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. 2018 Apr 4;38(14):3466–3479. doi: 10.1523/JNEUROSCI.2967-17.2018

Figure 2.

Figure 2.

Enzymatic conjugation of MCB to glutathione in the blood–CSF barrier. A, Method: GST-mediated conjugation of the substrate MCB with GSH produces a fluorescent metabolite that can be monitored over time. B–F, Ex vivo exposure of freshly isolated choroid plexuses to MCB led to the rapid formation of the fluorescent GSH-conjugated metabolite within epithelial cells (arrowhead in C and E). After 4 min, the fluorescence signal decreased in the choroidal epithelial cells (arrowheads in D and F), and seemingly concentrated in the underlying vessel containing stroma (arrows in E and F). B is a negative control. B–D and E, F show, respectively, low-magnification and high-magnification images of the choroidal tissue exposed to 10 μm (C–D) and 2.5 μm (E–F) MCB. In F, the dashed line delimits the apical membrane of epithelial cells. Scale bar, 20 μm. G–H, Choroid plexus epithelial cells cultured on a porous filter (G) form an enzymatic barrier preventing the basolateral to apical transfer of MCB. When MCB was added to the basolateral compartment at a concentration of 5 μm, the appearance of the parent compound in the apical compartment was fully impeded for 30 min and remained strongly limited thereafter (H, full line). When the cells were exposed to 50 μm MCB, the enzymatic barrier effect lasted only 10 min, and the MCB clearance rate strongly increased thereafter (H, dashed line). I, Excretion of the conjugate produced within the choroidal epithelium was highly polarized, 90% of the metabolite being found in the basolateral medium. The conjugate was not detected intracellularly (black bars, cells exposed to 5 μm MCB for 30 min). When cells were preincubated with 3 mm probenecid before MCB exposure, GS-MCB efflux was strongly reduced at both basolateral and apical membranes, and the metabolite accumulated in the epithelial cells (gray bars). Data are the mean ± SEM of four filters. **p < 0.01 and ***p < 0.001, statistically different from GS-MCB amount in the absence of probenecid, one-tailed Student's t test for unequal variance. nd, Not detected.