title: Endogenous 5-HT2C Receptors Phosphorylate the cAMP Response Element Binding Protein via Protein Kinase C-Promoted Activation of Extracellular-Regulated Kinases-1/2 in Hypothalamic mHypoA-2/10 Cells.
abstract: Serotonin 5-HT2C receptors (5-HT2CR) activate Gq proteins and are expressed in the central nervous system (CNS). 5-HT2CR regulate emotion, feeding, reward, or cognition and may serve as promising drug targets to treat psychiatric disorders or obesity. Owing to technical difficulties in isolating cells from the CNS and the lack of suitable cell lines endogenously expressing 5-HT2CR, our knowledge about this receptor subtype in native environments is rather limited. The hypothalamic mHypoA-2/10 cell line was recently established and resembles appetite-regulating hypothalamic neurons of the paraventricular nucleus (PVN), where 5-HT2CR have been detected in vivo. Therefore, we tested mHypoA-2/10 cells for endogenous 5-HT2CR expression. Serotonin or the 5-HT2CR preferential agonist WAY-161,503 initiated cAMP response element (CRE)-dependent gene transcription with EC50 values of 15.5 ± 9.8 and 1.1 ± 0.9 nM, respectively. Both responses were blocked by two unrelated 5-HT2CR-selective antagonists (SB-242,084, RS-102,221) but not by a 5-HT2AR (EMD-281,014) or 5-HT2BR (RS-127,455) antagonists. By single-cell calcium imaging, we found that serotonin and WAY-161,503 induced robust calcium transients, which were also blunted by both 5-HT2CR antagonists. Additionally we revealed, first, that 5-HT2CR induced CRE activation via protein kinase C (PKC)-mediated engagement of extracellular-regulated kinases-1/2 and, second, that intrinsic activity of WAY-161,503 was in the range of 0.3-0.5 compared with serotonin, defining the frequently used 5-HT2CR agonist as a partial agonist of endogenous 5-HT2CR. In conclusion, we have shown that hypothalamic mHypoA-2/10 cells endogenously express 5-HT2CR and thus are the first cell line in which to analyze 5-HT2CR pharmacology, signaling, and regulation in its natural environment.
authors.affiliation: Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Lauffer
authors.affiliation: Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Evi
authors.initials: E
authors.lastname: Glas
authors.affiliation: Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gudermann
authors.affiliation: Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, München, Germany andreas.breit@lrz.uni-muenchen.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Breit
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010286:Paraventricular Hypothalamic Nucleus / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D044405:Receptor, Serotonin, 5-HT2C / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D058826:Serotonin 5-HT2 Receptor Agonists / Q000494:pharmacology
chemical_list: C502777:8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino(1,2-a)quinoxalin-5(6H)-one; C097067:CP 94253; D011719:Pyrazines; D011725:Pyridines; D011810:Quinoxalines; D044405:Receptor, Serotonin, 5-HT2C; D058826:Serotonin 5-HT2 Receptor Agonists; D012701:Serotonin; D000242:Cyclic AMP; D011493:Protein Kinase C