Guarneri P, Russo D, Cascio C, De Leo G, Piccoli F, Guarneri R
Istituto di Biologia dello Sviluppo, C.N.R., Palermo, Italy.
Eur J Neurosci. 1998 May;10(5):1752-63. doi: 10.1046/j.1460-9568.1998.00191.x.
Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-D-aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1-100 microM) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 microM NMDA, but severe cell damage occurred by 60 min. When the GABAA (gamma-aminobutyric acid) receptor agonist muscimol (1-1000 microM), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 microM) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [3H] MK-801 and [3H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABAA receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.
在此，我们研究了N-甲基-D-天冬氨酸（NMDA）受体激活对神经甾体生成的可能调节作用，并探讨了神经甾体合成可能参与急性兴奋性毒性作用的假说。在离体视网膜中，暴露于NMDA会改变孕烯醇酮和硫酸孕烯醇酮的生成。这种效应呈剂量和时间依赖性，在暴露30分钟内，相对中等剂量的NMDA（1-100微摩尔）会增加合成，而在60分钟时或提高药物浓度时合成会降至对照值。NMDA刺激的神经甾体合成被（+）-5-甲基-10,11-二氢-5H-二苯并[a,d]环庚烯-5,10-亚胺马来酸氢盐（MK-801）和3-（2-羧基哌嗪-4-基）丙基-1-膦酸（CPP）阻断，依赖于细胞外钙，并可被谷氨酸重现。乳酸脱氢酶（LDH）释放和形态学分析显示，暴露于50微摩尔NMDA 30分钟后视网膜细胞活力未受到显著影响，但60分钟时会发生严重细胞损伤。当与已知可激活视网膜神经甾体生成的GABAA（γ-氨基丁酸）受体激动剂蝇蕈醇（1-1000微摩尔）一起添加NMDA时，未观察到神经甾体合成有额外增加，且NMDA诱导的LDH释放保持不变。然而，单独暴露于高浓度的蝇蕈醇（500微摩尔）会引发与NMDA相似程度的毒性。相比之下，荷包牡丹碱消除了神经甾体生成的增加和LDH释放。同样，用胆固醇侧链裂解细胞色素P450抑制剂R（+）-对氨基谷氨酰胺（AMG）预处理可减轻急性视网膜细胞损伤。AMG对NMDA刺激的神经甾体生成的抑制性质在观察中得到证实，即药物处理降低了孕烯醇酮含量，且不影响[3H]MK-801和[3H]蝇蕈醇的结合。结果表明，NMDA受体通过一种跨神经元机制调节神经甾体生成，这意味着GABAA受体被激活。早期NMDA介导的神经甾体合成刺激似乎在急性兴奋性毒性中起关键作用；因此，对其抑制可能会延迟神经元细胞死亡。
