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. 2020 Aug 6;6(8):2073–2096. doi: 10.1016/j.chempr.2020.06.017

Figure 2.

Figure 2

ARN23746 Restores [Cl−]i in DS Neurons, Does Not Inhibit NKCC2 in HEK Cells and Shows Improved Brain Penetration In Vivo in Comparison to Bumetanide

(A) Left, representative pseudo-color images (colored scale below) of the [Cl−]i measured with the MQAE Cl−-sensitive dye, in WT and Ts65Dn hippocampal neurons at 15 DIV, after treatment with control DMSO (0.1%) and the indicated compounds (10 μM). Scale bar: 70 μm. Right, quantification of the indicated compounds (10 μM) in modulating [Cl−]i in experiments such as those on the left. Data represent mean ± SEM from three independent experiments (two-way ANOVA, Finteraction (2,30)= 3.815, p = 0.033, Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

(B) Left, example traces obtained in the Cl− influx assay on NKCC2-transfected HEK293 cells for each compound (10 μM). The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC2-mediated Cl− influx. Right, quantification of the NKCC2 inhibitory activity in experiments such as those on the right. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from four independent experiments (Kruskal-Wallis one way ANOVA on Ranks, H = 16,962, DF = 2, p < 0.001, Dunn’s post hoc test, ∗∗∗p < 0.001).

(C) Left, example traces obtained in the Tl influx assay on KCC2-transfected HEK293 cells for each compound (10 μM). The arrows indicate the additions of TlSO4 and NaCl. Right, quantification of the KCC2 inhibitory activity in experiments such as those on the left. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from 4 independent experiments (one way ANOVA, F (2,51) = 10.676, p < 0.001, Dunnett's post hoc test ∗∗∗p < 0.001).

(D) Quantification of the level of bumetanide and ARN23746 in the brain at diverse time points (5, 15, 30, 60, 120 min) after injection in C57BL/6N mice (BioLASCO Taiwan). (Two-way ANOVA, Ftreatment (1,50) = 6.510, p = 0.014, Tukey’s post hoc test, ∗p < 0.05.).

(E) Quantification of the ratio between brain and plasma concentration of bumetanide and ARN2346 5 min after the injection (two-tailed t test, t = 7.915, p < 0.001). See also Figures S1E–S1G.