Wang J Y, Chi S I, Wang J Y, Hwang C P, Wang J Y
Department of Physiology, National Defense Medical Center, Taipei, Taiwan, ROC.
Chin J Physiol. 1996;39(4):227-33.
Using an in vitro primary cell culture model in which cortical neurons undergo a gradual and delayed neuronal death after a brief (5 min) challenge with glutamate receptor agonist N-methyl-D-aspartate (NMDA, 300 microM), the neuroprotective effects of various nitric oxide synthases (NOS) inhibitors were compared with that of the NMDA receptor antagonist dizocilpine maleate (MK-801). Our rat cortical cultures consisted of approximately 80-96% neurons and 5-20% astroglia as determined by immunocytochemical staining with antibodies against glial fibrillary acidic protein (GFAP) or neuron specific enolase (NSE). The delayed type of NMDA-induced neurotoxicity was examined by the morphological estimate of cell injury and was further confirmed by the activity of lactate dehydrogenase (LDH) in the extracellular fluid measured 24 hrs after the 5-min NMDA exposure. The accumulation of nitrite, the stable metabolite of nitric oxide (NO), was also measured 24 hrs after the 5-min NMDA exposure. The brief NMDA exposure caused about 60% neuronal death, as compared with persist (24 hr) NMDA exposure at 24 hr after NMDA exposure. Effects of drugs were studied by pretreating the cultures for 10 mins prior to the induction of NMDA neurotoxicity. Both the nonselective NOS inhibitor N alpha-nitro-L-arginine methyl ester (L-NAME, 100 microM) and the selective neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindozale (7-NI, 100 microM) suppressed nitrite accumulation and attenuated neuronal damage induced by NMDA. However, the selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG, 100 microM) exhibited no neuroprotective effects and no reduction in the nitrite production. The NMDA-induced neurotoxicity and nitrite production was abolished by pretreatment with the NMDA receptor antagonist MK-801 (100 microM). Thus the results indicate that a brief NMDA exposure leads to delayed neuronal damage with concomitant increase in NO production in cortical neuronal cultures. We suggest that the NO may originate primarily from nNOS. The neuroprotective effects of NOS inhibitors are weaker than that of MK-801.
利用体外原代细胞培养模型，其中皮质神经元在用谷氨酸受体激动剂N-甲基-D-天冬氨酸（NMDA，300微摩尔）短暂（5分钟）刺激后会经历逐渐且延迟的神经元死亡，将各种一氧化氮合酶（NOS）抑制剂的神经保护作用与NMDA受体拮抗剂马来酸氯氮平（MK-801）的作用进行了比较。通过用抗胶质纤维酸性蛋白（GFAP）或神经元特异性烯醇化酶（NSE）的抗体进行免疫细胞化学染色确定，我们的大鼠皮质培养物由约80-96%的神经元和5-20%的星形胶质细胞组成。通过细胞损伤的形态学评估来检查NMDA诱导的延迟性神经毒性，并在5分钟NMDA暴露后24小时测量细胞外液中乳酸脱氢酶（LDH）的活性进一步证实。在5分钟NMDA暴露后24小时也测量了一氧化氮（NO）的稳定代谢产物亚硝酸盐的积累。与NMDA暴露后24小时持续（24小时）暴露NMDA相比，短暂的NMDA暴露导致约60%的神经元死亡。在诱导NMDA神经毒性之前，通过对培养物进行10分钟预处理来研究药物的作用。非选择性NOS抑制剂Nα-硝基-L-精氨酸甲酯（L-NAME，100微摩尔）和选择性神经元一氧化氮合酶（nNOS）抑制剂7-硝基吲唑（7-NI，100微摩尔）均抑制亚硝酸盐积累并减轻NMDA诱导的神经元损伤。然而，选择性诱导型一氧化氮合酶（iNOS）抑制剂氨基胍（AG，100微摩尔）没有表现出神经保护作用，也没有降低亚硝酸盐的产生。用NMDA受体拮抗剂MK-801（100微摩尔）预处理可消除NMDA诱导的神经毒性和亚硝酸盐产生。因此，结果表明短暂的NMDA暴露会导致皮质神经元培养物中延迟性神经元损伤，并伴随NO产生增加。我们认为NO可能主要来源于nNOS。NOS抑制剂的神经保护作用比MK-801弱。
