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

Abstract

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. Gβγ subunits are a potential treatment target in numerous diseases. The present study investigated the mechanism whereby Gβγ subunits influence DOM-induced HTR. Main methodsThe effects of the Gβγ inhibitor 3′,4′,5′,6′-tetrahydroxyspiro[2-benzofuran-3,9′-xanthene]-1-one (gallein) and antagonistic peptide βARKct (β-adrenergic receptor kinase C-terminal fragment) on DOM-induced HTR were studied via an HTR test. The activation of the phospholipase C β (PLCβ)/inositol triphosphate (IP3)/calcium (Ca2+) signaling pathway and extracellular signal-regulated kinase (ERK) following Gβγ 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 Gβγ subunit-mediated regulation of cyclic adenosine monophosphate (cAMP) was assessed via a GloSensor™ cAMP assay. Key findingsThe Gβγ subunit inhibitors gallein and βARKct 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-4-yl]methanol (M100907), gallein inhibited PLCβ phosphorylation (pPLCβ), 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, PLCβ 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 Gαs 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. SignificanceThe Gβγ subunits potentially mediate the HTR after 5-HT2A receptor activation via the PLCβ/IP3/Ca2+/ERK1/2 and cAMP signaling pathways. Inhibitors targeting the Gβγ subunits potentially inhibit the hallucinogenic effects of 5-HT2A receptor agonists.