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体内大鼠背侧脊髓中选择性甘氨酸转运体-1抑制对N-甲基-D-天冬氨酸受体的正向调节作用

Positive N-methyl-D-aspartate receptor modulation by selective glycine transporter-1 inhibition in the rat dorsal spinal cord in vivo.

作者信息

Whitehead K J, Pearce S M, Walker G, Sundaram H, Hill D, Bowery N G

机构信息

Department of Pharmacology, Division of Neuroscience, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

出版信息

Neuroscience. 2004;126(2):381-90. doi: 10.1016/j.neuroscience.2004.04.006.

Abstract

In this study we have employed the selective glycine transporter-1 (GlyT-1) and GlyT-2 transporter inhibitors R-(-)-N-methyl-N-[3-[(4-trifluoromethyl)phenoxy]-3-phenyl-propyl]glycine (1:1) lithium salt (Org 24598) and 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)methyl]benzamide (Org 25543), respectively, and microdialysis perfusion to determine the effect of GlyT transporter inhibition on extracellular amino acid concentrations in the lumbar dorsal spinal cord of the halothane-anaesthetised rat. Reverse dialysis of Org 24598 (0.1-10 microM) induced a concentration-related increase in extracellular glycine accompanied by a progressive increase in citrulline, but not aspartate, glutamate or GABA, efflux. Org 25543 (10 microM) by the same route induced a similar increase in glycine levels without affecting the efflux of other amino acids quantified. To test the hypothesis that the increase in citrulline efflux resulted from activation of the N-methyl-D-aspartate receptor (NMDA-R)/nitric oxide synthase (NOS) signalling cascade, the sensitivity was determined of GlyT-1 inhibition-induced effects to NMDA-R antagonism or NOS inhibition. Co-administration by reverse dialysis of the selective NMDA-R channel blocker MK-801 (0.5 mM) or the selective antagonist of the strychnine-insensitive glycine site, 7-chlorokynurenic acid (1 mM), with Org 24598 (10 microM) did not affect the uptake inhibition-induced increase in glycine efflux, but did significantly attenuate the increase in extracellular citrulline. Similarly, co-administration with Org 24598 of the isoform non-selective and selective neuronal NOS inhibitors Nomega-nitro-L-arginine methyl ester (1 mM) or 1-(2-trifluoromethylphenyl)imidazole (0.2 mM), respectively, prevented Org 24598-induced citrulline efflux with no effect on increased glycine efflux. These data provide evidence that the observed increased in extracellular citrulline is a consequence of positive modulation of NMDA-R, secondary to increased extracellular glycine and support a protective role for GlyT-1 against fluctuations in extracellular glycine uptake at glutamatergic synapses in the dorsal spinal cord. Such a mechanism could be important to NMDA-R-mediated synaptic plasticity in the spinal cord and be of relevance to the clinical usage of GlyT-1 inhibitors.

摘要

在本研究中,我们分别使用了选择性甘氨酸转运体-1(GlyT-1)和甘氨酸转运体-2抑制剂R-(-)-N-甲基-N-[3-[(4-三氟甲基)苯氧基]-3-苯基丙基]甘氨酸(1:1)锂盐(Org 24598)和4-苄氧基-3,5-二甲氧基-N-[1-(二甲基氨基环戊基)甲基]苯甲酰胺(Org 25543),并采用微透析灌注法来确定抑制甘氨酸转运体对氟烷麻醉大鼠腰段背侧脊髓细胞外氨基酸浓度的影响。反向透析Org 24598(0.1 - 10 microM)会导致细胞外甘氨酸浓度呈浓度依赖性增加,同时瓜氨酸外流逐渐增加,但天冬氨酸、谷氨酸或γ-氨基丁酸的外流没有增加。通过相同途径给予Org 25543(10 microM)会使甘氨酸水平出现类似的增加,而不影响所检测的其他氨基酸的外流。为了验证瓜氨酸外流增加是由N-甲基-D-天冬氨酸受体(NMDA-R)/一氧化氮合酶(NOS)信号级联激活所致的假设,我们确定了GlyT-1抑制诱导的效应对于NMDA-R拮抗或NOS抑制的敏感性。将选择性NMDA-R通道阻滞剂MK-801(0.5 mM)或士的宁不敏感甘氨酸位点的选择性拮抗剂7-氯犬尿氨酸(1 mM)与Org 24598(10 microM)通过反向透析共同给药,并不影响摄取抑制诱导的甘氨酸外流增加,但显著减弱了细胞外瓜氨酸的增加。同样,分别将同工型非选择性和选择性神经元NOS抑制剂Nω-硝基-L-精氨酸甲酯(1 mM)或1-(2-三氟甲基苯基)咪唑(0.2 mM)与Org 24598共同给药,可阻止Org 24598诱导的瓜氨酸外流,而对增加的甘氨酸外流没有影响。这些数据表明,观察到的细胞外瓜氨酸增加是NMDA-R正向调节的结果,继发于细胞外甘氨酸增加,并支持GlyT-1对背侧脊髓谷氨酸能突触处细胞外甘氨酸摄取波动具有保护作用。这样一种机制可能对脊髓中NMDA-R介导的突触可塑性很重要,并且与GlyT-1抑制剂的临床应用相关。

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