Fig. 4

Ethanol-induced Cx43 hemichannel activity depends on TLR4 and IL-1β/TNF-α/p38 MAPK/iNOS-dependent signaling. (A) Averaged Etd uptake rate (AU/min) normalized with control condition (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 0.5 µM TAK-242, 100 ng/mL of IL-1ra + 100 ng/mL of sTNF-αR1, 1 µM SB203580, 1 µM L-N6, 20 nM 0.5 µM TAK-242 + 50 µM gap19 or 0.5 µM TAK-242 + 50 µM ¹⁰panx1. *p < 0.005, ethanol treatment compared to control conditions; ^#p < 0.01; ^##p < 0.005; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 30 cells analyzed for each repeat). (B-C) Averaged data of IL-1β (B) and TNF-α (C) released by astrocytes under control conditions (white circles) or treated for several time periods with 100 mM ethanol (red circles). *p < 0.05, **p < 0.01, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). (D-E) Representative fluorescence micrographs of basal NO production (DAF-FM, green) by astrocytes under control conditions (D) or treated for 24 h with 100 mM ethanol (E). Calibration Bar: 20 μm. (F) Average DAF-FM fluorescence normalized to control conditions (dashed line) by astrocytes treated with 100 mM ethanol for several time periods. *p < 0.05, **p < 0.005, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). (G) Average DAF-FM fluorescence normalized to control conditions (dashed line) by astrocytes treated for 72 h with 100 mM ethanol alone or in combination with the following agents: 0.5 µM TAK-242, 50 µM gap19, 50 µM ¹⁰panx1 or 0.5 µM TAK-242 + 50 µM gap19. ***p < 0.005, **p < 0.001, effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test)