title: Fluoxetine-mediated 5-HT2B receptor stimulation in astrocytes causes EGF receptor transactivation and ERK phosphorylation.
abstract: BACKGROUND
  Fluoxetine has relatively high affinity for Gq/11 protein-coupled 5-HT(2) receptors. Part of these receptors in brain are on astrocytes, where fluoxetine causes an increase in free cytosolic calcium concentration ([Ca(2+)](i)) and phosphorylation of extracellular regulated kinase 1 and 2 (ERK(1/2)).
  
  
  OBJECTIVE
  The objectives of the study are to identify subtype of the 5-HT(2) receptor involved, to establish whether ERK(1/2) phosphorylation is a result of 5-HT(2)-mediated transactivation of epidermal growth factor (EGF) receptors (EGFRs), and to determine signaling pathways up- and downstream of ERK(1/2).
  
  
  METHODS
  Primary cultures of mouse astrocytes, which express all three subtypes of the 5-HT(2) receptor but no 5-HT(2) transporter, were used. ERK(1/2) phosphorylation and c-Fos and FosB protein expression were determined with Western blotting, and c-fos and fosB mRNA expression with reverse transcription polymerase chain reaction. Receptor subtype was investigated with subtype-specific 5-HT antagonists and 5-HT(2B) receptor depletion and signaling pathways by EGFR phosphorylation, using immunoprecipitation and Western blotting, inhibition of protein kinase C (PKC), and [Ca(2+)](i) chelation by BAPTA/AM.
  
  
  RESULTS
  ERK(1/2) phosphorylation was abolished by SB204741, a universal 5-HT(2) receptor antagonist, and in 5-HT(2B) receptor-depleted cells, but unaffected by 5-HT(2A) or 5-HT(2C) receptor antagonists (M100907 and SB242084). Phosphorylation of ERK(1/2) and EGFRs was abolished by AG 1478, an inhibitor of EGFR tyrosine kinases, and GM 6001, an inhibitor of Zn-dependent metalloproteinases, suggesting growth factor "shedding" and transactivation of EGFRs. Chelation of [Ca(2+)](i) or PKC inhibition with GF 109203X abrogated ERK(1/2) phosphorylation. Up-regulated mRNA and protein expression of c-fos and fosB was abolished by SB204741, AG1478, and by U0126, an inhibitor of ERK phosphorylation by MAP kinase/ERK kinase.
authors.affiliation: Department of Clinical Pharmacology, China Medical University, Shenyang, People's Republic of China.
authors.forename: Baoman
authors.initials: B
authors.lastname: Li
authors.forename: Shiquen
authors.initials: S
authors.lastname: Zhang
authors.forename: Hongyan
authors.initials: H
authors.lastname: Zhang
authors.forename: Weiwei
authors.initials: W
authors.lastname: Nu
authors.forename: Liping
authors.initials: L
authors.lastname: Cai
authors.forename: Leif
authors.initials: L
authors.lastname: Hertz
authors.forename: Liang
authors.initials: L
authors.lastname: Peng