目的:(-)-2,5-二甲氧基-4-甲基苯丙胺(DOM)主要通过激活5-羟色胺5-羟色胺2A受体(5-HT2A受体)诱导小鼠头部抽搐反应(HTR)。然而,5-HT2A受体激活和HTR的潜在机制仍然难以捉摸。Gβγ亚基是许多疾病的潜在治疗靶标。本研究调查了Gβγ亚基影响DOM诱导的HTR的机制。
方法:Gβγ抑制剂3'的作用,4\',5\',通过HTR测试研究了DOM诱导的HTR上的6'-四羟基螺[2-苯并呋喃-3,9'-xanthene]-1-酮(gallein)和拮抗肽βARKct(β-肾上腺素能受体激酶C末端片段)。通过蛋白质印迹法检测Gβγ亚基抑制后磷脂酶Cβ(PLCβ)/三磷酸肌醇(IP3)/钙(Ca2)信号通路和细胞外信号调节激酶(ERK)的激活,均相时间分辨荧光(HTRF)肌醇磷酸(IP1)测定和荧光成像板读数器(FLIPR)钙6测定。通过GloSensor™cAMP测定评估环磷酸腺苷(cAMP)的Gβγ亚基介导的调节。
结果:Gβγ亚基抑制剂gallein和βARKct降低了DOM诱导的C57BL/6J小鼠的HTR。像5-HT2A受体选择性拮抗剂(R)-[2,3-二(甲氧基)苯基]-[1-[2-(4-氟苯基)乙基]哌啶-4-基]甲醇(M100907),Gallein抑制PLCβ磷酸化(pPLCβ),IP1生产,Ca2+瞬变,DOM在人5-HT2A受体稳定或瞬时转染的人胚肾(HEK)293T细胞中诱导ERK1/2磷酸化(pERK1/2)和cAMP积累。此外,PLCβ蛋白抑制剂1-[6-[[(8R,9S,13S,14S,17S)-3-甲氧基-13-甲基-6,7,8,9,11,12,14,15,16,17-十氢环戊并[a]菲-17-基]氨基]己基]吡咯-2,5-二酮(U73122)(10nmol/mouse),细胞内Ca2+阻断剂6-[6-[6-[5-乙酰氨基-4,6-二羟基-2-(磺氧基甲基)氧杂-3-基]氧基-2-羧基-4-羟基-5-磺氧基氧杂-3-基]氧基-2-(羟甲基)-5-(磺氨基)-4-磺氧基-3-基]氧基-3,4-二羟基-鼠2-L型Ca2通道阻断剂3-O-(2-甲氧基乙基)5-O-丙-2-基2,6-二甲基-4-(3-硝基苯基)-1,4-二氢吡啶-3,5-二羧酸(尼莫地平)(4mg/kg),丝裂原细胞外调节激酶1/2(MEK1/2)抑制剂(Z)-3-氨基-3-(4-氨基苯基)硫基-2-[2-(三氟甲基)苯基]丙-2-烯腈(SL327)(30mg/kg),和Gαs蛋白选择性拮抗剂4,4',4″,4-(羰基双-(亚氨基-5,1,3-苯三基双(羰基亚氨基)))四苯-1,3-二磺酸(NF449)(10nmol/小鼠)降低了DOM诱导的C57BL/6J小鼠的HTR。
结论:Gβγ亚基可能通过PLCβ/IP3/Ca2+/ERK1/2和cAMP信号通路介导5-HT2A受体活化后的HTR。靶向Gβγ亚基的抑制剂潜在地抑制5-HT2A受体激动剂的致幻作用。
OBJECTIVE: (-)-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.
METHODS: The 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.
RESULTS: The 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.
CONCLUSIONS: The 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.