ABCG4, LRP-1, or RAGE that have been implicated in Aβ transport [55–58]. Although nicardipine is an effective inhibitor of ABCG2/BCRP [59], expression of this transport protein has not been reported in SK-N-SH and is absent or minimal CHO cells [60–62]. Therefore, the observations described in Figure 4 are deemed to be specific to P-gp and not confounded by activity of other ABC transporters.

μ μ μ **Figure 4.** P-gp activity in CHO-APP and SK-N-SH cells. P-gp efflux activity was measured using the fluorogenic Pgp substrate calcein-AM. CHO-APP cells were treated with (**a**) verapamil at 3, 10 or 30 µM or DMSO control, or (**b**) nicardipine at 1, 3, or 10 µM or DMSO control, immediately prior to the addition of 0.1 µM calcein-AM. Similarly, SK-N-SH cells were treated with varying concentrations of either (**c**) verapamil or (**d**) nicardipine or DMSO control. Fluorescence measurements were obtained at 485/535 nm every minute over twenty minutes. Data are presented as the mean ± SEM of three independent experiments, with each condition conducted with six replicates.
