Jourdi Hussam, Lu Xiaoying, Yanagihara Ted, Lauterborn Julie C, Bi Xiaoning, Gall Christine M, Baudry Michel
Neuroscience Program and Department of Biology, University of Southern California, Los Angeles, CA 90089-2520, USA.
J Pharmacol Exp Ther. 2005 Jul;314(1):16-26. doi: 10.1124/jpet.105.083873. Epub 2005 Mar 22.
Prolonged exposure of cultured hippocampal slices to CX614 [2H,3H,6aH-pyrrolidino[2'',1''-3',2']1,3-oxazino[6',5'-5,4]-benzo[e]1,4-dioxan 10-one], a positive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAr) modulator, decreases receptor response to synaptic stimulation, an effect that could reflect reduced receptor expression. The present study investigates this down-regulation and its underlying mechanisms using cultured rat hippocampal slices. Chronic treatment with CX614 gradually reduced levels of glutamate receptor (GluR)1 and GluR2/3 AMPAr subunits and of their anchoring proteins synapse-associated protein 97 (SAP97) and glutamate receptor interacting protein 1 (GRIP1) through 48 h. Decline in SAP97 and GRIP1 levels was associated with increased abundance of lower molecular weight bands, suggesting degradation of these proteins. CX614 effects were partially reversible after drug removal. GluR1 and GluR2/3 down-regulation and their slow recovery were associated with similar changes in SAP97 and GRIP1 levels. Treatment with CX614 for 48 h significantly reduced AMPAr mRNA levels in hippocampus, whereas 8-h exposure did not. Blockade of ionotropic glutamate receptors prevented CX614-induced decrease in AMPAr subunits and mRNA, with regional selectivity, although an AMPAr blocker was more efficacious than an N-methyl-D-aspartate receptor blocker. Blockade of calpain activity reduced CX614-induced degradation of SAP97 and GRIP1 and prevented decreases in AMPAr subunit but not mRNA levels. Treatment with CX614 alone or in combination with glutamate receptor blockers or calpain inhibitor III did not modify lactate dehydrogenase release into culture medium, implying the absence of cell toxicity. We conclude that CX614-induced AMPAr protein loss is primarily mediated by AMPAr activation and involves calpain-dependent proteolysis of SAP97 and GRIP1. CX614-induced suppression of AMPAr gene expression is, however, calpain-independent, and all these effects are not associated with cell damage.
将培养的海马切片长时间暴露于CX614（2H,3H,6aH-吡咯烷并[2'',1''-3',2']1,3-恶嗪并[6',5'-5,4]-苯并[e]1,4-二恶烷-10-酮），一种阳性α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体（AMPAr）调节剂，会降低受体对突触刺激的反应，这种效应可能反映了受体表达的降低。本研究使用培养的大鼠海马切片来研究这种下调及其潜在机制。用CX614进行慢性处理在48小时内逐渐降低了谷氨酸受体（GluR）1和GluR2/3 AMPAr亚基及其锚定蛋白突触相关蛋白97（SAP97）和谷氨酸受体相互作用蛋白1（GRIP1）的水平。SAP97和GRIP1水平的下降与较低分子量条带丰度的增加相关，表明这些蛋白质发生了降解。药物去除后，CX614的作用部分可逆。GluR1和GluR2/3的下调及其缓慢恢复与SAP97和GRIP1水平的类似变化相关。用CX614处理48小时可显著降低海马中AMPAr mRNA水平，而暴露8小时则不会。离子型谷氨酸受体的阻断以区域选择性的方式阻止了CX614诱导的AMPAr亚基和mRNA的减少，尽管一种AMPAr阻断剂比N-甲基-D-天冬氨酸受体阻断剂更有效。钙蛋白酶活性的阻断减少了CX614诱导的SAP97和GRIP1的降解，并阻止了AMPAr亚基水平的降低，但未阻止mRNA水平的降低。单独使用CX614或与谷氨酸受体阻断剂或钙蛋白酶抑制剂III联合处理均未改变乳酸脱氢酶释放到培养基中的情况，这意味着不存在细胞毒性。我们得出结论，CX614诱导的AMPAr蛋白损失主要由AMPAr激活介导，并且涉及SAP97和GRIP1的钙蛋白酶依赖性蛋白水解。然而，CX614诱导的AMPAr基因表达的抑制不依赖于钙蛋白酶，并且所有这些效应均与细胞损伤无关。
