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Clinical evidence suggest astrocytic abnormality in major depression (MD) while treatment with anti-psychotic drugs affects astroglial functions. Astroglial cells are involved in pH homeostasis of the brain by transporting protons (through sodium-proton transporter 1, NHE1, glutamate transporters EAAT1/2 and proton-lactate co-transporter MCT1) and bicarbonate (through the sodium-bicarbonate co-transporter NBC or the chloride-bicarbonate exchanger AE). Here we show that chronic treatment with fluoxetine increases astroglial pHi by stimulating NHE1-mediated proton extrusion. At a clinically relevant concentration of 1 μM, fluoxetine significantly increased astroglial pHi from 7.05 to 7.34 after 3 weeks and from 7.18 to 7.58 after 4 weeks of drug treatment. Stimulation of NHE1 is a result of transporter phosphorylation mediated by several intracellular signaling cascades that include MAPK/ERK1/2, PI3K/AKT and ribosomal S6 kinase (RSK). Fluoxetine stimulated phosphorylation of ERK1/2, AKT and RSK in a concentration dependent manner. Positive crosstalk exists between two signal pathways, MAPK/ERK1/2 and PI3K/AKT activated by fluoxetine since ERK1/2 phosphrylation could be abolished by inhibitors of PI3K, LY294002 and AKT, triciribine, and AKT phosphorylation by inhibitor of MAPK, U0126. As a result, RSK phosphorylation was not only inhibited by U0126 but also by inhibitor of LY294002. The NHE1 phoshorylation resulted in stimulation of NHE1 activity as revealed by the NH4Cl-prepulse technique; the increase of NHE1 activity was dependent on fluoxetine concentration, and could be inhibited by both U0126 and LY294002. Our findings suggest that regulation of astrocytic pHi and brain pH may be one of the mechanisms underlying fluoxetine action.

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