谷氨酸促进大鼠海马神经元的内钙升高

谷氨酸能影响大鼠海马神经元胞内钙信号的变化,进而影响海马神经元神经冲动的发放和学习记忆过程。运用荧光测钙技术实时监测了大鼠海马神经元内钙信号的动态变化,同时分析了谷氨酸对其胞内钙信号的影响。试验表明：谷氨酸能够显著提高胞内游离钙离子的浓度;细胞外钙离子的存在、谷氨酸刺激时间及刺激频率的增加都能引起胞内钙信号不同程度的升高;但谷氨酸的过度刺激会引起钙离子浓度的超负荷,从而导致神经元结构和功能的损坏。     

应用激光共聚焦扫描技术对海马脑片神经元内钙的观察

用微量注射法将荧光剂Ｆｌｕｏ－３注入大鼠海马,对神经元进行在体荧光标记,可清晰地标记多个神经细胞。联合应用激光共聚焦扫描显微镜,观察大鼠海马脑片ＣＡ１锥体细胞在青霉素,谷氨酸模拟致痫及缺糖缺氧时胞内钙的变化。结果显示：无镁时,谷氨酸和青霉素可致海马ＣＡ１锥体细胞胞内钙的缓慢增加;离体缺糖缺氧时ＣＡ１锥体细胞胞内钙亦增多。     

对羟基苯甲醛对神经元缺氧所致钙超载的抑制作用

通过复制大鼠原代皮层神经元缺糖缺氧/复糖复氧(OGD/Rep)损伤模型,采用MTT法检测细胞存活率、硝酸还原酶法检测NO释放量、流式细胞仪检测胞内钙离子浓度、Western Blot法及RT-PCR法检测神经元L型钙通道、NMDA型钙通道、TRPM7通道蛋白和基因的表达,评价天麻成分对羟基苯甲醛对OGD/Rep损伤致大鼠原代皮层神经元钙通道过度开放的影响,探讨天麻成分对羟基苯甲醛的神经保护作用是否通过抑制钙通道的过度开放减轻钙超载,结果表明天麻成分对羟基苯甲醛可提高大鼠原代皮层神经元OGD/Rep损伤的存活率、减少NO的释放、降低胞内钙离子浓度、降低L型钙通道蛋白的表达。     

钙调蛋白及其结合蛋白对神经递质释放的调控作用

钙离子（Ca2+）是调节突触前神经递质的胞吐释放的关键离子信号.作为胞内最普遍存在的钙离子感受器的钙调蛋白（CaM）被发现能通过与多种蛋白的相互作用,调控着突触小泡的生发、运输及再填充,从而传递胞内Ca2+浓度变化的信号,对神经递质的释放及突触电生理活动起到至关重要的调控作用.本文综述了CaM及其结合蛋白是如何参与对突触小泡的胞吐释放和胞吞恢复的调控,并探讨了其中可能的分子机制.     

钙离子:进入细胞浆的途径与血管平滑肌收缩机制

血管平滑肌收缩所需的钙离子源于细胞外流入和细胞内释放。钙流入途径主要有膜电位依赖式和与受体耦联的钙通道。释放钙离子机制受除极IP_3、cIP_3和钙离子作用而激发。进入细胞浆的钙离子与钙受体蛋白结合而引起收缩。血管平滑肌没有肌钙蛋白C,由钙调蛋白或Leiotonin C代之。钙调蛋白通过使肌球蛋白磷酸化;而Leiotonin C则通过直接激活肌动蛋白,引起血管收缩。     

酿酒酵母细胞中钙离子信号传导途径的研究进展

酿酒酵母（SaccharomycescP增v括fdP）细胞可以通过ca2＋／钙调磷酸酶信号途径来应对许多外界环境胁迫。在交配信息素、盐或者其他环境压力存在的条件下,钙离子会通过细胞质膜上的未鉴定的钙转运蛋白x和M或者由Cchl和Midl组成的钙通道进入细胞质。胞质内钙离子浓度的增加会激活细胞质里的钙调磷酸酶（calcineurin）。钙调磷酸酶的一个非常重要的作用是去磷酸化细胞质内的转录因子Crzl,造成它快速地从细胞质转移到细胞核,从而诱导包括液泡膜上钙泵蛋白基因PMCl以及内质网膜和高尔基体膜上钙泵蛋白基因尸脚,在内的目标基因的表达。这两个钙泵蛋白和液泡膜上的Ca2＋／H＋交换蛋白Vcxl一起作用,将细胞质内的钙离子浓度控制在50～200nmol／L的正常生理浓度内．使细胞能够正常生长。该综述主要论述了酿酒酵母细胞内Ca2＋／钙调磷酸酶信号途径的最新研究进展。     

牛磺酸对大鼠脑神经细胞内钙稳态的影响

目的和方法 :利用激光扫描共聚焦显微镜和双波长荧光分光光度计 ,分别观察牛磺酸对无血清培养的单个海马神经细胞和分散的新生大鼠脑神经细胞内Ca2 浓度 ([Ca2 ]i)的影响 ,并探讨牛磺酸调节神经细胞内钙稳态的作用机理。结果 :牛磺酸主要通过刺激细胞内钙库释放 ,在一定剂量范围内 (0 .0 2～ 6 .4mmol/L)使神经细胞[Ca2 ]i 轻微升高 ,在 0 .4mmol/L时的升钙作用最大 (升钙百分率为 12 .2 0 %± 1.2 4% )。在测定介质中加入钙离子载体A2 3187(10 μmol/L) ,使神经细胞内的钙离子浓度升高 ,若此时加入牛磺酸 (1.6mmol/L) ,则神经细胞内钙离子的浓度下降。结论 :牛磺酸对细胞内钙离子有双向调节作用 ,牛磺酸可能是通过对钙稳态的调节作用来阻止细胞内钙超载 ,保护神经细胞、并发挥其增强动物学习记忆等方面功能的。     

突触后钙通路有助于视锥与L型水平细胞间的突触可塑性

我们实验室以前发现,视网膜视锥与亮度型水平细胞（luminosity—type horizontal cell,LHC）之间的突触传递效率具有可塑性。重复性刺激红敏视锥增加了LHC对红光的超极化反应幅度,而且这种增强作用是可逆的。在本文中,我们运用细胞内记录技术和药理学分析的方法来考察重复性红光刺激引起的反应增强的可能机制。当通过胞内注射Ca^2＋的螯合剂EGTA来降低LHC内的Ca^2＋浓度后,重复性红光引起的反应增强被抑制,提示突触后钙信号是反应增强的一个重要因素。另外,反应增强现象还可以被钙离子通透的AMPA受体（Ca^2＋-permeable AMPA receptor,CP-AMPAR）的拈抗剂阻断,说明通过钙离子通透的谷氨酸受体内流的Ca^2＋与胞内Ca^2＋浓度的改变有关。进一步发现,胞外灌流ryanodine或caffeine也可以消除反应增强现象,说明由钙诱导的钙释放（calcium—induced calcium release,CICR）引起的钙信号可能也参与了反应增强现象的产生。结果提示,CICR和CP—AMPAR与重复性红光刺激引起的LHC对红光的反应增强有关。     

人白血病细胞系P2X7受体的表达及其介导的细胞内钙浓度变化

采用半定量RT-PCR和流式细胞术,在基因和蛋白水平研究了白血病细胞系U937、HL60和Ramos细胞P2X7受体的表达。荧光染料Fura-2／AM负载后,用荧光分光光度计测定P2X7受体激动剂三磷酸腺苷(adenosine 5′-triphosphate,ATP)和苯甲酰苯甲酸ATP(2′,3′-O-(4-benzoyl)benzoyl-ATP,BzATP)刺激前后细胞内钙离子浓度的变化,以确认其功能。结果表明：U937和HL60细胞系表达P2X7受体的mRNA和蛋白,Ramos不表达;在激动剂的刺激下,可引发U937和HL60细胞胞内钙浓度的显著升高,但对Ramos没有作用。当去除胞外钙离子时,ATP和BzATP刺激均不能引起U937和HL60细胞胞内钙离子浓度的升高。提示U937和HL60细胞表达P2X7受体的基因和功能蛋白,Ramos细胞则不表达该受体。     

钙通道与钙释放通道

1.Ca~(2+)的重要生理作用胞内游离钙浓度([Ca~(2+)])的变化调节着细胞的代谢、基因表达等细胞共有的活动,以及始动兴奋、收缩或出胞分泌以及激活和失活离子通道等细胞不同的反应。[Ca~(2+)]的升高主要依赖于胞外钙经质膜上的钙通道内流或/和胞内储存钙的释放。释放的内钙也是藉细胞器膜的钙释放通道进入胞浆。可见通道启闭活动的正常是维持[Ca~(2+)]正常的一个重要保证。2.离子通道及其分类离子通道是贯穿于质膜或细胞器膜的大分子蛋白质,其中央形成能通过离子的亲水性孔道(pores)。离子的跨膜转运是通过膜上通道蛋白的功能来完     

Elevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen-glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca(2+)](i)) response in the rat hippocampal neurons. The levels of NO and [Ca(2+)](i) in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca(2+)](i) responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca(2+)](i) response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca(2+)](i) responses to hypoxia and OGD, indicating an involvement of L-type Ca(2+) channels in the NO-mediated mechanisms. In addition, the [Ca(2+)](i) responses were attenuated by ODQ or KT5823, inhibitors of the cGMP-PKG pathway, and by acivicin, an inhibitor of gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca(2+) currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca(2+) currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca(2+) current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca(2+) via the hippocampal L-type Ca(2+) channel by S-nitrosylation during an initial phase of energy deprivation.     

Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence

The extremely short biological half-life of endothelial-derived nitric oxide (NO) has impeded real-time measurements of NO synthesis. We used the membrane-permeable fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) to study determinants of NO synthesis in bovine aortic endothelial cells (BAECs). A step increase in shear stress (SS) from 0.3 to 3.4 dyne/cm(2) triggered an increase in DAF-2 fluorescence starting 3.0 +/- 0.5 min after the flow rise and peaking at 44.7 +/- 7.2 min. This was abolished by intracellular Ca(2+) chelation, but was unaffected by blocking extracellular Ca(2+) influx or by inhibiting SS-related changes in intracellular pH. The increase in DAF-2 fluorescence occurred significantly earlier in BAECs transfected with either superoxide dismutase (SOD) or catalase (CAT), indicating concomitant reactive oxygen species (ROS) generation by SS and "competition" between ROS- and DAF-2-NO interactions. These data provide novel insights into several NO signaling determinants and reveal that DAF-2 can assess real-time SS-stimulated NO synthesis in endothelial cells. This should facilitate the analysis of NO-signaling pathways.     

Impairment of endothelial nitric oxide production by acute glucose overload

We examined the effects of acute glucose overload (pretreatment for 3 h with 23 mM D-glucose) on the cellular productivity of nitric oxide (NO) in bovine aortic endothelial cells (BAEC). We had previously reported (Kimura C, Oike M, and Ito Y. Circ Res, 82: 677-685, 1998) that glucose overload impairs Ca(2+) mobilization due to an accumulation of superoxide anions (O(2)(-)) in BAEC. In control cells, ATP induced an increase in NO production, assessed by diaminofluorescein 2 (DAF-2), an NO-sensitive fluorescent dye, mainly due to Ca(2+) entry. In contrast, ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload, which was restored by superoxide dismutase, but not by catalase or deferoxamine. Furthermore, pyrogallol, an O(2)(-) donor, also attenuated ATP-induced increase in DAF-2 fluorescence. In contrast, a nonspecific intracellular Ca(2+) concentration increase induced by the Ca(2+) ionophore A-23187, which depletes the intracellular store sites, elevated DAF-2 fluorescence in both control and high D-glucose-treated cells in Ca(2+)-free solution. These results indicate that glucose overload impairs NO production by the O(2)(-)-mediated attenuation of Ca(2+) entry.     

Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron

Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependent dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72+/-0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99+/-0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1+/-6.6 and 3.2+/-1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1+/-5.8, 2.6+/-0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure.     

Functional implications of Ca2+ mobilizing properties for nitric oxide production in aortic endothelium

We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.     

Ca2+- and phosphatidylinositol 3-kinase-dependent nitric oxide generation in lung endothelial cells in situ with ischemia

Endothelial cells generate nitric oxide (NO) in response to agonist stimulation or increased shear stress. In this study, we evaluated the effects of abrupt cessation of shear stress on pulmonary endothelial NO generation and its relationship to changes in intracellular Ca(2+). In situ endothelial generation of NO and changes in intracellular Ca(2+) in isolated, intact rat lungs were evaluated using fluorescence microscopy with diaminofluorescein diacetate, an NO probe, and Fluo-3, a Ca(2+) probe. The onset of increased NO generation in endothelial cells of subpleural microvessels in situ occurred between 30 and 90 s after onset of ischemia and was preceded by an increase in intracellular Ca(2+) due to both influx of extracellular Ca(2+) and release from intracellular stores. Flow cessation-induced NO generation in endothelial cells in situ was Ca(2+)-, calmodulin-, and PI3-kinase-dependent. The similarity of endothelial cell response (increased NO generation) to either increased flow or cessation of flow suggests that cells respond to an imposed alteration from a state of adaptation. This response to flow cessation may constitute a compensatory vasodilatatory mechanism and may play a role in signaling for cell proliferation and vascular remodeling.     

Hemichannels in cardiomyocytes open transiently during ischemia and contribute to reperfusion injury following brief ischemia

The aim of this study was to investigate changes in hemichannel activity during in vitro simulated ischemia [oxygen-glucose deprivation (OGD)] and the contribution of hemichannels to ischemia-reperfusion injury in rat neonatal cardiomyocytes. Dye uptake assays showed that hemichannels opened as OGD progressed, peaking after 1 h, and then closed, returning to the pre-OGD state after 2 h of OGD. The increase in dye uptake after 1 h of OGD was inhibited by hemichannel blockers (lanthanum chloride and a connexin 43 mimetic peptide, Gap26). During OGD, intracellular Ca(2+) concentration ([Ca(2+)](i)) began to increase after 1 h and reached several micromolar after 2 h. After 1 h of OGD, Gap26 inhibited the increases in hemichannel activity and [Ca(2+)](i). In contrast, dantrolene [an endo(sarco)plasmic reticulum Ca(2+) release inhibitor] suppressed the increase in [Ca(2+)](i), but not in hemichannel activity. After 2 h of OGD, the combined administration of 2',4'-dichlorobenzamil and dantrolene reduced [Ca(2+)](i) to <1 microM and increased hemichannel activity to the level attained after 1 h of OGD. Simulated ischemia-reperfusion, induced by 1 h of OGD followed by 2 h of recovery, reduced cell viability to 54% of the control level. The addition of Gap26 to OGD medium improved viability to 80% of the control level. In conclusion, this study demonstrated that 1) hemichannels open transiently during OGD, 2) closure of hemichannels, but not their opening, is regulated by an increase in [Ca(2+)](i) during OGD, and 3) open hemichannels contribute to cell injury during recovery from OGD.     

Mechanism of guanosine-induced neuroprotection in rat hippocampal slices submitted to oxygen-glucose deprivation

Guanine derivates have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. Guanine derivatives are spontaneously released to the extracellular space from cultured astrocytes during oxygen-glucose deprivation (OGD) and may act as trophic factors, glutamate receptors blockers or glutamate transport modulators, thus promoting neuroprotection. The aim of this study was to evaluate the mechanisms involved in the neuroprotective role of the nucleoside guanosine in rat hippocampal slices submitted to OGD, identifying a putative extracellular binding site and the intracellular signaling pathways related to guanosine-induced neuroprotection. Cell damage to hippocampal slices submitted to 15 min of OGD followed by 2 h of reperfusion was decreased by the addition of guanosine (100 microM) or guanosine-5'-monophosphate (GMP, 100 microM). The neuroprotective effect of guanosine was not altered by the addition of adenosine receptor antagonists, nucleosides transport inhibitor, glutamate receptor antagonists, glutamate transport inhibitors, and a non-selective Na(+) and Ca(2+) channel blocker. However, in a Ca(2+)-free medium (by adding EGTA), guanosine was ineffective. Nifedipine (a Ca(2+) channel blocker) increased the neuroprotective effect of guanosine and 4-aminopyridine, a K(+) channel blocker, reversed the neuroprotective effect of guanosine. Evaluation of the intracellular signaling pathways associated with guanosine-induced neuroprotection showed the involvement of PKA, PKC, MEK and PI-3 K pathways, but not CaMKII. Therefore, this study shows guanosine is acting via K(+) channels activation, depending on extracellular Ca(2+) levels and via modulation of the PKA, PKC, MEK and/or PI-3 K pathways.     

Chondroitin sulfate reduces cell death of rat hippocampal slices subjected to oxygen and glucose deprivation by inhibiting p38, NFκB and iNOS

The glycosaminoglycan chondroitin sulfate (CS) is a major constituent of the extracellular matrix of the central nervous system where it can constitute part of the perineuronal nets. Constituents of the perineuronal nets are gaining interest because they have modulatory actions on their neighbouring neurons. In this study we have investigated if CS could afford protection in an acute in vitro ischemia/reoxygenation model by using isolated hippocampal slices subjected to 60min oxygen and glucose deprivation (OGD) followed by 120min reoxygenation (OGD/Reox). In this toxicity model, CS afforded protection of rat hippocampal slices measured as a reduction of lactate dehydrogenase (LDH) release; maximum protection (70% reduction of LDH) was obtained at the concentration of 3mM. To evaluate the intracellular signaling pathways implicated in the protective effect of CS, we first analysed the participation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2 by western blot. OGD/Reox induced the phosphorylation of p38 and dephosphorylation of ERK1/2; however, CS only inhibited p38 but had no effect on ERK1/2. Furthermore, OGD/Reox-induced translocation of p65 to the nucleus was prevented in CS treated hippocampal slices. Finally, CS inhibited iNOS induction caused by OGD/Reox and thereby nitric oxide (NO) production measured as a reduction in DAF-2 DA fluorescence. In conclusion, the protective effect of CS in hippocampal slices subjected to OGD/Reox can be related to a modulatory action of the local immune response by a mechanism that implies inhibition of p38, NFκB, iNOS and the production of NO.     

Calcium-Dependent Protein Kinase C Activation in Acutely Isolated Neurons During Oxygen and Glucose Deprivation

Glutamate excitotoxicity and necrotic cell death are characteristic features of ischemic neuronal injury. In the penumbral area, glutamate exposure is less pronounced and neuronal death is delayed. Recent studies suggest that delayed neuronal death is propagated by intracellular signalling pathways. Protein kinase C (PKC) activation may initiate apoptosis, but its role in ischemia is still not clear. In this study the PKC activity was investigated during non-excitotoxic ischemia in acutely dissociated rat CA1 neurons. During oxygen and glucose deprivation (OGD) the PKC activity measured with the fluorescent dye Fim-1 increased rapidly reaching a maximum of 31+/-8% (P < 0.05) after 5 min. When extracellular Ca2+ was depleted, the fluorescence intensity increased by 20+/-8% (P<0.05), but with a slower onset. In neurons treated with thapsigargin in a Ca2+ depleted solution, however, OGD did not trigger PKC activation. The results suggest that the PKC activation is mainly triggered by Ca2+ release from endogenous stores.