Localization and mobility of omega-conotoxin-sensitive Ca2+ channels in hippocampal CA1 neurons

Voltage-dependent Ca2+ channels (VDCCs) are modulators of synaptic plasticity, oscillatory behavior, and rhythmic firing in brain regions such as the hippocampus. The distribution and lateral mobility of VDCCs on CA1 hippocampal neurons have been determined with biologically active fluorescent and biotinylated derivatives of the selective probe omega-conotoxin in conjunction with circular dityndallism, digital fluorescence imaging, and photobleach recovery microscopy. On noninnervated cell bodies, VDCCs were found to be organized in multiple clusters, whereas after innervation the VDCCs were concentrated and immobilized at synaptic contact sites. On dendrites, VDCC distribution was punctate and was interrupted by extensive bare regions or abruptly terminated. More than 85% of the dendritic VDCCs were found to be immobile by fluorescence photobleach recovery. Thus, before synaptic contact, specific mechanisms target, segregate, and immobilize VDCCs to neuronal cell bodies and to specialized dendritic sites. Regulation of this distribution may be critical in determining the firing activity and integrative properties of hippocampal CA1 neurons.     

Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity

Activity shapes the structure of neurons and their circuits. Two-photon imaging of CA1 neurons expressing enhanced green fluorescent protein in developing hippocampal slices from rat brains was used to characterize dendritic morphogenesis in response to synaptic activity. High-frequency focal synaptic stimulation induced a period (longer than 30 minutes) of enhanced growth of small filopodia-like protrusions (typically less than 5 micrometers long). Synaptically evoked growth was long-lasting and localized to dendritic regions close (less than 50 micrometers) to the stimulating electrode and was prevented by blockade of N-methyl-D-aspartate receptors. Thus, synaptic activation can produce rapid input-specific changes in dendritic structure. Such persistent structural changes could contribute to the development of neural circuitry.     

Prolonged Ca2+-dependent afterhyperpolarizations in hippocampal neurons of aged rats

The possibility that calcium is elevated in brain neurons during aging was examined by quantifying afterhyperpolarizations induced by spike bursts in CAl neurons of hippocampal slices from young and aged rats. The afterhyperpolarizations result from Ca2+-dependent K+ conductance increases and are blocked in medium low in Ca2+ and prolonged in medium high in Ca2+. The afterhyperpolarization and associated conductance increases were considerably prolonged in cells from aged rats, although inhibitory postsynaptic potentials did not differ with age. Since elevated intracellular Ca2+ can exert deleterious effects on neurons, the data suggest that altered Ca2+ homeostasis may play a significant role in normal brain aging.     

Muscarinic depression of long-term potentiation in CA3 hippocampal neurons

Behavioral studies have suggested that muscarinic cholinergic systems have an important role in learning and memory. A muscarinic cholinergic agonist is now shown to affect synaptic plasticity in the CA3 region of the hippocampal slice. Long-term potentiation (LTP) of the mossy fiber-CA3 synapse was blocked by muscarine. Low concentrations of muscarine (1 micromolar) had little effect on low-frequency (0.2 hertz) synaptic stimulation but did significantly reduce the magnitude and probability of induction of LTP. Experiments under voltage clamp showed that muscarine blocked the increase in excitatory synaptic conductance normally associated with LTP at this synapse. These results suggest a possible role for cholinergic systems in synaptic plasticity.     

硝普钠诱导体外培养的海马神经元凋亡的研究

目的：观察一氧化氮（NO）供体硝普钠（SNP）对体外培养的海马神经元凋亡的影响。方法：用终浓度分别为0、25、50、100、200、400、600μmol/L的SNP处理海马神经元24h，用MTT比色法分析细胞存活率，倒置显微镜、Hoechst 33258荧光染色观察凋亡的形态学改变，DNA琼脂糖凝胶分析凋亡的生化特征。结果：SNP可剂量依赖性的降低神经元的存活率，当SNP浓度为50μmol/L时，其存活率为56.2%；倒置显微镜观察可见神经元胞体固缩，突起断裂，网络消失；荧光显微镜可见染为高亮蓝色的典型凋亡小体，其细胞核明显固缩、凝聚和断裂，且随SNP剂量的增加，出现凋亡小体的细胞明显增多；50、100、200μmol/L SNP处理海马神经元，电泳图谱显示清晰的DNA梯度。结论：SNP可诱导培养的海马神经元凋亡。     

Inactivation and block of calcium channels by photo-released Ca2+ in dorsal root ganglion neurons

Calcium channels are inactivated by voltage and intracellular calcium. To study the kinetics and the mechanism of calcium-induced inactivation of calcium channels, a "caged" calcium compound, dimethoxy-nitrophen was used to photo-release about 50 microM calcium ion within 0.2 millisecond in dorsal root ganglion neurons. When divalent cations were the charge carriers, intracellular photo-release of calcium inactivated the calcium channel with an invariant rate [time constant (tau) approximately equal to 7 milliseconds]. When the monovalent cation sodium was the charge carrier, photorelease of calcium inside or outside of the cell blocked the channel rapidly (tau approximately equal to 0.4 millisecond), but the block was greater from the external side. Thus the kinetics of calcium-induced calcium channel inactivation depends on the valency of the permeant cation. The data imply that calcium channels exist in either of two conformational states, the calcium- and sodium-permeant forms, or, alternatively, calcium-induced inactivation occurs at a site closely associated with the internal permeating site.     

Interaction of brain synaptic vesicles induced by endogenous Ca2+ -dependent phospholipase A2

Endogenous phospholipase A2 activity of brain synaptic vesicles was Ca2+ -dependent and was increased by prostaglandin F2 alpha, calmodulin, adenosine 3', 5' -monophosphate, and adenosine triphosphate, whereas the activity was inhibited by prostaglandin E2 in the absence or presence of calmodulin. Light-scattering measurements demonstrated that stimulation of the enzyme's activity correlated with the induction of vesicle-vesicle aggregation. The effects of these compounds on endogenous synaptic vesicle phospholipase A2 activity may imply a common end point of their purported neuromodulatory actions, and indicate that synaptic vesicle phospholipase A2 may play a central role in presynaptic neurotransmission.     

Extensive dendritic sprouting induced by close axotomy of central neurons in the lamprey

Massive dendritic sprouting was induced in identified giant reticulospinal neurons of the lamprey by axotomy close to the soma. An axonal lesion slightly farther from the cell body induced new growth from both dendrites and axon. The amount of new growth per cell was the same whether it originated from the dendrites alone or from axonal and dendritic compartments. The location of the axonal lesion therefore determines where, in the neuron, membrane is inserted to produce the new neurites. The dendritic tree of a differentiated vertebrate central neuron was shown to have sufficient plasticity to extend new growth for several millimeters beyond the normal dendritic domain.     

The role of excitatory amino acids and NMDA receptors in traumatic brain injury

Brain injury induced by fluid percussion in rats caused a marked elevation in extracellular glutamate and aspartate adjacent to the trauma site. This increase in excitatory amino acids was related to the severity of the injury and was associated with a reduction in cellular bioenergetic state and intracellular free magnesium. Treatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist dextrophan or the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid limited the resultant neurological dysfunction; dextrorphan treatment also improved the bioenergetic state after trauma and increased the intracellular free magnesium. Thus, excitatory amino acids contribute to delayed tissue damage after brain trauma; NMDA antagonists may be of benefit in treating acute head injury.     

Ca2+-CaM系统在生长素诱导莴苣侧根原基形成中的作用

采用不同浓度的CaCl2、EGTA\,CPZ\,TFP和La(NO3)3处理莴苣幼苗种子根,结果表明,4 mg*L-1的氯化钙对莴苣幼苗侧根原基形成有促进作用,侧根原基数目为对照的2.1倍,并对生长素的促根效应有加合效应,可达17%以上;而钙离子专一性螯合剂乙醇双乙胺醚-N,N-四乙酸(EGTA)则起抑制作用,且其抑制作用能在一定程度上被氯化钙所恢复;钙调素抑制剂氯丙嗪(CPZ)和三氟拉嗪(TFP)也起抑制作用,抑制率最高可达40%左右;而钙离子通道阻断剂镧离子在很低浓度下却有促进作用,且与生长素处理有加合效应,其效应与钙离子类似.     

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.     

Ca2+参与硝普钠(SNP)对干旱胁迫下小麦幼苗叶片NO水平的调控

研究干旱胁迫下外源一氧化氮(NO)供体硝普钠(SNP)处理对小麦幼苗叶片NO水平的影响及其与Ca2+的关系.在15%PEG-6000胁迫下测定NO含量和NO合成酶活性,结果表明,干旱胁迫下小麦幼苗叶片一氧化氮合酶(NOS)活性显著增加,且钙依赖型cNoS(CaM的组成型NOS)快速调控NO产生,但是随着胁迫时间的延长,不依赖钙iNOS(CaM的诱导型NOS)的活性在NOS活性比例缓慢增加,而硝酸还原酶(NR)产生NO的能力只占总NR提取物活性的很小一部分;0.1 mmol/L SNP处理可显著提高干旱胁迫下小麦幼苗叶片NOS和NR活性,诱导NO水平提高,显著缓解膜脂过氧化;用质膜Ca2+通道抑制剂LaCl3与SNP共处理,显著减弱或抵消SNP促进NO合成作用.SNP显著提高干旱胁迫下小麦幼苗叶片NO合成酶活性和NO含量,有效缓解膜的氧化损伤,而Ca2+参与SNP对干旱胁迫下小麦幼苗叶片NO水平的调控.     

PCV2感染通过胞内Ca2+超载诱导仔猪腹股沟淋巴结及脾脏淋巴细胞凋亡

为探讨猪圆环病毒Ⅱ型(PCV2)感染仔猪的淋巴结和脾脏淋巴细胞内钙信号变化在细胞凋亡中的作用,选取19头PCV2抗原和抗体均为阴性的5周龄健康断奶仔猪,随机分成对照组(4头)和试验组(15头),试验组仔猪通过滴鼻接种PCV2,并于接种后14、21和35 d分别扑杀5头,对照组仔猪滴鼻同量PBS后当天扑杀。所有仔猪扑杀时均取腹股沟淋巴结及脾脏,流式细胞术检测细胞凋亡率,荧光分光光度法检测细胞内Ca2+浓度,孔雀绿比色测定法检测Ca2+-ATP酶的活性,荧光定量RT-PCR检测钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)mRNA转录水平。结果表明:所有接种病毒仔猪腹股沟淋巴结及脾脏细胞凋亡率均极显著高于对照组(P<0.01),Ca2+浓度均极显著高于对照组(P<0.01);接种病毒仔猪腹股沟淋巴结Ca2+-ATP酶活性在21 d时显著下降(P<0.05),脾脏中Ca2+-ATP酶活性在21 d和35 d时显著下降(P<0.05);腹股沟淋巴结CaMKⅡmRNA转录在整个试验过程中无明显变化,但脾脏中CaMKⅡmRNA在PCV2接种后21 d和35 d比对照组显著上调(P<0.05)。结论:细胞内Ca2+超载是PCV2感染引起仔猪淋巴结及脾脏淋巴细胞凋亡的重要机制。     

鱿鱼墨黑色素吸附Ca2+的活性研究

[目的]为鱿鱼墨在饲料、保健品等领域的应用提供理论依据。采用高速离心法精制的鱿鱼墨黑色素与Ca~(2+)作用,探究酸度、温度、时间、黑色素添加量、Ca~(2+)浓度及盐度等因素对鱿鱼墨黑色素吸附Ca~(2+)的影响。[结果]当pH为4、温度为40℃、黑色素添加量为0.02g、吸附时间为10h时,鱿鱼墨黑色素对钙的吸附量最大,达到65%。不同浓度的氯化钠和氯化镁对黑色素吸附Ca~(2+)的影响较小,氯化铁对黑色素吸附钙的影响很大,随着氯化铁浓度的增大,吸附量急剧增加。[结论]鱿鱼墨黑色素对Ca~(2+)的吸附能力并不理想,吸附量也不多。     

Ca(2+)-induced Ca2+ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose

Calcium-induced calcium release (CICR) may function widely in calcium-mediated cell signaling, but has been most thoroughly characterized in muscle cells. In a homogenate of sea urchin eggs, which display transients in the intracellular free calcium concentration ([Ca2+]i) during fertilization and anaphase, addition of Ca2+ triggered CICR. Ca2+ release was also induced by the CICR modulators ryanodine and caffeine. Responses to both Ca2+ and CICR modulators (but not Ca2+ release mediated by inositol 1,4,5-trisphosphate) were inhibited by procaine and ruthenium red, inhibitors of CICR. Intact eggs also displayed transients of [Ca2+]i when microinjected with ryanodine. Cyclic ADP-ribose, a metabolite with potent Ca(2+)-releasing properties, appears to act by way of the CICR mechanism and may thus be an endogenous modulator of CICR. A CICR mechanism is present in these nonmuscle cells as is assumed in various models of intracellular Ca2+ wave propagation.     

ABP1参与BY2细胞生长素响应的Ca2+信号转导过程

将Ca2+响应实时荧光报告系统引入雌二醇诱导生长素结合蛋白(ABP1)调控表达的BY2细胞中,获得了 ABP1过表达(ABP1-ox)、抑制表达(ABP1-anti)同时对Ca2+标记的几个BY2细胞株.对这些细胞株进行雌二醇诱导调控其ABP1过表达或抑制表达后,分析ABP1的表达量,并通过在细胞外添加IAA处理,实时...     

The Role of Extracellular Ca2+Influx, Intracellular Ca2+ Release and Calmodulin in Mouse Egg Fertilization

The effects of various Ca^2 -modifying drugs on moue egg fertilization were studied.Ca^2 chelator,ethylen glycol-bis-(2-aminoethyl)-tetracetic acid(EGTA),and calmodulin(CaM) antagonist,trifluoperzaine (TFP),inhibited fertilization in a dose-dependent manner,whild Ca^2 channel bolcker,verspamil,did not have any effect.When intracellular Ca^2 release was blocked by 8-(N,N-diethylamino) octy 1-3,4,5-trimethoxy-benzonate(TME-8) or the Ca^2 oscillations were inhibited by an inhibitor of endoplasmic reticulum Ca^2 -At-Pase,thapsigargin,the second polar body emission and pronuclear formation were significantly decreased.In contrast,inhibition of intracellular Ca^2 release via bolckage of inositol 1,4,5-triphosphate (IP3) production by neomycin or lithium did not affect fertilization.The results sugest that both extracellular influx,intracellular Ca^2 release and CaM activation are required for mormal fertilization.However,extracellular influx through voltage-gated Ca^2 channel and intracellular release induced by IP3 and not the only pathways for producing Ca^2 transients in moue eggs.     

抗病毒剂VA诱导烟草对TMV的抗性与Ca2+的关系

以枯斑三生烟为材料,抗病毒剂VA为诱导剂,用细胞化学沉淀法测定了VA对烟草叶片细胞中Ca^2 分布的影响,并探讨了Ca^2 在植物诱导抗病性中的作用。结果发现,枯斑三生烟健叶中的Ca^2 主要分布于细胞间隙和液泡,喷施VA可以增加健叶细胞间隙中的Ca^2 。接种TMV后,细胞间隙中的Ca^2 逐渐向细胞内转移,在接种后48h,Ca^2 主要分布于过敏性坏死细胞和近坏死区细胞,远坏死区细胞间隙缺Ca^2 ;以后由于枯斑停止发展,远坏死区细胞逐渐恢复钙稳态,VA TMV处理比水 TMV处理恢复钙稳态快,在接种后72h就可恢复,而水 TMV处理的远坏死区要在接种后96h才恢复钙稳态。     

肉鸡腹水综合征患鸡肺脏组织钙沉积和Ca2＋-ATPase活性变化

肺动脉收缩和重塑在肉鸡AS发生过程中起着重要作用.本实验经采用右心导管法研究发现AS患鸡PASP 、PADP和mPAP极显著高于对照组(P <0.01).同时,采用焦锑酸钾沉淀法、电镜酶细胞化学法研究AS患鸡肺脏组织Ca2+和Ca2+-ATPase活性变化,发现AS患鸡肺脏组织中钙沉积量显著增多,且Ca2+-ATPase的电子密度颗粒显著减少或缺失.表明AS患鸡具有明显的肺动脉高压,其可能与肺脏组织,特别是肺动脉平滑肌细胞钙处理能力异常导致的钙离子浓度升高和肌浆网Ca2+-ATPase酶活性降低有关.