Xiao Lin, Feng Chunzhi, Chen Yizhang
Institute of Neuroscience, Second Military Medical University, 800 XiangYin Road, Shanghai 200433, People's Republic of China.
Mol Endocrinol. 2010 Mar;24(3):497-510. doi: 10.1210/me.2009-0422. Epub 2010 Feb 16.
Glucocorticoid (GC) has been shown to affect the neuronal survival/death through a genomic mechanism, but whether or not it does through a nongenomic mechanism is unknown. Using a previously identified GR-deficient primary hippocampal neuron culture, we show here that a 15-min coexposure of N-methyl-D-aspartate (NMDA) with corticosterone at a stress-induced level significantly enhances neuronal death compared to NMDA alone. This enhancing effect of GC can be mimicked by the BSA-conjugated corticosterone, which is plasma membrane impermeable and cannot be blocked by RU38486 spironolactone. Furthermore, using a calcium-imaging technique, we found that B could increase both the percentage of neurons showing a significant increment of intracellular free calcium (Ca2+) due to NMDA stimulation and the amplitude of Ca2+ increment in the individual responsive cells. Interestingly, this boosting effect of GC on Ca2+ increment could be blocked by the NMDA receptor subunit 2A (NR2A)-specific antagonist [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) but not by the NMDA receptor subunit 2B (NR2B)-specific antagonist Ro25-6981. Moreover, we also found that GC can dramatically attenuate the NMDA-induced activation of ERK1/2 without affecting that of p38; and that the NMDA-induced ERK1/2 activation and its attenuation by GC both can be occluded by the NVP-AAM077 but not by Ro25-6981. Consistently, the enhancing effect of GC on NMDA neurotoxicity can also be blocked by NVP-AAM077 and the ERK1/2 inhibitor PD98059 but not by Ro25-6981 and p38 inhibitor SB203580. Indeed, the NMDA neurotoxicity itself can be blocked by Ro25-6981 or SB203580, whereas it is increased by NVP-AAM077 and PD98059. Therefore, it is probable that NMDA triggers a prodeath signaling through the NR2B-p38 MAPK pathway, and a prosurvival signaling through the NR2A-ERK1/2 MAPK pathway, whereas the latter was negatively regulated by rapid GC action. Taken together, the present data suggest a nongenomic action by GC that enhances NMDA neurotoxicity through facilitating Ca2+ increment and attenuating the NR2A-ERK1/2-mediated neuroprotective signaling, implicating a novel pathway underlying the regulatory effect of GC on neuronal survival/death.
糖皮质激素（GC）已被证明可通过基因组机制影响神经元的存活/死亡，但它是否通过非基因组机制发挥作用尚不清楚。我们利用先前鉴定的GR缺陷型原代海马神经元培养物，在此表明，在应激诱导水平下，N-甲基-D-天冬氨酸（NMDA）与皮质酮共同暴露15分钟，与单独使用NMDA相比，显著增强了神经元死亡。GC的这种增强作用可被与牛血清白蛋白（BSA）结合的皮质酮模拟，后者不能透过质膜，且不能被RU38486螺内酯阻断。此外，使用钙成像技术，我们发现GC可增加因NMDA刺激而导致细胞内游离钙（[Ca2+]i）显著增加的神经元百分比，以及单个反应性细胞中[Ca2+]i增加的幅度。有趣的是，GC对[Ca2+]i增加的这种促进作用可被NMDA受体亚基2A（NR2A）特异性拮抗剂[(R)-[(S)-1-(4-溴苯基)-乙氨基]-(2,3-二氧代-1,2,3,4-四氢喹喔啉-5-基)-甲基]-膦酸（NVP-AAM077）阻断，但不能被NMDA受体亚基2B（NR2B）特异性拮抗剂Ro25-6981阻断。此外，我们还发现GC可显著减弱NMDA诱导的ERK1/2激活，而不影响p38的激活；并且NMDA诱导的ERK1/2激活及其被GC的减弱作用均可被NVP-AAM077阻断，但不能被Ro25-6981阻断。一致地，GC对NMDA神经毒性的增强作用也可被NVP-AAM077和ERK1/2抑制剂PD98059阻断，但不能被Ro25-6981和p38抑制剂SB203580阻断。实际上，NMDA神经毒性本身可被Ro25-6981或SB203580阻断，而被NVP-AAM077和PD98059增强。因此，NMDA可能通过NR2B-p38丝裂原活化蛋白激酶（MAPK）途径触发促死亡信号，并通过NR2A-ERK1/2 MAPK途径触发促存活信号，而后者受到GC快速作用的负调控。综上所述，目前的数据表明GC存在一种非基因组作用，即通过促进[Ca2+]i增加和减弱NR2A-ERK1/2介导的神经保护信号来增强NMDA神经毒性，这暗示了GC对神经元存活/死亡调节作用的一种新途径。
