G¦ subunit inhibitor decreases DOM-induced head twitch response via the PLC/IP3/Ca²⁺/ERK and cAMP signaling pathways

Aims: (-)-2,5-dimethoxy-4-methylamphetamine (DOM) induces the head-twitch response (HTR) primarily by activating the serotonin 5-hydroxytryptamine 2A receptor (5-HT2A receptor) in mice. However, the mechanisms underlying 5-HT2A receptor activation and the HTR remain elusive. G beta gamma subunits are a potential treatment target in numerous diseases. The present study investigated the mechanism whereby G beta gamma subunits influence DOMinduced HTR. Main methods: The effects of the G beta gamma inhibitor 3',4',5',6'-tetrahydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one (gallein) and antagonistic peptide beta ARKct (beta-adrenergic receptor kinase C-terminal fragment) on DOM-induced HTR were studied via an HTR test. The activation of the phospholipase C beta (PLC beta)/inositol triphosphate (IP3)/ calcium (Ca2+) signaling pathway and extracellular signal-regulated kinase (ERK) following G beta gamma subunit inhibition was detected by western blotting, Homogeneous Time-Resolved Fluorescence (HTRF) inositol phosphate (IP1) assay and Fluorometric Imaging Plate Reader (FLIPR) calcium 6 assay. The G beta gamma subunit-mediated regulation of cyclic adenosine monophosphate (cAMP) was assessed via a GloSensor T cAMP assay. Key findings: The G beta gamma subunit inhibitors gallein and beta ARKct reduced DOM-induced HTR in C57BL/6J mice. Like the 5-HT2A receptor-selective antagonist (R)-[2,3-di(methoxy)phenyl]-[1-[2-(4-fluorophenyl)ethyl]piperidin-4yl]methanol (M100907), gallein inhibited PLC beta phosphorylation (pPLC beta), IP1 production, Ca2+ transients, ERK1/2 phosphorylation (pERK1/2) and cAMP accumulation induced by DOM in human embryonic kidney (HEK) 293T cells stably or transiently transfected with the human 5-HT2A receptor. Moreover, PLC beta protein inhibitor 1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a] phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione (U73122) (10 nmol/mouse), intracellular Ca2+ blocker 6[6-[6-[5-acetamido-4,6-dihydroxy-2-(sulfooxymethyl)oxan-3-yl]oxy-2-carboxy-4-hydroxy-5-sulfooxyoxan-3-yl] oxy-2-(hydroxymethyl)-5-(sulfoamino)-4-sulfooxyoxan-3-yl]oxy-3,4-dihydroxy-5-sulfooxyoxane-2-carboxylic acid (heparin) (5 nmol/mouse), L-type Ca2+ channel blocker 3-O-(2-methoxyethyl) 5-O-propan-2-yl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nimodipine) (4 mg/kg), mitogen extracellular regulating kinase 1/2 (MEK1/2) inhibitor (Z)-3-amino-3-(4-aminophenyl)sulfanyl-2-[2-(trifluoromethyl) phenyl]prop-2-enenitrile (SL327) (30 mg/kg), and Gas protein selective antagonist 4,4',4.,4.-(Carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF449) (10 nmol/mouse) reduced DOM-induced HTR in C57BL/6J mice. Significance: The G beta gamma subunits potentially mediate the HTR after 5-HT2A receptor activation via the PLC beta/IP3/ Ca2+/ERK1/2 and cAMP signaling pathways. Inhibitors targeting the G beta gamma subunits potentially inhibit the hallucinogenic effects of 5-HT2A receptor agonists.