Effects of Ethanol on NMDA Receptors in Brain: Possibilities for Mg2+ -Ethanol Interactions

The major excitatory neurotransmitter in the CNS is L-glutamate, and one of the subtypes of L-glutamate receptors, the N -methyl-D-aspartate (NMDA) subtype, has been found to be quite sensitive to inhibition by low concentrations of ethanol (5–50 mm). The NMDA receptor-ion channels are unique in that they exhibit a voltage-dependent blockade by physiological concentrations of Mg²⁺, a blockade that is relieved as the cell membrane is depolarized. Several lines of evidence also suggest that the activity of this receptor-channel complex may be regulated through a high-affinity Mg²⁺ site, which is distinct from the channel-blocking site and could even be located on the extracellular domain of the protein. This high-affinity Mg²⁺ site has been shown to increase the binding of N -[1-(2-thienyl) cyclohexyl]piperidine within the ion channel, as well as the binding of competitive antagonist such as 3-(±)-carboxypiperazine-4-yl)-[1,2]-propyl-1-phosphonic acid and the receptor coactivator glycine. The relationship between the acute effects of ethanol on receptor activation and the regulatory properties of Mg²⁺ is not yet known, although the hypomagnesemia that occurs in chronic alcoholism could certainly have implications for receptor function. A significant amount of molecular characterization of the multiple isoforms of the NMDA receptor-ion channel will be required before the role of Mg²⁺ can be clarified and any relationship between Mg²⁺ regulation and ethanol inhibition established.     

Differential sparing of somatostatin-neuropeptide Y and cholinergic neurons following striatal excitotoxin lesions

We previously found that quinolinic acid striatal excitotoxin lesions result in a relative sparing of somatostatin and neuropeptide Y neurons. In the present study we examined dose-response effects of excitotoxins acting at the three subtypes of glutamate receptors: N-methyl-D-aspartate (AA1), quisqualate (AA2), and kainic acid (AA3). Concentrations of both somatostatin-like immunoreactivity (SLI) and neuropeptide a Y-like immunoreactivity (NPYLI) were compared with those of substance P-like immunoreactivity (SPLI) and GABA. Kainic acid (AA3), quisqualic acid (AA2), and AMPA (AA2) resulted in dose-dependent reductions in all four neurochemical markers examined, while N-methyl-D,L-aspartate (AA1) and quinolinic acid (AA1) resulted in relative sparing of SLI and NPYLI. At doses of each excitotoxin which resulted in comparable 50% reductions in both GABA and SPLI only N-methyl-D,L-aspartate and quinolinic acid had no significant effect on concentrations of SLI and NPYLI. The relative sparing of somatostatin-neuropeptide Y neurons was confirmed histologically by using histochemical staining for NADPH-diaphorase neurons combined with either Nissl stains, or immunohistochemical staining for enkephalin. Lesions with N-methyl-D-aspartate agonists resulted in preferential sparing of NADPH-diaphorase neurons while these neurons were more vulnerable than other neurons to kainic acid or AMPA. Choline acetyltransferase neurons were relatively spared, as compared with other neurons, by agents acting at all three glutamate receptor subtypes. N-methyl-D,L-aspartate lesions were blocked with MK-801, while there was no effect on quisqualic acid or kainic acid lesions. The relative sparing of somatostatin-neuropeptide Y neurons following striatal excitotoxin lesions with N-methyl-D-aspartate (AA1) agonists probably reflects a paucity of AA1 receptors on these neurons. Since these neurons are also spared in Huntington's disease, excitotoxins acting at the N-methyl-D-aspartate (AA1) site provide an improved neurochemical model of this illness.     

Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors

A series of ω-phosphono-α-car☐ylic acids were tested as antagonists of excitatory amino acid depolarizations and long-term potentiation (LTP) in region CA1 of rat hippocampal slices. The 5- and 7-phosphono compounds (±AP5and±AP7) blocked N-methyl-D-aspartate (NMDA) depolarizations and prevented the induction of LTP of the synaptic field potential and population spike components of the Schaffer collateral response.±AP5and±AP7 did not reduce kainate or quisqualate depolarizations and did not affect unpoten synaptic response amplitude.±AP5, ±AP6and±AP8 did not block amino acid excitant responses or LTP.These results demonstrate that NMDA receptors present in hippocampal region CA1 are not necessary for normal synaptic transmission, but are involved in the initiation of long-term synaptic plasticity.     

Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells

We examined the effects of riluzole, a neuroprotective drug, on voltage–dependent Na channels, nicotinic receptors, and voltage-dependent Ca channels, as well as catecholamine secretion, in comparison with those of verapamil and nicardipine, in primary cultures of bovine adrenal chromaffin cells. Riluzole inhibited veratridine-induced ²²Na influx via voltage-dependent Na channels even in the presence of ouabain, an inhibitor of Na,K-ATPase. Blockade of Na channels by riluzole was concentration-dependent with an IC₅₀ of 5.3 μM. It was associated with a similar concentration-related reduction of veratridine-induced ⁴⁵Ca influx via voltage-dependent Ca channels, and of catecholamine secretion. Riluzole had no effect on ⁴⁵Ca influx caused by high K, which directly activates voltage-dependent Ca channels, and on nicotine-induced ²²Na influx, which passes through the nicotinic receptors. Verapamil and nicardipine attenuated ²²Na influx caused by veratridine or nicotine at the same concentrations as they suppressed high K-induced ⁴⁵Ca influx. The inhibitory effect of riluzole on veratridine-induced ²²Na influx disappeared at high concentrations of veratridine. A potentiation of veratridine (site 2 toxin)-induced ²²Na influx caused by α-scorpion venom (site 3 toxin), β-scorpion venom (site 4 toxin), or brevetoxin PbTx-3 (site 5 toxin), occurred in the presence of riluzole in the same manner as in control cells. These results suggest that riluzole binds to the veratridine site in voltage–dependent Na channels. It does not impair the cooperative interaction between the functional peptide segments of Na channels, but selectively inhibits gating of Na channels, thereby reducing Ca influx via Ca channels and catecholamine secretion. In contrast, verapamil and nicardipine suppress Na influx both Na channels and nicotinic receptors. Received: 4 November 1997 / Accepted: 11 February 1998     

水溶性脂聚体介导NR2B siRNA治疗神经病理性痛的实验研究

目的 探讨水溶性脂聚体(WSLP)介导含2B亚基的N-甲基-D-天冬氨酸受体小干扰RNA(NR2B siRNA)治疗大鼠神经病理性痛的可行性.方法 健康雄性SD大鼠100只,周龄6周,体重180～200 g,采用随机数字表法,将大鼠随机分为5组(n=20):对照组(C组)、假手术组(S组)、神经病理性痛组(NP组)、WSLP-NR2B siRNA组(W组)、WSLP-阴性对照NR2B siRNA(WN组).采用坐骨神经分支部分结扎法制备大鼠神经病理性痛模型.C组不予任何处理;S组仅暴露坐骨神经,不牵拉和损伤神经;NP组于制备模型后即刻鞘内注射生理盐水20μl;W组和WN组于制备模型后即刻分别鞘内注射相应的siRNA 20μl.于模型制备前1 d及制备后3、7、14和21 d时测定机械缩足反应阈值(MWT)及热缩足反应持续时间(TWD),模型制备后3 d,痛阈测定结束后,每组取10只大鼠,取L4-6节段背根神经节,测定NR2B mRNA及其蛋白的表达水平.结果 与S组比较,NP组、W组和WN组MWT降低,TWD延长,NR2B mRNA及其蛋白表达上调(P＜0.05或0.01),C组上述指标差异无统计学意义(P＞0.05);与NP组比较,W组MWT升高,TWD缩短,NR2B mRNA及其蛋白表达下调(P＜0.01),WN组上述指标差异无统计学意义(P＞0.05).结论 WSLP不仅成功介导NR2B siRNA,抑制NR2B的表达,还可减轻大鼠神经病理性痛.

Abstract：

Objective To investigate the feasibility of NR2B small interference RNA(NR2B siRNA)carried by water-soluble lipopolymer(WSLP)for treatment of neuropathic pain in rats.Methods One hundred healthy male SD rats weighing 180-200 g were randomly divided into 5 groups(n=20 each):normal control group (group C),sham operation group(group S),neuropathic pain group(group NP),group WSLP-NR2B siRNA (group W)and group WSLP-negative NR2B siRNA(group WN).Neuropathic pain was induced by partial ligation of sciatic nenre.WSLP-NR2B siRNA complex was formed by binding WSLP and NR2B siRNA.Normal saline.WSLP-NR2B siRNA complex and WSLP-negative NR2B siRNA 20μl were injected intrathecally after operation in NP,W and WN groups respectively.Mechanical withdrawal threshold(MWT)and thermal withdrawal duration (TWD)were measured before(baseline)and at 3,7,14 and 21 days after operation.Ten animals in each group were sacrificed on the 3rd day after operation and the lumbar segment(L4-6)of the dorsal root ganglia was removed for determination of the expression of NR2B mRNA and protein using RT-PCR and Western blot analysis.Results Sciatic nerve ligation significantly decreased MWT and prolonged TWD and increased NR2B mRNA and protein expression in group NP as compared with group C.WSLP-NR2B siRNA complex significantly reduced sciatic nerve ligation-induced hyperalgesia and decreased NR2B mRNA and protein expression in group W as compared with group NP.Conclusion WSLP not only mediates NR2B siRNA successfully and inhibits the expression of NR2B,but also reduces neuropathic pain in rats.     

荛花酚对H2O2和兴奋性神经递质诱导的大鼠皮层神经元细胞损伤的保护作用

目的从中药祖师麻中发现有效的神经保护成分,阐明其治疗神经退行性疾病的药效物质基础。方法使用体外培养的大鼠皮层神经元细胞作为筛选系统,综合运用各种色谱技术进行活性跟踪指导下的分离,对发现的活性成分在体外细胞水平研究其神经保护机制。结果我们发现中药祖师麻的甲醇提取物可显著地减轻由谷氨酸（L-glutamate）、海人藻酸（kainic acid,KA）和过氧化氢（H2O2）诱导的大鼠皮层神经元细胞损伤。从祖师麻中分离纯化了一系列化合物,其中活性最强的是荛花酚（wikstromol）。荛花酚在50μM的浓度下可显著地降低H2O2诱导的细胞损伤。荛花酚显著地减少了细胞内谷胱甘肽、超氧化物歧化酶等抗氧化物质的下降,并减轻由兴奋性神经递质（谷氨酸、海人藻酸）诱导的神经毒性损伤,但不能减轻由N-甲基-D-天冬氨酸（NMDA）诱导的神经毒性损伤。结论本研究确证了荛花酚可作为一种天然神经细胞保护剂,推测其神经保护机制可能是通过抗氧化作用实现的。     

Adenosinergic system is involved in development of diazepam tolerance in mice

In the present study the effect of adenosinergic system on the development of diazepam tolerance to motor disturbances in mice was investigated. Diazepam tolerance was obtained by administration of diazepam at a dose of 5.0 mg/kg, s.c. for ten consecutive days. On the 1st and the 10th day of the experiment motor impairments were measured in two behavioural tests: rota-rod and chimney test. We showed that acute diazepam injection produced significant motor impairments in mice and that effect was decreased by repeated diazepam treatment, confirming the development of tolerance to the motor impairing effect of diazepam. We demonstrated that adenosine A₁ and/or A_2A receptor agonists: CPA (0.025 and 0.05 mg/kg, i.p.), CGS 21680 (0.1 and 0.2 mg/kg, i.p.), NECA (0.005 and 0.01 mg/kg, i.p.) pretreatment with diazepam were able to attenuate the development of diazepam tolerance and adenosine receptor antagonists: DPCPX (1.0 and 3.0 mg/kg, i.p.), DMPX (3.0 and 6.0 mg/kg, i.p.) and caffeine (10.0 and 20.0 mg/kg, i.p.) induced the opposite effect. The most apparent effects were obtained by non-selective agonist (NECA) and antagonist (caffeine) of adenosine receptors. We conclude that adenosinergic system plays an important role in mechanisms underlying the development of benzodiazepine tolerance.     

硫酸镁减轻瑞芬太尼诱发的术后痛觉过敏

目的：评价N-甲基-M-天冬氨酸（NMDA）受体拮抗剂硫酸镁对持续输注瑞芬太尼引起术后痛觉过敏的预防作用。方法：行脊柱侧弯矫形手术患者75名,年龄10～25岁,ASA分级I～II级,随机被分为3组,每组25例：S组（术中持续以0．05μg·kg^-1·min^-1速率输注瑞芬太尼）,R组（术中持续以0．4μg·kg^-1·min^-1速率输注瑞芬太尼）,M组（术中持续以0．4μg·kg^-1·min^-1速率输注瑞芬太尼,并在输注前给予硫酸镁40mg／kg静滴,继以20mg·kg^-1·h^-1的速率持续输注至术毕）。术后用芬太尼进行病人自控镇痛（PCA）。观察病人术后48h内（PCA）药液使用量和疼痛评分。结果：术后36h和48h内,R组病人的术后芬太尼需求量显著高于S组（P〈0．05）。术后24、36和48h内,M组病人的术后芬太尼需求量显著少于R组（P〈0．01）。术后3组病人疼痛评分无显著差别。结论：瑞芬太尼术中持续输注（0．4μg·kg^-1·min^-1）会诱发术后痛觉过敏,术中输注硫酸镁可以抑制瑞芬太尼导致的痛觉过敏。     

美金刚对缺血性血管性痴呆大鼠神经元谷氨酸能信号通路的影响

目的 研究美金刚对缺血性血管性痴呆(VD)大鼠神经功能的影响及作用机制.方法 70只SD大鼠随机均分为VD组、美金刚5 mg·kg-1 ·d-1组(M1组)、美金刚10 rng· kg-1 ·d-1组(M2组)、美金刚20 mg·kg1 ·d-1组(M3组)和假手术组(C组).术后4周,采用Morris水迷宫观察大鼠的空间学习能力,尼氏染色法观察海马组织形态学,免疫组化法检测谷氨酸2B受体(NR2B)免疫阳性细胞的表达,生化法检测海马组织匀浆的谷氨酸、一氧化氮含量及总一氧化氮合酶、诱导型一氧化氮合酶活性.结果 与C组相比,VD组学习记忆能力降低,CA3区椎体细胞数及CA1、CA3区NR2B细胞表达减少,谷氨酸含量增高,一氧化氮含量减少(P＜0.05或P＜0.01);而美金刚能改善VD组上述指标的变化,其中M3组最为显著(P＜0.05).结论 美金刚通过调节VD大鼠神经元谷氨酸能信号通路,从而改善慢性缺血导致的认知功能障碍.