Fig. 5. MAP kinase phosphorylation is not required for VRAC activity.

(a) Intracellular ATP levels in cells exposed to control Basal medium, Basal medium +vehicle (0.05% DMSO), or 0.1 µM Trametinib (Tram.). Data are the means ± SEM of 3 different experiments per group in two astrocytic cultures, normalized to control ATP levels. (b) Western blot analysis of Erk1/2 activation in Basal, 30% hypoosmotic, or Basal medium supplemented with 10% FBS. Cells were pretreated with vehicle, 0.1 µM Trametinib (TR), or 20 µM PD98059 (PD) for 20 min and then exposed to Basal, hypoosmotic, or serum-containing media supplemented with the same inhibitors, as indicated. Erk1/2 phosphorylation was quantified as the ratio of pErk1/2 to total Erk immunoreactivity in the same lysates. Data are the means ± SEM of three Western blot analyses in two cell culture preparations. *p<0.05, **p<0.01, vs. no inhibitor under the same conditions. Insets show representative immunoreactivity images for pErk1/2 and total Erk1/2. Molecular weight standard in the first lane =37 kD. (c) Kinetics of swelling-activated d-[³H]aspartate release in astrocytes pretreated with vehicle or 0.1 µM Trametinib and perfused with isosmotic and hypoosmotic (HYPO) media. Data are the means ± SEM of 4 experiments per group performed in one astrocytic culture.