Fluoxetine Causes Astrocytic ERK Phosphorylation By 5-HT(2B) Receptor-Mediated EGF Receptor Transactivation

IntroductionThe 5-HT₂ receptor have three subtypes,5-HT_2A,5-HT_2B and 5-HT_2C receptor.All three 5-HT₂ receptors are G_q protein-coupled receptor(GPCR).And their stimulation activates phospholipase C,leading to an increase of free cytosolic calcium concentration.Moreover,G_q receptor-mediated epidermal growth factor receptor (EGFR) transactivation can activate MAPK(including ERK) signaling pathway. Primary cultures of mouse astrocytes express 5-HT_2A,5-HT_2B and 5-HT_2C receptors. Fluoxetine has relatively high affinity for G_q protein-coupled 5-HT₂ receptors.And it causes an increase in free cytosolic calcium concentration by a direct 5-HT₂ receptors action in cultured mouse astrocytes.Objective1,To investigate the effect of fluoxetine on astrocytic ERK_1/2 phosphorylation.2,To identify subtype of the 5-HT₂ receptor involved.3,To establish whether ERK_1/2 phosphorylation is a result of 5-HT₂-mediated transactivation of epidermal growth factor receptors(EGFRs).Materials and methodsPrimary cultures of mouse astrocytes,which express all three subtypes of the 5-HT₂ receptor but no 5-HT₂ transporter,were used.ERK_1/2 phosphorylation were determined with Western blotting.The 5-HT₂ receptor subtype was investigated with subtype-specific 5-HT₂ antagonists.And the signaling pathways of transactivation of epidermal growth factor receptors were identified with inhibitor of the EGFR tyrosine kinase and broad-based inhibitor of Zn²⁺-activaed metalloproteinases.Results1,Five micromolars of fluoxetine induced a statistically significant increase of ERK phosphorylation.The stimulated ERK phosphorylation reached about 200%of the control value when the concentrations of fluoxetine were increased to 40μM-60μM (P＜0.05).A statistically significant increase of ERK phosphorylation was seen after 20 min,and it lasted until 40 min of fluoxetine(10μM) treatment(P＜0.05).2,In the presence of SB204741,an antagonist of all three 5-HT₂ receptors, fluoxetine-induced ERK phosphorylation was inhibited by 40%at 200 nM and abolished at 400 nM and higher concentrations(P＜0.05).In contrast,no inhibition was seen with the selective 5-HT_2A receptor antagonist M100907(0.01 nM-3μM) or the selective 5-HT_2C receptor antagonist SB242084(0.5 nM-5μM).3,Both AG 1478(1μM) and GM 6001(10μM) virtually abolished the effects of 10μM fluoxetine on ERK phosphorylation(P＜0.05).ConclusionFluoxetine causes EGF receptor transactivation and ERK phosphorylation by a direct 5-HT_2B receptors action in cultured mouse astrocytes,and these effects show time dependence and concentration dependence.