OBJECTIVE Respiratory depression hinders the use of anaesthetics and sedative hyp-notics.To explore the mechanism of LCX001 on protection against respiratory depression,a novel AMPA receptor modulator LCX001,synthesized by our Institute of Medicinal Chemistry,is expected to relieve suppressed respiration. METHODS LCX001 was tested to alleviate respiratory depression triggered by opioid(fentanyl and TH-030418),propofol and pentobarbital in the plethysmography recording.The acetic acid writhing and hot-plate tests were conducted to evaluate analgesic effect of LCX001.Binding assay and whole-cell recording were used to analyze the property of LCX001 on positive modulation. The function of AMPA receptors were determined by location of receptors in the membrane and state of channel opening, and both processes were impressed by AMPA receptor regulatory proteins. Ac-cording to the theory,the effect of LCX001 on the expression of stargazin was measured firstly by west-ern blotting. The variation of receptor surface location was observed by live cell imaging. The regula-tion on neuronal Ca2+and cell function was investigated intensively by Ca2+imaging to clarify mecha-nism of LCX001. RESULTS LCX001 effectively rescued and prevented opioid (fentanyl and TH-030418), propofol, and pentobarbital-induced respiratory depression by strengthening respiratory fre-quency and minute ventilation in rats. The acetic acid writhing test and hot-plate test revealed potent anti-nociceptive efficacy of LCX001,in contrast to some ampakines that did not affect analgesia. Fur-thermore,LCX001 potentiated[3H]AMPA and L-glutamate binding affinity to AMPA receptors,and facili-tated glutamate-evoked inward currents in HEK293 cells stably expressing GluA2(R).Importantly,appli-cation of LCX001 generated a significant increase in GluA2(R) surface expression in a mechanism of stargazin up-regulation,and restrained opioid-induced abnormal intracellular Ca2+load,which might par-ticipate in breathing modulation. CONCLUSION The novel pharmacological effect and potential new mechanism of LCX001 might promote ampakines to be a therapeutic option for protection against respi-ratory depression.

哺乳动物脑中的快速兴奋性突触信号传递主要通过离子型谷氨酸受体——α-氨基-3-羟基-5-甲基-4-异(噁)唑丙酸(AMPA)受体来介导,该过程涉及AMPA受体与各种辅助亚基形成蛋白-蛋白复合物而发生效应,并在突触可塑性中起作用,形成学习和记忆的基础.Stargazin是目前研究较为成熟的相关跨膜AMPA受体调节蛋白家族成员之一,可直接与AMPA受体相互作用,通过两种不同的机制调控AMPA受体的突触靶向,进而影响突触可塑性,其涉及调节通道门控和受体药理学,与疼痛、视觉剥夺及精神分裂症等密切相关,故已成为研究突触传递效能的重要新靶点.未来,对AMPA受体的研究从stargazin着手将成为研究思路之一.

目的 研究Stargazin-亚硝基化修饰在脑缺血再灌注后突触可塑性中的作用,并探讨NO调控AMPAR"Trafficking"的分子机理.方法 采用线栓法阻塞大脑中动脉复制局灶性脑缺血再灌注(MCAO/R)损伤大鼠作为模型组,分别在每只大鼠MCAO/R模型内给予NMDAR抑制剂MK801、氧化还原剂DTT干预作为实验组.采用mNSS评分标准检测大鼠神经功能,TTC染色检测脑部缺血损伤情况,TUNEL染色检测以及WB检测缺血侧海马神经元凋亡情况,Griess法检测缺血侧海马组织中NO的含量,此外Western Blot检测海马组织中Stargazin-亚硝基化修饰水平以及AMPAR蛋白的表达和活化情况.结果 给予MK801和DTT处理后MCAO/R模型中Stargazin-亚硝基化修饰水平(P<0.01)以及NO含量(P<0.01)下降;AMPAR亚基GluR2磷酸化水平降低(P<0.0001);抑制Stargazin亚硝基化修饰能够改善MCAO/R引起的神经损伤与凋亡(P<0.001).结论 在MCAO/R模型中,抑制内源性NO和Stargazin亚硝基化水平可促进神经突触重塑,其机制可能是干预了Stargazin辅助蛋白与AMPAR亚基GluR2亲和力有关.

Chronic pain patients often have anxiety disorders,and some of them suffer from anxiety even after analgesic administration.In this study,we investigated the role of AMPAR-mediated synaptic transmission in the ventromedial prefrontal cortex(vmPFC)in chronic pain-induced persistent anxiety in mice and explored potential drug targets.Chronic inflammatory pain was induced in mice by bilateral injection of complete Freund's adjuvant(CFA)into the planta of the hind paws;anxiety-like behaviours were assessed with behavioural tests;S-nitrosylation and AMPAR-mediated synaptic transmission were examined using biochemical assays and electrophysiological recordings,respectively.We found that CFA induced persistent upregulation of AMPAR membrane expression and function in the vmPFC of anxious mice but not in the vmPFC of non-anxious mice.The anxious mice exhibited higher S-nitrosylation of stargazin(an AMPAR-interacting protein)in the vmPFC.Inhibition of S-nitrosylation by bilaterally infusing an exogenous stargazin(C3025)mutant into the vmPFC rescued the surface expression of GluA1 and AMPAR-mediated synaptic transmission as well as the anxiety-like behaviours in CFA-injected mice,even after ibuprofen treatment.Moreover,administration of ZL006,a small molecular inhibitor disrupting the interaction of nNOS and PSD-95(20 mg·kg-1·d-1,for 5 days,i.p.),significantly reduced nitric oxide production and S-nitrosylation of AMPAR-interacting proteins in the vmPFC,resulting in anxiolytic-like effects in anxious mice after ibuprofen treatment.We conclude that S-nitrosylation is necessary for AMPAR trafficking and function in the vmPFC under chronic inflammatory pain-induced persistent anxiety conditions,and nNOS-PSD-95 inhibitors could be potential anxiolytics specific for chronic inflammatory pain-induced persistent anxiety after analgesic treatment.