中枢神经系统钙稳态失调和老龄脑功能

脑的老化表现为记忆力的减退。脑老化的钙假说认为脑的老化与中枢神经系统「Ｃａ＾２＋」ｉ的调节机制紊乱有关,衰老可以通过多种因素导致「Ｃａ＾２＋」ｉ升高,影响突触传导,神经递质释放,信号转导等导致记忆障碍,本文综述了近年来的进展。     

血小板激活时胞质游离钙浓度与两种颗粒数变化的相关性

用电镜形态计量法检测血小板α颗粒（αＧ）和致密颗粒（ｄＧ）的数密度,用钙荧光指示剂Ｆｕｒａ２检测血小板胞质游离Ｃａ＾２＋浓度（「Ｃａ＾２＋」ｉ）,观察到在钙离子导体Ａ２３１８７作用下,血小板「Ｃａ＾２＋」ｉ明显升高。凝血酶与ＡＤＰ也都分别引起「Ｃａ＾２＋」ｉ升高,且有浓度依赖性,选用三种激动剂的不同量以反映血小板不同程度激活时,测定「Ｃａ＾２＋」与颗粒数密度,分析两者间的相关性,发现αＧ和ｄＧ的数     

氨对脑细胞胞浆游离钙含量的影响

目的与方法：采用Ｆｕｒａ－２／ＡＭ探针技术观察ＮＨ４Ｃｌ对离体急性分离之Ｗｉｓｔａｒ乳鼠大脑细胞胞头游离钙「Ｃａ＾２＋」ｉ含量的影响。结果：ＮＨ４＾＋浓度为２．５ｍｍｏｌ／Ｌ时脑细胞内「Ｃａ＾２＋」ｉ含量升高。在一定范围内,随着ＮＨ４＾＋浓度的加大,细胞内Ｃａ＾２＋持续升高。ＮＨ４＾＋的升钙作用主要被Ｎｉｃａｒｄｉｐｉｎｅ所阴断,其变化特征类以ＫＣｌ。结论：ＮＨ４＾＋主要通过影响电压依赖性钙离子通     

内皮素对培养心肌细胞内游离钙浓度的作用

实验用培养新生ＳＤ大鼠心室肌细胞,以Ｆｕｒａ－２／ＡＭ荧光指示剂负载检测收肌细胞内游离钙浓度（「Ｃａ＾２＋」）的变化,探讨内皮素－１（ＥＴ－１）对「Ｃａ＾２＋」ｉ的作用及其机制。结果显示：ＥＴ－１引起心肌细胞「Ｃａ＾２＋」ｉ升高有两个时相,瞬时相持续相。ＥＴ－１诱导的瞬时相「Ｃａ＾２＋」ｉ升高呈浓度依赖性,预先用ＥＴＡ特异性受阻断剂ＢＱ１２３处理,可阻断ＥＴ－１引起的「Ｃａ＾２＋」ｉ升高,揭示上述     

衰老性记忆障碍与脑突触体内游离[Ca~(2 )]_i的相关性

采用行为观察和生化检测相结合的方法 ,在过去工作的基础上 ,研究了 12月龄和 18月龄小鼠学习记忆能力的变化和 18月龄小鼠四个脑区 (海马、大脑皮层、四叠体和小脑 )突触体内 [Ca2 ]i 的水平 ,同时还比较了老年记忆保持良好组与记忆障碍组小鼠的脑钙水平。结果表明 ,随着年龄的增长 ,小鼠的学习记忆能力显著下降 ,上述脑区 (除大脑皮层外 )突触内 [Ca2 ]i 均明显升高 ,其中老年记忆障碍小鼠脑钙水平升高最为显著。提示 ,小鼠衰老性记忆障碍可能与其脑突触体内 [Ca2 ]i 的超载有关。     

两组不同月龄小鼠学习记忆能力与脑突触体内游离[Ca^2＋]i的相关性

本文采用Ｙ－迷宫和一次被动回避反应模型,观察了１,６月龄小鼠的学习记忆行为,并运用Ｃａ~（２＋）的荧光探针和ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统,检测了两组小鼠四个主要脑区（海马、皮层、小脑、中脑四叠体）突触体内游离钙离子浓度（［Ｃａ~（２＋）］_ｉ）的变化。结果表明随着年龄的增长,小鼠的分辨学习及记忆能力均下降,同时海马突触体内游离［Ｃａ~（２＋）］_ｉ升高显著。     

α1-肾上腺素受体亚型介导细胞内游离Ca2+增高的信号转导途径

本文探讨了α１ａ,α１ｂ,α１ｄ三种亚型肾上腺素受体激动时细胞内Ｃａ６２＋浓度升高的信号转导途径。在稳定表达三亚型α１－ＡＲ的ＨＥＫ２９３细胞２系中,用ｆｕｒａ－２方法细胞内Ｃａ＾２＋信号强弱的变化。结果显示,百日咳毒素对去甲肾上腺素激动三亚型α１－ＡＲ而引起的「Ｃａ＾２＋」ｉ升高无影响,Ｕ－７３１２２和ＰＭＡ明显抑制「Ｃａ＾２＋」ｉ升高．     

M型受体在大鼠肾上腺嗜铬细胞内钙库释放和激素分泌中的作用

在单个大鼠肾上腺嗜铬细胞上,用显微荧光测量和碳纤电极记录方法,测量可激活毒蕈碱（ｍｕｓｃａｒｉｎｅ,Ｍ）受体的激动剂乙酰甲胆碱（ｍｅｔｈａｃｈｏｌｉｎｅ,ＭＣｈ）对胞内游离钙浓度「Ｃａ＾２＋」ｉ和儿茶酚胺激素分泌的影响。在细胞外液含２ｍｍｏｌ／Ｌ Ｃａ＾２＋时,用含钙或不含钙的ＭＣｈ（１ｍｍｏｌ／Ｌ）刺激细胞,均引起「Ｃａ＾２＋」ｉ的升高或钙振荡,并诱发激素的分泌。     

茶碱改善东莨菪碱诱发的大鼠记忆障碍

用高效液相色谱测定了不同年龄ＳＤ大鼠与记忆有关脑区的腺苷和乙酰胆碱水平。结果表明,１８～２０月龄鼠的脑内腺苷含量明显高于３～６月龄鼠,而乙酰胆碱（ＡＣｈ）含量却显著低于３～６月龄鼠。经腹腔给大鼠注射东莨宕碱建立近期记忆障碍模型,同时经脑室给予茶碱后,其跳台成绩明显对照组,且脑内ＡＣｈ含量亦显著升高。提示腺苷含量的随龄增加可能是老年记忆障碍的一个重要因素,茶碱作为腺苷受体阻断剂可能通过提高脑内ＡＣｈ     

应用流式细胞仪研究抗IL——8单抗对IL——8激活人颗粒细胞活力的中和作用

以ＩＬ－８免疫的ＢＡＬＢ／Ｃ小鼠脾细胞与Ｓｐ２／０或６５３小鼠骨髓瘤细胞融合构建了淋巴细胞杂交瘤克隆Ｉ８－Ｓ２和Ｉ８－６３。ＥＬＩＳＡ叠加试验（ＥＬＩＳＡ Ａｄｄｉｔｉｖｉｔｙ Ｔｅｓｔ）表明这两杂交瘤克隆分泌的单抗分别识别ＩＬ－８分子的不同表位。ＩＬ－８能激活人颗粒细胞,引起细胞内Ｃａ＾２＋浓度（「Ｃａ＾２＋」）上升。通过流式细胞仪分析「Ｃａ＾２＋」的变化,发现两个克隆单抗对ＩＬ－８激活细胞的活     

Na(+)/Ca(2+) exchanger 2 is neuroprotective by exporting Ca(2+) during a transient focal cerebral ischemia in the mouse

Na(+)/Ca(2+) exchanger (NCX), by mediating Na(+) and Ca(2+) fluxes bi-directionally, assumes a role in controlling the Ca(2+) homeostasis in the ischemic brain. It has been suggested that the three isoforms of NCX (NCX1, 2 and 3) may be differentially involved in permanent cerebral ischemia. However, the role of NCX2 has not been defined in ischemic reperfusion injury after a transient focal cerebral ischemia. Furthermore, it is not known whether NCX2 imports or exports intracellular Ca(2+) ([Ca(2+)](i)) following ischemia and reperfusion. To define the role of NCX2 in ischemia and reperfusion, we examined mice lacking NCX2, in vivo and in vitro. After an in vitro ischemia, a significantly slower recovery in population spike amplitudes, a sustained elevation of [Ca(2+)](i) and an increased membrane depolarization were developed in the NCX2-deficient hippocampus. Moreover, a transient focal cerebral ischemia in vivo produced a larger infarction and more cell death in the NCX2-deficient mouse brain. In particular, in the wild type brain, NCX2-expressing neurons were largely spared from cell death after ischemia. Our results suggest that NCX2 exports Ca(2+) in ischemia and thus protects neuronal cells from death by reducing [Ca(2+)](i) in the adult mouse brain.     

Mutual Antagonism of κ-Opiate and α2-Adrenoceptor Agonist Effects on Intrasynaptosomal Free [Ca2+]i

Synaptosomes prepared from rat cerebral cortices on Percoll discontinuous density gradients were loaded with the fluorescent EGTA analogue Quin 2 to allow measurement of intracellular free [Ca2+]i. When either kappa-opiate or alpha 2-adrenoceptor agonists were incubated with the synaptosomes, there was a highly significant (p less than 0.004, p less than 2.7 X 10(-6), respectively) reduction in intrasynaptosomal free [Ca2+]i relative to controls. As these synaptosomes are not depolarised, the data suggest that both alpha 2-adrenoceptor agonists and kappa-opiate agonists inhibit neurotransmitter release, decreasing the availability of intraneuronal [Ca2+]i rather than altering Ca2+ entry. However, when these two agonists were coincubated, there was a complete abolition of the effects of either agonist; in fact, there was an apparent increase in the intrasynaptosomal free [Ca2+]i. Neither morphine nor [D-Ala2-D-Leu5]enkephalin, mu and delta opiate agonists respectively, had any significant effect on intrasynaptosomal free [Ca2+]i. These results show that the individual effects of clonidine and dynorphin A1-13 are in keeping with the role of these substances at autoreceptors controlling neurotransmitter release. The mutual antagonism of their effects on [Ca2+]i is more difficult to explain but it may be a mechanism that prevents the occurrence of excessive inhibition of neuronal systems.     

External calcium, intrasynaptosomal free calcium and neurotransmitter release

In a physiological medium the resting membrane potential of synaptosomes from guinea-pig cerebral cortex, estimated from rhodamine 6G fluorescence measurements, was nearly -50mV. This agreed with calculations using the Goldman-Hodgkin-Katz equation. With external [Ca2+] less than or equal to 3 mM veratridine depolarisation (to -30 mV) was accompanied by increases in intrasynaptosomal free calcium concentrations (monitored by entrapped quin2) and parallel increases in total acetylcholine release. With external [Ca2+] greater than 3 mM both intrasynaptosomal free calcium concentrations and transmitter release were paradoxically reduced, providing further evidence for a close correlation between the two events. To support an explanation of these findings based on divalent cation screening of membrane surface charge (increasing the voltage gradient within the membrane and closing voltage-inactivated channels) surface potential measurements were made on synaptic lipid liposomes by using a fluorescent surface-bound pH indicator. These experiments provided evidence for the presence of screenable surface charge on synaptosomes, and it was further shown in depolarised synaptosomes themselves that total external [Ca2+ + Mg2+], and not [Ca2+] alone, set the observed peak in intrasynaptosomal free calcium.     

PnTx3-6 a spider neurotoxin inhibits K+-evoked increase in [Ca2+](i) and Ca2+-dependent glutamate release in synaptosomes

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.     

Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.     

Synaptosome behaviour is unaffected by weak pulsed electromagnetic fields

The present study examined the effect on rat cortical synaptosomes of a 2 h exposure to 50-Hz electromagnetic fields (EMFs) with a peak magnetic field of 2 mT. We measured modifications of synaptosomal mitochondrial respiration rate, ATP production, membrane potential, intrasynaptosomal Ca(2+) concentration and free iron release. The O(2) consumption remained unvaried in exposed synaptosomes at about 2 nM O(2)/min/mg proteins; ATP production was also unchanged. The intrasynaptosomal Ca(2+) concentration decreased slowly and there was a slight, but non-significant, depolarisation of the synaptosomal membrane. Finally, the free iron release by synaptosomal suspensions, a useful predictor of neuro-developmental outcome, remained unchanged after EMF exposure. On the whole, our results indicate that the physiological behaviour of cortical synaptosomes is not affected by weak pulsed EMFs.     

Pattern of compensatory expression of voltage-dependent Ca2+ channel alpha1 and beta subunits in brain of N-type Ca2+ channel alpha1B subunit gene-deficient mice with a CBA/JN genetic background.

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice with a CBA/JN genetic background show no apparent behavioral or anatomical-histological abnormality, presumably owing to compensation by other Ca(2+) channels. In this study, we examined the mRNA expression of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of alpha(1B)-deficient mice. We found that the mRNA expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits were the same in the olfactory bulbs of wild, heterozygous and homozygous alpha(1B)-deficient mice. In the cerebral cortex, alpha(1A) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits was found among wild, heterozygous and homozygous mice. In hippocampus and cerebellum, beta(4) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2) and beta(3) subunits was found among wild, heterozygous and homozygous mice. These results suggest that the compensatory mechanisms differ in different brain regions of alpha(1B)-deficient mice with a CBA/JN genetic background.     

Hypoxia affects the physiological behavior of rat cortical synaptosomes

Nerve cells, especially synaptosomes, are very susceptible to hypoxia and the subsequent oxidative stress. In this paper, we examined the effects of hypoxia (93% N(2):2% O(2):5% CO(2), v/v/v) on rat cortical synaptosomes by evaluating modifications of synaptosomal mitochondrial respiration rate and ATP production, membrane potential, intrasynaptosomal mitochondrial Ca(2+) concentration ([Ca(2+)](i)), and desferoxamine-chelatable free iron and esterified F2-isoprostane levels after different periods of hypoxia and after 30 min of reoxygenation. Oxygen consumption decreased significantly during 120 min of hypoxia and was restored after reoxygenation. At the same time, ATP production decreased and remained significantly lower even after reoxygenation. This involved a depolarization of the synaptosomal mitochondrial membrane, although the [Ca(2+)](i) remained practically unchanged. Indeed, iron and F2-isoprostane levels, representing useful prediction markers for neurodevelopmental outcome, increased significantly after hypoxia, and there was a strong correlation between the two variables. On the whole our results indicate that synaptosomal mitochondria undergo mitoptosis after 2 h of hypoxia.     

Selective age-related changes in the PKC-sensitive, calmodulin-binding protein, neurogranin, in the mouse brain

Brain ageing is associated with a dysregulation of intracellular calcium (Ca(2+)) homeostasis which leads to deficits in Ca(2+)-dependent signalling pathways and altered neuronal functions. Given the crucial role of neurogranin/RC3 (Ng) in the post-synaptic regulation of Ca(2+) and calmodulin levels, age-dependent changes in the levels of Ng mRNA and protein expression were analysed in 3, 12, 24 and 31-month-old mouse brains. Ageing produced significant decreases in Ng mRNA expression in the dorsal hippocampal subfields, retrosplenial and primary motor cortices, whereas no reliable changes were seen in any other cortical regions examined. Western blot indicated that Ng protein expression was also down-regulated in the ageing mouse brain. Analysis of Ng immunoreactivity in both hippocampal CA1 and retrosplenial areas indicated that Ng protein in aged mice decreased predominantly in the dendritic segments of pyramidal neurones. These data suggest that age-related changes of post-synaptic Ng in selected brain areas, and particularly in hippocampus, may contribute to altered Ca(2+)/calmodulin-signalling pathways and to region-specific impairments of synaptic plasticity and cognitive decline.