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Fluoxetine-mediated 5-HT2B receptor stimulation in astrocytes causes EGF receptor transactivation and ERK phosphorylation

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Abstract

Rationale

Fluoxetine has relatively high affinity for Gq/11 protein-coupled 5-HT2 receptors. Part of these receptors in brain are on astrocytes, where fluoxetine causes an increase in free cytosolic calcium concentration ([Ca2+]i) and phosphorylation of extracellular regulated kinase 1 and 2 (ERK1/2).

Objective

The objectives of the study are to identify subtype of the 5-HT2 receptor involved, to establish whether ERK1/2 phosphorylation is a result of 5-HT2-mediated transactivation of epidermal growth factor (EGF) receptors (EGFRs), and to determine signaling pathways up- and downstream of ERK1/2.

Materials and methods

Primary cultures of mouse astrocytes, which express all three subtypes of the 5-HT2 receptor but no 5-HT2 transporter, were used. ERK1/2 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-HT2B receptor depletion and signaling pathways by EGFR phosphorylation, using immunoprecipitation and Western blotting, inhibition of protein kinase C (PKC), and [Ca2+]i chelation by BAPTA/AM.

Results

ERK1/2 phosphorylation was abolished by SB204741, a universal 5-HT2 receptor antagonist, and in 5-HT2B receptor-depleted cells, but unaffected by 5-HT2A or 5-HT2C receptor antagonists (M100907 and SB242084). Phosphorylation of ERK1/2 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 [Ca2+]i or PKC inhibition with GF 109203X abrogated ERK1/2 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.

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Acknowledgements

This study was supported by Grant Nos. 30670651 and 30770667 from the National Natural Science Foundation of China. Sanofi-Aventis, Chilly-Mazarin, France is thanked for a gift of M100907.

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Authors and Affiliations

  1. Department of Clinical Pharmacology, China Medical University, Shenyang, People’s Republic of China

    Baoman Li, Shiquen Zhang, Hongyan Zhang, Weiwei Nu, Leif Hertz & Liang Peng

  2. Laboratory of Molecular Biology, Liaoning University of Traditional Chinese Medicine, Shenyang, People’s Republic of China

    Liping Cai

  3. College of Basic Medical Sciences, China Medical University, No. 92 Beier Road, Heping District, Shenyang, People’s Republic of China

    Liang Peng

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  1. Baoman Li
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  2. Shiquen Zhang
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  3. Hongyan Zhang
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  4. Weiwei Nu
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  6. Leif Hertz
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  7. Liang Peng
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Correspondence to Liang Peng.

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Baoman Li and Shiquen Zhang contributed equally to the article.

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Li, B., Zhang, S., Zhang, H. et al. Fluoxetine-mediated 5-HT2B receptor stimulation in astrocytes causes EGF receptor transactivation and ERK phosphorylation. Psychopharmacology 201, 443–458 (2008). https://doi.org/10.1007/s00213-008-1306-5

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  • DOI: https://doi.org/10.1007/s00213-008-1306-5

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