“不完全氧糖剥夺”对A型氨基丁酸受体介导的抑制性突触后膜电流的增强作用

摘要: 氧糖剥夺模型作为研究脑缺血的离体模型被广泛使用,该模型模拟了局灶性脑缺血的病理变化。然而在缺血病灶中心区与正常脑组织之间的称为缺血半暗带的区域,脑血流也有程度不一的降低。为了模拟这种病理变化,我们发展了一种中等程度氧糖剥夺的离体脑片模型,该模型满足两个条件,氧气部分剥夺而葡萄糖完全剥夺。临床上通过增强A型γ-氨基丁酸受体介导的抑制活动可以阻止海马神经元的坏死,但其机制不清楚。因此我们采用全细胞膜片钳的记录方法,研究中等程度的氧糖剥夺对海马脑片CA1区神经元的A型γ-氨基丁酸受体介导的抑制性突触后膜电流(IPSCs)的影响。我们发现中等程度的氧糖剥夺使A型γ-氨基丁酸受体电流(IPSCs)的峰值增加而衰减时程延长。进一步研究发现该电流的峰值增加是由于A型γ-氨基丁酸受体－氯离子通道的电导增加所致,而与氯离子的反转电位无关。这些发现提示神经系统在中等程度脑缺血的早期阶段可以通过内稳态机制来维持兴奋性系统和抑制性系统之间的平衡。关键词: 中等程度脑缺血, A型γ-氨基丁酸受体, 抑制性突触后膜电位, 峰值     

尼氟灭酸对γ-氨基丁酸激活DRG神经元电流的作用

目的:观察尼氟灭酸(NFA)对大鼠背根神经节(DRG)神经元γ-氨基丁酸(GABA)激活电流的调制作用。方法:在新鲜分离的大鼠DRG神经元,应用全细胞膜片钳技术记录NFA和GABA激活电流。结果:部分DRG神经元(21/48,43.75%)外加NFA(0.1～100μmol/L)能引起浓度依赖性的外向电流,而大多数DRG神经元(150/159,94.32%)外加GABA(0.1～100μmol/L)则引起明显的浓度依赖性的内相电流。NFA-(100μmol/L)和GABA-(100μmol/L)激活电流的幅值分别是(0.27±0.06)nA(n=12)和(1.29±0.72)nA(n=53)。然而,预使用NFA(0.1～100μmol/L)能明显的抑制GABAA受体介导的内向电流。NFA的这一抑制作用也具有明显的浓度依赖性。但NFA没有改变GABA激活内向电流的EC50(大约30μmol/L)和翻转电位(大约-10mV)(P>0.05)。结论:预加NFA对GABA激活电流的峰值有明显的浓度依赖性的抑制作用。     

细胞外Ca2+对爪蟾脑片神经元微抑制性突触后电流的调制

应用盲法膜片钳全细胞记录技术,以爪蟾视顶盖神经元微抑制性突触后电流(miniature inhibitory postsyn-aptic currents,mIPSCs)为指标,观察了细胞外Ca^2 对爪蟾脑片神经元突触后mIPSC的调制。结果表明：用细胞外无钙或无钙含乙二醇双乙胺醚-N,N′-四乙酸(EGTA)(200nmol／L—2mmol／L)溶液灌流,均可使mIPSCs的发放频率降低;非特异性钙离子拮抗剂氯化铬(100μmol／L)也可使mIPSCs的频率降低;内质网钙泵抑制剂thapsigargin(TG)以及内质网ryanodine受体(RyR)激动剂ryanodine均可使mIPSCs频率升高,内质网RyR拮抗剂普鲁卡因则可降低mIPSCs的频率;磷脂酶C抑制剂U73122也可降低mIPSCs的频率,对三磷酸肌醇(inositol 1,4,5-triphosphate,IP3)水平有抑制作用的咖啡因亦可显著地降低mIPSCs,甚至完全抑制mIPSCs。从而表明：对突触前神经元及其末梢,细胞外钙离子可通过细胞膜上的钙通道进入细胞内,使细胞内钙浓度升高,突触前神经末梢释放出更多的神经递质。进而可能使突触后mIPSCs的频率增加;突触前细胞内钙储池上的Rya和IP3R均可介导钙从其中释放,并也可使突触前细胞内的钙离子浓度升高,进而可能使突触后mIPSCs的发放频率增加。     

血管紧张素1-7对氧糖剥夺/复氧海马神经元的保护作用

血管紧张素1-7（angiotensin 1-7, Ang1-7）在神经系统中发挥重要作用。已有研究发现,Ang1-7在脑缺血动物模型中发挥保护作用,但至今未见有关Ang1-7对氧糖剥夺/复氧（oxygen-glucose deprivation/ reoxygenation, OGD/R）损伤神经元的保护作用及其机制的研究报道。本研究以厌氧培养及不含葡萄糖的EBSS培养基培养、建立新生大白鼠原代培养的海马神经元OGD/R模型模拟脑缺血环境,实验分为3组：正常对照组、实验对照组和Ang1-7处理组。倒置显微镜观察神经元形态显示,Ang1-7处理组的神经元形态明显改善|CCK8试剂盒检测发现,Ang1-7处理组的细胞活性提高|流式细胞术研究发现,Ang1-7处理组的神经元凋亡和坏死率降低、神经元内Ca2+及NO水平降低|Western印迹结果发现,Ang1-7处理组Bax表达降低,Bcl-2表达增加。以上结果说明,Ang1-7可降低OGD/R神经元中NO和Ca2+水平,降低Bax蛋白、增加Bcl-2蛋白的表达,减少OGD/R神经元凋亡和坏死率,对OGD/R神经元发挥了保护作用。本研究为进一步在神经元水平上研究Ang-1-7的保护机制奠定基础,对中风等脑缺血疾病的防治具有重要意义。     

A型γ-氨基丁酸受体α1亚基Cys~(166)-Leu~(296)片段的配体结合和结构性质

研究A型γ 氨基丁酸受体 (γ aminobutyricacidtypeA ,GABAAreceptor)α1亚基Cys166 Leu2 96片段的苯并二氮杂 (benzodiazepine ,BZ)结合位点及其结构特性 ,了解该片段结构与功能的关系 .利用PfuDNA多聚酶依赖的点突变技术将该片段的每一残基用丙氨酸替代 ,通过E .coli体系过表达 ,纯化得到各种突变蛋白 .运用圆二色性 (circulardichroism ,CD)技术测定突变蛋白的二级结构 ,借助荧光各向异性 (fluorescenceanisotropy ,FA)、荧光共振能量转移 (fluorescenceresonanceenergytrans fer,FRET)技术测定其与BZ荧光配基Bodipy FLRo 1986 (BFR)的结合强弱 .通过与野生型的比较 ,确定其残基是否与结构和或结合相关 .结果显示 ,突变体R191A、G2 12A、S2 13A、R2 14A及V2 79A的结合能力减弱 2～ 3倍 ,除V2 79A显著增加α螺旋外均无二级结构的改变 .E193A、S2 78A、V2 79A和P2 80A的α螺旋显著增多 ,N2 75A和R2 76A的α螺旋则显著减少 .推测Cys166 Leu2 96的Arg191,Gly2 12 ,Ser2 13 和Arg2 14 可能位于BZ的结合袋 ,其第 4个环区 (Glu2 10 Asn2 16)与结合密切相关 .Glu193 、Ser2 78和Pro2 80 参与维持β折叠结构 ,而Asn2 75和Arg2 76参与维持α螺旋结构 .Cys166 Leu2 96的第 9个环区 (Asn2 75 Pro2 80 )对其结     

大鼠脑内γ-氨基丁酸对电针镇痛和吗啡镇痛的对抗作用

给200g 左右的大鼠腹腔注射γ-氨基丁酸(GABA)合成和释放抑制剂3-巯基丙酸(3MP)25、20和10mg/kg,可以加强电针和吗啡的镇痛作用。这种加强作用可被 GABA 降解酶抑制剂氨氧乙酸(AOAA)所对抗。腹腔注射 AOAA 25mg/kg 能提高脑内 GABA 含量,同时降低电针和吗啡的镇痛作用,这种降低作用可被 GABA 合成酶抑制剂异烟肼和 GABA 受体阻断剂氯甲基荷包牡丹碱所对抗。这些实验结果表明,脑内 GABA 能系统对于电针和吗啡镇痛具有对抗作用。文中讨论了 GABA 上述作用的可能机理。     

miR-182-5p靶向XBP1对氧糖剥夺/复氧诱导的神经细胞损伤的影响

该文探究mi R-182-5p靶向内质网应激相关蛋白剪接型X盒结合蛋白1(X-box bindingprotein1,XBP1)对氧糖剥夺/复氧(oxygen-glucosedeprivation/reoxygenation,OGD/R)诱导的神经细胞损伤的影响。选择大鼠神经细胞(PC-12)对研究对象,随机将其分为:Control组、OGD/R组、mi R-NC组(OGD/R+转染mi R-NC)、mi R-182-5pmimics组(OGD/R+转染mi R-182-5pmimics)、mi R-182-5pmimics+pc DNA-NC组(OGD/R+转染mi R-182-5pmimics+pc DNANC)、mi R-182-5pmimics+pc DNA-XBP1组(OGD/R+转染mi R-182-5pmimics+pc DNA-XBP1)。除Control组常规培养外,其余各组均进行OGD/R诱导。q RT-PCR法检测细胞mi R-182-5p、XBP1m RNA表达情况;CCK8检测细胞增殖情况;流式细胞仪检测细胞凋亡率;ELISA法检测细胞SOD、MDA、IL-6、TNF-α水平; Western blot检测细胞中PCNA、caspase-9、Bax、XBP1蛋白的表达情况;双荧光素酶实验检测mi R-182-5p与XBP1互作情况。结果显示,与Control组相比, OGD/R组mi R-182-5p表达、存活率、克隆数、SOD水平、PCNA蛋白表达水平降低(P<0.05),凋亡率、MDA、IL-6、TNF-α水平以及Bax、caspase-9、XBP1蛋白表达水平升高(P<0.05);与OGD/R组、mi R-NC组相比,mi R-182-5pmimics组mi R-182-5p表达、存活率、克隆数、SOD水平及PCNA蛋白表达水平升高(P<0.05),凋亡率、MDA、IL-6、TNF-α水平以及Bax、caspase-9、XBP1蛋白表达水平降低(P<0.05);与mi R-182-5p mimics组、mi R-182-5p mimics+pc DNA-NC组相比,mi R-182-5pmimics+pc DNA-XBP1组存活率、克隆数、SOD水平及PCNA蛋白表达水平降低(P<0.05),凋亡率、MDA、IL-6、TNF-α水平以及Bax、caspase-9、XBP1蛋白表达水平升高(P<0.05);与mi R-NC+XBP1-WT组相比,mi R-182-5pmimics+XBP1-WT组双荧光素酶活性明显降低(P<0.05)。总之,mi R-182-5p可能通过靶向抑制XBP1表达,进而抑制OGD/R诱导的神经细胞损伤。     

Inhibitory Postsynaptic Currents Induced by Activation of Interneurons in Cultured Rat Hippocampal Neurons

Using the patch-clamp technique in the whole-cell configuration, we studied the characteristics of a series of action potentials (APs) induced by a 500-msec-long current pulse applied to a pre-synaptic unit, as well as the kinetic characteristics of post-synaptic currents (PSCs) evoked by the APs in a post-synaptic unit, in synaptically connected pairs of cultured hippocampal neurons. Presynaptic inhibitory units were identified as GABA-ergic interneurons; they were divided into two groups according to the size of the soma and the number of processes. The kinetic characteristics of PSCs, which were induced in the post-synaptic neuron by a series of the APs generated in the pre-synaptic cell, demonstrated a certain dependence on the morphological characteristics of these cells. In interneurons with large-sized somata, the kinetics of the currents were more fast, and the reversal potential was close to the equilibrium Cl potential. In interneurons with small-sized somata, currents were slower, and the reversal potential was shifted. We conclude that under conditions of culturing, a pre-synaptic cell not only directly provokes the development of PSC in a post-synaptic neuron and determines the amplitude of this current but also significantly influences the kinetics of this current. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 116–123, March–April, 2005.     

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A(2) (PLA(2))/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca(2+)-mobilization and enhanced bradykinin-promoted Ca(2+)-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPARgamma agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.     

Partial site-specific assignment of a uniformly C, N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR

Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz ¹⁵N-¹³CO-¹³Cα and ¹⁵N-¹³Cα-¹³CX correlation spectra, using uniformly ¹³C, ¹⁵N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic ¹⁵N-¹³Cα-¹³Cβ side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively ¹³C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Cα and Cβ resonances indicated the presence of typical α-helical secondary structure, consistent with previous studies.     

Enhancement of lignan biosynthesis in suspension cultures of <Emphasis Type="Italic">Linum nodiflorum</Emphasis> by coronalon,indanoyl-isoleucine and methyl jasmonate

The effect of the two synthetic elicitors coronalon and indanoyl-isoleucine and of methyl jasmonate (MeJA) on the accumulation and biosynthesis of lignans by cell suspension cultures of Linum nodiflorum (Linaceae) was investigated. The production of 6-methoxypodophyllotoxin (MPTOX) could be increased more than tenfold, the maximal content reaching up to over 2.5% of the cell dry weight. The highest yield was achieved by administering 50 μM of the synthetic elicitors on the fourth day and extracting the products on the tenth day of the culture period. An additional lignan accumulated in elicitor-treated cultures. Its structure was elucidated by extensive 1D and 2D NMR measurements, revealing its identity as 5′-demethoxy-MPTOX (5′-dMPTOX). Its average content amounted up to over 5% of the cell dry weight. Growth was only slightly affected by the addition of the elicitors. Methyl jasmonate exerted a moderate stimulating effect on the L. nodiflorum cells with MPTOX and 5′-dMPTOX contents going up to 1.4 and 2.1% of the cell dry weight, respectively. The activities of deoxypodophyllotoxin 6-hydroxylase and β-peltatin 6-O-methyltransferase, two enzymes involved in MPTOX biosynthesis, were increased up to 21.9-fold and 14.6-fold, respectively, in the treated cultures.