神经生长因子对小鼠突触体内Ca2+水平的调节作用

观察了多次海马内微注射NGF对小鼠突触体内游离钙水平的影响,并在离体情况下观察NGF对EGTA和CaCl₂分别造成突触体内低钙和高钙状态的调节作用。结果如下:(1)在体实验表明,一定剂量的NGF可显著降低老年小鼠海马突触体内游离钙水平(P<0.05);(2)离体实验表明,当突触体游离钙水平降低时,适当剂量的NGF具有升高游离钙水平的作用;而突触体内游离钙水平升高时,则NGF有降低游离钙水平的作用。提示NGF对游离钙水平的双向调节作用可能是NGF改善老年性记忆衰退的作用机制。     

AMG－1对突触体内游离钙水平及大鼠尾动脉收缩力的影响

观察了ＡＭＧ－１对高钾所致大鼠脑突触体内游离钙浓度［Ｃａ２＋］ｉ升高，及去甲肾上腺素引起的离体大鼠尾动脉环收缩力的影响。结果表明，ＡＭＧ－１１０和１００μｍｏｌ·Ｌ－１可对抗高钾（３０ｍｍｏｌ·Ｌ－１）引起的［Ｃａ２＋］ｉ升高，使分别降低１９±１１％及５７±１２％；在无钙Ｋｒｅｂｓ－Ｈｅｎｓｌｅｌｅｉｔ液中，ＡＭＧ－１能抑制去甲肾上腺素引起大鼠尾动脉收缩力的作用。     

海马内钙离子水平对小鼠记忆巩固的影响

采用一次性被动回避反应模型，观察了海马（双侧）定位注射ＣａＣｌ２（２．９μｇ·只－１）和ＥＧＴＡ（１ｎｇ·只－１）对记忆巩固过程的影响。电击（０．３ｍＡ，５ｓ）后立即注射上述化合物，２４ｈ后检测，并运用Ｃａ２＋指示剂Ｆｕｒａ－２作为细胞内钙离子的荧光探针，采用精密的ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统，检测了分离的单个神经细胞内游离钙及其变化。结果表明，与生理盐水对照组相比，海马内注射ＣａＣｌ２或ＥＧＴＡ都使小鼠的步入潜伏期缩短，说明记忆保持下降。以３Ｈ－亮氨酸为标记物进行的高体实验表明：ＣａＣｌ２或ＥＧＴＡ都使３Ｈ－亮氨酸参入海马突触体蛋白质的总量降低，但从ＳＤＳ－聚丙烯酰胺凝胶电泳条带来看，受这两种化合物影响的突触体蛋白质分子量范围不完全一致。提示海马内游离Ｃａ２＋水平过高或过低都能损害记忆巩固过程，其作用机理与Ｃａ２＋影响突触体蛋白质合成有关。     

缺氧缺糖条件下大鼠脑皮质神经元内游离钙浓度的变化及神经生长因子的作用

以Ｆｕｒａ２／ＡＭ为细胞内钙离子的荧光指示剂，用双波长荧光分光光度计测定了缺氧缺糖时体外培养的大鼠胎鼠神经细胞内游离钙（［Ｃａ２＋］ｉ）的变化，并观察了神经生长因子（ＮＧＦ）的影响。结果表明，脑皮质细胞缺氧缺糖培养１６～２４ｈ时，细胞大量死亡。ＮＧＦ剂量依赖地减少神经元缺氧缺糖培养２４ｈ时乳酸脱氢酶（ＬＤＨ）的释放，提高细胞生存力。细胞缺氧缺糖早期引起［Ｃａ２＋］ｉ减少，而后期引起［Ｃａ２＋］ｉ显著升高，导致细胞损害。ＮＧＦ５０μｇ·Ｌ－１在缺氧缺糖早期提高［Ｃａ２＋］ｉ到正常水平，降低后期［Ｃａ２＋］ｉ的升高。提示，ＮＧＦ通过稳定［Ｃａ２＋］ｉ或降低后期的胞内钙升高保护了脑皮质神经元免受缺氧缺糖的损害。     

Role of tumor necrosis factor and nitric oxide in the protection of N-methyl-N-(3,4-methylenedioxybenzoyl) methyl-acetamide against immunological liver injury in mice

研究了Ｎ－甲基－Ｎ－（３，４－亚甲二氧基苯甲酰）甲基－乙酰胺（ＳＹ－６４０）的保肝作用及其机理．给小鼠ｉｖ活卡介苗（ＢＣＧ）１２ｄ后再ｉｖ脂多糖（ＬＰＳ）诱导小鼠血浆一氧化氮（ＮＯ），肿瘤坏死因子（ＴＮＦ），谷丙转氨酶（ＧＰＴ），谷草转氨酶（ＧＯＴ）剧烈升高及严重的肝损伤．以ＳＹ－６４０给小鼠ｉｇ（每日一次，连续１０ｄ），显著降低ＢＣＧ＋ＬＰＳ诱导的肝损伤小鼠血浆ＧＰＴ，ＧＯＴ和ＴＮＦ水平的升高，血浆ＮＯ水平的升高更加显著，肝损伤减轻．以单甲基精氨酸抑制ＮＯ的生成，ＳＹ－６４０的上述作用被抵消．ＳＹ－６４０对正常小鼠血浆ＮＯ，ＧＰＴ，ＧＯＴ水平无影响．可见，ＳＹ－６４０的保肝作用与其升高血浆ＮＯ，降低血浆ＴＮＦ水平有关．     

神经生长因子改善衰老性记忆障碍及突触机制的探讨

目的　探讨神经生长因子(NGF)改善衰老性记忆障碍的机制。方法　采用开场行为和一次性被动回避反应模型,观察海马内微量注射NGF对衰老小鼠自发活动和记忆巩固过程的影响, 同时用Ca²⁺荧光探针Fura-2/AM和AR-CM-MIC阳离子测定系统测定海马突触体游离钙水平,以³H-Leu为标记物测定海马突触体总蛋白的合成量。结果　NGF使衰老小鼠在新异环境中的自发活动和探究行为明显增多,并显著延长电击后24 h的步入潜伏期(STL); NGF显著降低衰老小鼠海马突触体内的高钙水平,并使海马突触体蛋白质的³H-Leu参入量显著增加。结论　NGF改善衰老性记忆障碍与其降低海马突触体内高钙水平,并由此促进海马突触体蛋白质的合成有关。     

神经生长因子在培养的皮质神经细胞中抑制谷氨酸毒性

目的：研究ＮＧＦ是否防止原代培养的神经细胞中谷氨酸引起的损伤，方法：采用皮质神经细胞体外培养及形态学观察，测定神经细胞的生存力和ＬＤＨ的释入来分析ＮＧＦ的作用，并利用钙指示剂Ｆｕｒａ－２来检测（Ｃａ＾２＋）的变化，结果：ＮＧＦ阻止（Ｃａ＾２＋）的增加，并且对谷氨酸引起的皮质细胞的损伤具有结拮抗作用，最大拮抗剂量为６０μｇ．Ｌ＾－１，结论：ＮＧＦ通过稳定（Ｃａ＾２＋）水平，或阻止（Ｃａ＾２＋）的升高     

褪黑激素对小鼠脑细胞内游离钙浓度的抑制作用

目的 研究褪黑激素（Ｍｅｌ）对老年小鼠大脑皮层突触体内钙含量以及激动剂动诱发的新生小鼠脑细胞（Ｃａ＾２＋）升高的影响,以探讨Ｍｅｌ抗衰老的作用机理。方法 钙离子荧光染料Ｆｕｒａ－２ＡＭ负载已制备的突触体或细胞,ＲＦ－５０００型双波长荧光分光光度计测定（Ｃａ＾２＋）ｉ,结果：长期使用Ｍｅｌ抑制老年小鼠大脑皮层Ｃａ＾２＋超负荷,Ｍｅｌ也降低钙通道激活剂Ｂａｙ－Ｋ８６４４,高浓度氯化钾（ＫＣｌ）和谷氨酸     

钙拮抗剂TMB-8抑制BHQ,NE和KCl引起的培养乳牛基底动脉单个平滑肌细胞内游离钙的升高

用ＡＲＣＭＭＩＣ阳离子测定系统，测量单个细胞内游离钙浓度（［Ｃａ２＋］ｉ），研究８（Ｎ，Ｎ二乙胺）ｎ辛基３，４，５三甲氧基苯甲酸酯（ＴＭＢ８）对培养乳牛基底动脉平滑肌［Ｃａ２＋］ｉ的作用。在细胞外钙浓度为１３ｍｍｏｌ·Ｌ－１时，ＴＭＢ８（３０μｍｏｌ·Ｌ－１）可明显抑制ＢＨＱ，ＮＥ及ＫＣｌ引起［Ｃａ２＋］ｉ的升高。在细胞外钙为零＋ＥＧＴＡ０１ｍｍｏｌ·Ｌ－１时，ＴＭＢ８（１０，３０及１００μｍｏｌ·Ｌ－１）可浓度依赖性地降低静息［Ｃａ２＋］ｉ，ＴＭＢ８（３０μｍｏｌ·Ｌ－１）可几乎完全阻断ＢＨＱ及ＮＥ引起［Ｃａ２＋］ｉ的增加。研究表明ＴＭＢ８降低培养乳牛基底动脉平滑肌［Ｃａ２＋］ｉ的机制，主要是抑制肌浆网Ｃａ２＋的释放，或增加肌浆网对Ｃａ２＋的摄入，并由此间接地抑制细胞外钙的内流。     

Inhibitory effects of melatonin on free intracellular calcium in mouse brain cells

目的：研究褪黑激素（Ｍｅｌ）对老年小鼠大脑皮层突触体内钙含量以及激动剂诱发的新生小鼠脑细胞［Ｃａ２＋］ｉ升高的影响,以探讨Ｍｅｌ抗衰老的作用机理．方法：钙离子荧光染料Ｆｕｒａ２ＡＭ负载已制备的突触体或细胞,用ＲＦ５０００型双波长荧光分光光度计测定［Ｃａ２＋］ｉ．结果：长期使用Ｍｅｌ抑制老年小鼠大脑皮层Ｃａ２＋超负荷,Ｍｅｌ也降低钙通道激活剂ＢａｙＫ８６４４,高浓度氯化钾（ＫＣｌ）和谷氨酸钠诱发的分离的新生小鼠脑细胞［Ｃａ２＋］ｉ升高．结论：Ｍｅｌ对中枢神经元［Ｃａ２＋］ｉ超载的抑制作用与其抗衰老作用有关．     

Effect of ethanol on phospholipid turnover and calcium mobilization in chick embryos

Effects of ethanol on [3H]inositol and [14C]choline incorporation into phosphatidylinositol (PI) and phosphatidylcholine (PC), free intrasynaptosomal Ca2+ ([Ca2+]i) and synaptosomal 45Ca2+ uptake, were investigated in the brain and heart of 17-day-old chick embryos to which a 10% ethanol solution had been injected on the 3rd day of embryogenesis. In brain synaptosomes, ethanol increased the incorporation of [3H]inositol and [14C]choline into PI and PC, increased [Ca2+]i, and decreased 45Ca2+ uptake. On the other hand, in heart synaptosomal membrane, ethanol decreased the incorporation of [3H]inositol and [14C]choline into PI and PC, decreased [Ca2+]i, and increased 45Ca2+ uptake. Ethanol stimulated in vitro [3H]inositol and [14C]choline incorporation into PI and PC in the brain and heart in both the control and ethanol-treated groups. However, addition of ethanol did not affect the release of 45Ca2+ from the synaptosomal membrane of either organ in either group. Addition of ethanol inhibited 45Ca2+ uptake in a dose-dependent manner in the brain but not in the heart. In both organs, there was a relationship between phospholipid turnover and [Ca2+]i after ethanol.     

Effect of ethanol on calcium-uptake and phospholipid turnover by stimulation of adrenoceptors and muscarinic receptors in mouse brain and heart synaptosomes

The effect of ethanol treatment on mouse brain and heart synaptosomal 45Ca uptake and the incorporation of [3H]inositol and [14C]choline into phosphatidylinositol (PI) and phosphatidylcholine (PC) were investigated. Ethanol in drinking water (15%) was given to mice for 3 weeks. The consumption of ethanol increased gradually during treatment but food intake was almost the same as control. The body weight of ethanol-treated mice was slightly less than that of control. The synaptosomal lipid peroxidation level of ethanol-treated mice was almost the same as control in the brain and heart. On the other hand, the synaptosomal glutathione level of ethanol-treated mice was higher than control in both brain and heart. The 45Ca uptake of brain and heart from ethanol-treated mice was 87% and 216% of control mice, respectively. Not only ethanol but also norepinephrine (NE), carbachol (Carb), or isoproterenol (IsoPro) added in vitro increased 45Ca uptake in all cases. The incorporation of [3H]inositol into PI in the brain and heart synaptosomes of ethanol-treated mice was 150% and 113% of control, respectively. The incorporation of [14C]choline into PC in the brain and heart of ethanol-treated mice was 104% and 125% of control, respectively. In vitro addition of ethanol, NE, Carb or IsoPro to brain synaptosomes increased the incorporation of [3H]inositol and [14C]choline into PI and PC, respectively, in both control and ethanol-treated mice. In the case of heart synaptosomes, NE and Carb increased the incorporation of [3H]inositol and [14C]choline into phospholipids in control mice but not ethanol-treated mice. However, IsoPro increased the incorporation by both control and ethanol-treated heart synaptosomes. These results suggest that alpha-adrenoceptors and the cholinergic system of the heart play important roles in modulating the toxic effects of ethanol.     

脑室注射NGF对大鼠学习记忆以及自发行为的影响

目的：观察脑室注射神经生长因子（NGF）对大鼠学习记忆以及自发行为的影响。方法：以18月龄大鼠为材料，侧脑室多次注射NGF（累计剂量55μg·只(-1)）．同龄对照组注射等体积人工脑积液（ACSF）。采用开场行为和Y-迷宫实验检测了大鼠自发活动和学习记忆能力的改变。行为实验后分离主要脑区（皮层、海马、小脑、间脑）的突触体，使用3H－Leu为标记物检测了离体突触体总蛋白的合成量。结果：NGF能提高大鼠的自发活动和学习记忆能力，并能提高与学习、运动相关脑区的突触体蛋白质合成。结论：提示NGF对大鼠自发活动和学习记忆的影响与学习、运动相关脑区的突触部位蛋白质合成能力提高有关。     

Protective effect of nerve growth factor on neurons after traumatic brain injury

The protective effect of nerve growth factor (NGF) on neurons after traumatic brain injury (TBI) was investigated. A brain trauma model of fluid-percussion in rats was established, and 7s NGF was infused continuously in its cerebral ventricle. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and [Ca2+]i overloading in brain tissues was observed after giving exogenous NGF postinjury. We found that the activity of SOD, GSH-Px, and CAT was markedly higher in NGF-treated group than in the simple trauma group (P < 0.01). Although the level of [Ca2+]i in the NGF-treated group increased, the value was significantly lower than that in the simple trauma and control groups (P < 0.01). These findings suggest that exogenous NGF can (a) increase the activity of the major antioxidant enzymes in brain tissues and attenuate the injuries to neurons induced by oxygen-free radicals, (b) reduce the severe overload of [Ca2+]i and stabilize its homeostasis, and (c) provide clear protective effects on neurons after traumatic brain injury.     

缺氧缺糖条件下大鼠脑皮质神经元内游离钙浓度的变化及神经生长因子的作用

以Fura-2/AM为细胞内钙离子的荧光指示剂,用双波长荧光分光光度计测定了缺氧缺糖时体外培养的大鼠胎鼠神经细胞内游离钙([Ca²⁺]i)的变化,并观察了神经生长因子(NGF)的影响。结果表明,脑皮质细胞缺氧缺糖培养16～24h时,细胞大量死亡。NGF剂量依赖地减少神经元缺氧缺糖培养24h时乳酸脱氢酶(LDH)的释放,提高细胞生存力。细胞缺氧缺糖早期引起[Ca²⁺]i减少,而后期引起[Ca²⁺]i显著升高,导致细胞损害。NGF50μg·L^-1在缺氧缺糖早期提高[Ca²⁺]i到正常水平,降低后期[Ca²⁺]i的升高。提示,NGF通过稳定[Ca²⁺]i或降低后期的胞内钙升高保护了脑皮质神经元免受缺氧缺糖的损害。     

Cellular mechanisms of the acute increase of glutamate release induced by nerve growth factor in rat cerebral cortex

Nerve growth factor (NGF) was found to increase glutamate release in the developing visual cortex. We investigated the cellular mechanisms of this effect and its dependence on extracellular and intracellular Ca2+. The NGF-induced enhancement of glutamate release from superfused rat visual cortex synaptosomes required mild depolarization. Removal of external Ca2+ during depolarization with 15 mM K+ only halved the effect of NGF on glutamate release. NGF increased [Ca2+]i in K+-depolarized synaptosomes preloaded with fura-2AM both in the presence and in the absence of external Ca2+. The effects of NGF on glutamate release and [Ca2+]i elevation were prevented by an anti-TrkA receptor monoclonal antibody. NGF increased synaptosomal inositol (1,4,5)-triphosphate (InsP3) during depolarization and the InsP3 receptor antagonist heparin abolished the effect of NGF on evoked glutamate release both in the presence and in the absence of external Ca2+. The effect of NGF on the evoked glutamate release in Ca2+-free medium was abolished by dantrolene, a ryanodine receptor blocker, by CGP 37157, a blocker of the mitochondrial Na+/Ca2+ exchanger and by pretreatment of synaptosomes with caffeine. NGF significantly increased the depolarization-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the subsequent phosphorylation of synapsin I in the absence of external Ca2+ and the NGF effect on evoked glutamate release was inhibited by the CaMKII inhibitors KN-93 and CaMKII 281-309 peptide but not by the MAP kinase inhibitor PD 98059. Thus, the effect of NGF on evoked glutamate release is linked to an increase in [Ca2+]i contributed by both Ca2+ entry and mobilization from InsP3-sensitive, ryanodine-sensitive and mitochondrial stores and to the subsequent activation of CaMKII.     

三氟拉嗪对大鼠脑突触体内Ca2＋水平和Ca2＋，Mg2＋─ATP酶活性的作用

用Ｑｕｉｎ２法测得大鼠脑突触体内静息游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）为８５±１３μｍｏｌ·ｇ－１ｐｒｏｔｅｉｎ．三氟拉嗪（ＴＦＰ）１，５和１０μｍｏｌ·Ｌ－１对静息突触体［Ｃａ２＋］ｉ无明显影响，但能以剂量依赖方式增高６５ｍｍｏｌ·Ｌ－１ＫＣｌ所致突触体［Ｃａ２＋］ｉ升高，从１９２±５８μｍｏｌ·ｇ－１ｐｒｏｔｅｉｎ分别达到２３３±６３，４３１±９９和６６１±１７３μｍｏｌ·ｇ－１ｐｒｏｔｅｉｎ．ＴＦＰ５，１０和５０μｍｏｌ·Ｌ－１分别使突触体Ｃａ２＋，Ｍｇ２＋－ＡＴＰ酶活性降低３１％，４１％和４５％；使Ｍｇ２＋－ＡＴＰ酶活性降低３０％，３６％和３９％，提示ＴＦＰ可能是通过抑制钙调素，进而抑制Ｃａ２＋，Ｍｇ２＋－ＡＴＰ酶活性，使突触体［Ｃａ２＋］ｉ升高，促进神经末梢释放递质     

钙调素拮抗剂E6对大鼠神经细胞内钙的影响

目的　观察钙调素拮抗剂E6对神经细胞静息[Ca2 +]i、KCl (5 0mmol·L-1)和谷氨酸 (glutamicacid ,Glu ,0 1mmol·L-1)引起的神经细胞 [Ca2 +]i 升高的影响。方法　用Ca2 +敏感荧光指示剂Fura 2 /AM负载大鼠脑细胞 ,测定神经细胞内游离Ca2 +浓度 ([Ca2 +]i)。结果　E6可升高神经细胞静息 [Ca2 +]i,EC50 为 6 6 8μmol·L-1;无外Ca2 +条件下 ,也升高 [Ca2 +]i,EC50 为 1 30 μmol·L-1,说明E6主要是通过促进细胞内贮存Ca2 +释放引起内Ca2 +升高。E6抑制KCl升高细胞 [Ca2 +]i 的IC50 为 6 0 μmol·L-1;抑制Glu激发的细胞 [Ca2 +]i 升高的IC50 为 0 15 μmol·L-1。结论　E6可能是通过与细胞内钙调素 (Calmodulin ,CaM)结合后 ,影响兴奋性氨基酸受体的开放和电压依赖性钙通道的活性状态 ,表现出对由去极化或受体激动剂引起的内Ca2 +升高的抑制作用