高糖对缺氧神经元的影响及钙相关机制研究

研究高糖对神经元缺氧的影响,并探讨其钙相关机制.利用SD大鼠大脑皮质神经元体外缺氧模型,通过对细胞活力的检测,观察浓度分别为22.7(对照组),30,40,50,60(高糖组)mmol/L的葡萄糖对神经元缺氧的影响;以Fura-2/Am为荧光指示剂,测定细胞内游离钙离子浓度([Ca2+]i.结果发现当培养基中葡萄糖浓度达到60 mmol/L时,高糖可引起缺氧神经元损伤;与对照组相比,有钙介质与无钙介质中静息[Ca2+]i均升高,P＜0.01;但对照组与高糖组在有钙介质中对氯化钾(KCl)、谷氨酸(Glu)刺激引起的[Ca2+]i升高无明显差异,P＞0.05;在无钙介质中,对氯化钙(CaCl2)引起的[Ca2+]i增高率无明显差别,P＞0.05.本研究表明:高糖对神经元的损伤作用可能与其促进细胞内钙离子释放,诱发细胞内钙超载有关.     

大鼠小直径痛觉三叉神经节神经元同时存在三磷酸肌醇敏感性钙库和阿诺碱敏感性钙库？P2X和P2Y嘌呤受体介导细胞内钙信号转导通路的变化

背景： 面部创伤或面部手术等可能损伤三叉神经系统而导致三叉神经疼痛,由于其疼痛剧烈难忍、易复发,长期以来一直为口腔临床治疗的一大难题。现有大量研究发现嘌呤类受体与三叉神经痛相关,目前对其作用机制知之甚少。 目的：探讨在小直径三叉神经节神经元中嘌呤类受体介导钙信号途径。 方法：用Fura-2荧光染料通过显微镜荧光测定技术实时检测急性分离成年大鼠小直径三叉神经节神经元的细胞内钙离子浓度。 结果：用正常外液或去除细胞外Ca2+灌流细胞,分别给予thapsigargin（1 μmol/L）,内质网钙泵ATP酶抑制剂,和咖啡因（20 mmol/L）,ryanodine受体激动剂,均能够引起细胞内游离钙离子浓度（[Ca2+]i）不同程度地升高。ATP（100 μmol/L）也能够产生类似的效应。在去除细胞外Ca2+条件下,ATP引起的[Ca2+]i升高可被thapsigargin可逆性地抑制,而不能被咖啡因抑制;然而在正常外液环境中,ATP引起的细胞内[Ca2+]i 升高不能完全地被thapsigargin抑制。 结论：在痛觉三叉神经节神经元中,嘌呤类受体介导的[Ca2+]i升高有两条途径,一种途径是通过代谢型P2Y受体作用于三磷酸肌醇敏感性钙库;另一种途径是通过离子型受体P2X受体引起外钙内流。     

不同温度对谷氨酸诱发原代培养皮质神经元损伤的保护机制

目的探讨不同温度对谷氨酸诱发皮质神经元损伤的保护机制。方法体外培养新生24 h内Wistar大鼠的皮质神经元,毒性剂量的谷氨酸(200μmol·L-1)诱致皮质神经元损伤,随机分为37℃组(常温)和33℃组(亚低温),两组再根据谷氨酸持续作用时间点(10 min,3 h,6 h,24 h)分为4个亚组,标记为T1,T2,T3,T4。两组细胞均在损伤后24 h,通过激光扫描共聚焦显微镜测量细胞内Ca2+浓度([Ca2+]i)、线粒体内Ca2+浓度([Ca2+]m)及线粒体跨膜电位(ΔΨm)。结果 37℃组和33℃组的[Ca2+]i及[Ca2+]m均随损伤时间的延长而显著性增加,同时伴有ΔΨm显著下降(P0.05);33℃组较37℃组[Ca2+]i及[Ca2+]m的增加明显减少,同时显著减低ΔΨm下降幅度(P0.05)。结论 33℃可抑制谷氨酸诱发皮质神经元损伤时的[Ca2+]m内流,减少[Ca2+]m,维持ΔΨm。     

大鼠星形胶质细胞的内质网钙库特征

用显微荧光测量技术研究了纯化培养的大鼠神经胶质细胞的内质网钙库的组成、特征.用IP3-敏感的和非IP3-敏感的钙库的激活剂乙酰甲胆碱(Acetyl-β-Methylcholine Chloride,MCh)和咖啡因(Caffeine,CAF)及线粒体解偶剂FCCP组成不同的刺激条件作用于细胞,观察胞内钙信号的变化.结果表明MCh和CAF均可使[Ca2+]i出现与刺激浓度依赖性的钙升高.快速重复的刺激可导致钙库的耗竭,而足够长的间隔时间可使钙库的Ca2+储存得到不同程度的恢复;用Thapsigargin耗竭内质网钙库后,MCh和CAF不能引起[Ca2+]i升高;线粒体的解偶剂FCCP可诱发中度的[Ca2+]i升高,并与MCh和CAF敏感的钙库相对独立.研究显示星形胶质细胞内的钙库包括:MCh和CAF敏感的两种内质网钙库,它们或相对独立或密切相关;线粒体钙库参与胞质内Ca2+缓冲系统.     

丁咯地尔抑制NE和Glu引起的单个脑细胞游离钙增高

目的：研究丁咯地尔对去甲肾上腺素(NE)和谷氨酸(Glu)引起大鼠单个脑细胞内游离钙增高的影响。方法：应用AR-CM-MIC阳离子测定系统测量细胞内游离钙([Ca2+]i)。结果：细胞外钙为1．3 mmol·L,丁咯地尔0．1,1．0,1 0．0μmol·L-1对细胞静息[Ca2+]i无明显影响,对NE诱导的[Ca2+]i增高明显抑制,对Glu诱导的[Ca2+]i增高具有一定的抑制作用。结论：丁咯地尔能抑制NE和Glu引起的单个脑细胞游离钙增高。     

缺氧抑制脑动脉Kv2.1通道作用机制的研究

目的探讨缺氧对脑动脉Kv通道抑制作用的机制是否由内源性15-羟二十碳四烯酸(15-HETE)介导。方法选取健康Wistar大鼠,通过酶法分离培养脑动脉和颈内动脉平滑肌细胞,分为正常对照组、缺氧组和去甲二氢愈创木酸(NDGA)缺氧组,正常对照组平滑肌细胞常规培养48h、缺氧组在缺氧箱内培养48h、NDGA缺氧组加入50μmol/L的NDGA后在缺氧箱内培养48h,使用RT-PCR和Western blotting技术检测大鼠脑动脉和颈内动脉平滑肌细胞上Kv2.1通道mRNA及蛋白质的表达情况。结果缺氧可以抑制大鼠脑动脉和颈内动脉平滑肌细胞上Kv2.1通道的表达,缺氧组与正常对照组相比,Kv2.1通道mRNA及蛋白质的表达下调(P<0.05);采用NDGA抑制15-脂氧化酶(15-LOX)后,导致脑动脉和颈内动脉平滑肌细胞上Kv2.1通道的表达上调,NDGA可以保护缺氧对于Kv通道的抑制,NDGA缺氧组与缺氧组相比,Kv2.1通道mRNA及蛋白质的表达明显上调(P<0.05)。结论缺氧可能通过内源性15-HETE发挥对Kv通道的抑制作用,15-HETE可能是影响脑动脉张力的重要中介因素。     

全脑缺血再灌注损伤大鼠海马细胞周期依赖性蛋白激酶-5的变化

目的 探讨急性全脑缺血-再灌注(isehemia-reperfusion,I-R)损伤时大鼠海马细胞周期依赖性蛋白激酶-5(Cdk-5)的变化.方法 用三血管结扎法建立急性全脑I-R大鼠模型.将大鼠随机分为假手术对照组、脑缺血组、I-R组及尼莫地平(nimodipine,NIM)处理组.用免疫沉淀法测定Cdk-5酶活性,用Fura-2负载及荧光测定细胞内游离Ca2+浓度{[Ca2+]I}.结果 与假手术对照组比较,脑缺血组及除I-R 5 h亚组外的其余I-R亚组大鼠海马神经元[Ca2+]I升高(P＜0.05)且伴细胞内Cdk-5活性升高(P＜0.05).NIM处理组大鼠海马细胞[Ca2+]I低于缺血组及I-R 30 min组(P＜0.05,P＜0.05),而Cdk-5的活性也低于I-R 30 min组(P＜0.05).结论 急性全脑缺血和/或再灌注时大鼠海马神经元Cdk-5活性升高,神经元[Ca2+]I升高对Cdk-5活性升高可能起重要作用.     

大鼠急性感染性脑水肿的脑保护作用研究

目的探讨百日咳菌液诱导的急性感染性脑水肿的发生机制和类型,并观察尼莫地平和MK-801的作用.方法测定脑组织水分含量和EB含量,Fura-2/AM测定突触体[Ca2+]i,放射性配基结合法测定NMDA受体的结合量.结果 [Ca2+]i随脑水肿时间的延长而持续进行性升高,伴随脑组织水分含量和EB含量增加.MK-801预处理和尼莫地平治疗后,脑组织含水量和EB含量及[Ca2+]i明显下降(P＜0.05).PB组与NS组相比Kd值有显著性差异,而Bmax差异无显著性.结论感染性脑水肿是混合性脑水肿,它的发生与Ca2+通道开放和NMDA受体介导的迟发性钙离子内流及BBB通透性增加密切相关.MK-801和尼莫地平通过缓解细胞内钙离子超载和改善BBB的通透性,从而减轻脑水肿.     

石杉碱甲对血管性痴呆小鼠模型海马神经细胞静息态[Ca^2＋]i的影响

目的观察血管性痴呆(VD)小鼠海马神经细胞内静息态游离Ca2+浓度([Ca2+]i)变化,以及石杉碱甲(哈伯因)对VD的治疗效果和[Ca2+]i的影响,进而探讨[Ca2+]i的改变在VD发病机制中的作用.方法采用双侧颈总动脉线结、反复缺血-再灌注法,制作小鼠血管性痴呆动物模型,并设假手术组作为对照,服用石杉碱甲者为治疗组.分别于术后第29天、第30天进行学习、记忆和行为学测试;然后快速制取海马活细胞,以Fluo-3/AM为荧光探针,在激光扫描共聚焦显微镜下观察各组海马神经细胞静息态[Ca2+]i变化.结果 (1)模型组的学习和记忆成绩低于假手术组及治疗组(P<0.05),治疗组及假手术组间无显著性差别;(2)模型组神经细胞静息态[Ca2+]i显著高于假手术组、治疗组(P<0.05),治疗组及假手术组间无显著性差别.结论石杉碱甲作为中枢神经系统特异性胆碱酯酶抑制剂和NMDA受体拮抗剂,可降低血管性痴呆小鼠海马静息态[Ca2+]i,并改善其临床症状;因此提示海马神经细胞静息态[Ca2+]i过高参与了血管性痴呆的发病过程.     

Intracellular Ca2+ changes in cultured vascular smooth muscle cells by treatment with various spasmogens.

In order to evaluate various spasmogens, which are candidates for cerebral vasospasm after subarachnoid haemorrhage, the intracellular calcium mobilizations were examined in cultured vascular smooth muscle cells preloaded with a fluorescent Ca2+ probe fura-2. Endothelin, oxyhaemoglobin, 5-hydroxytryptamine, norepinephrine, prostaglandin F2 alpha, leukotrienes C4 and D4 produced dose-dependent increases in intracellular Ca2+ concentration ([Ca2+]i). However, bilirubin did not induce any significant [Ca2+]i elevation. The maximal levels of [Ca2+]i peak attained by endothelin or oxyhaemoglobin were higher than those of other compounds. Endothelin was the most potent in that it induced a high sustained [Ca2+]i elevation at much lower concentrations compared with others. The combination of oxyhaemoglobin and endothelin induced a transient increase in [Ca2+]i followed by a sustained lower plateau, then the [Ca2+]i level was again increased slowly followed by a sustained higher plateau which lasted for more than 10 min after the exposure. These results suggest that endothelin and/or oxyhaemoglobin may play a crucial role in contraction of vascular smooth muscle after subarachnoid haemorrhage.     

Bradykinin-induced intracellular Ca2+ elevation in neuroblastoma X glioma hybrid NG108-15 cells; relationship to the action of inositol phospholipids metabolites

The effect of bradykinin on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator quin 2. Bradykinin induced two phases of change in [Ca2+]i. Bradykinin induced a spike phase of [Ca2+]i increase which was detectable within 15 s and decayed to near-basal concentration in 3 min and then a prolonged plateau phase of [Ca2+]i increase which continued for 15 min. The bradykinin-induced spike phase was not diminished by decreasing extracellular Ca2+ concentration ([Ca2+]o) to 1 microM. On the contrary, the plateau phase was dependent on [Ca2+]o and inhibited by Ca2+ blockers, verapamil (50 microM), nifedipine (1 microM). The iontophoretic injection of inositol-trisphosphate (IP3) into the single cell induced the increase of [Ca2+]i, which was independent of [Ca2+]o. These results indicate that the bradykinin-induced spike phase is mediated by the release of intracellular Ca2+ stores induced by IP3, while the plateau phase is mediated by influx of extracellular Ca2+ probably through voltage-sensitive Ca2+ channels.     

Mechanism of hemolysate-induced [Ca2+]i elevation in cultured fibroblasts.

Erythrocyte lysate (hemolysate) released from blood clot after subarachnoid hemorrhage is the causative agent for chronic cerebral vasospasm, a prolonged contraction of cerebral arteries. Fibroblasts, the outer layer cells of vessel wall that in contact with blood clot directly, may contribute to cerebral vasospasm. However, the effect of hemolysate on intracellular Ca2+ ([Ca2+]i) mobilization in fibroblasts has not been studied. We investigated hemolysate-induced [Ca2+]i mobilization in cultured neonatal human dermal and canine middle cerebral arterial fibroblasts by using fura-2 microfluorimetry. Hemolysate increased [Ca2+]i by releasing internal Ca2+ stores and promoting Ca2+ entry. Tyrosine kinase inhibitors partially but significantly reduced the effect of hemolysate. The major components of hemolysate, oxyhemoglobin (OxyHb) and adenosine triphosphate (ATP) failed to mimic the effect of hemolysate. In cultured canine middle cerebral arterial fibroblasts, hemolysate produced similar Ca2+ mobilization to that of dermal cells. OxyHb and ATP failed again to reproduce the effect of hemolysate. We conclude that hemolysate increases [Ca2+]i in fibroblasts and this effect of hemolysate is not mediated by OxyHb or ATP but by some unknown factors.     

Effect of endothelins on cytosolic free calcium concentration in neuroblastoma NG108-15 and NCB-20 cells

The effects of endothelin(ET)-1, ET-2, ET-3 and Big ET on intracellular level of Ca2+ ([Ca2+]i) were studied in neuroblastoma NG108-15 and NCB-20 cells. All ETs, except Big ET, induced an increase in [CA2+]i in NG108-15 cells in a dose-dependent manner, with EC50: 6.7, 11.2 and 71 nM, respectively. However, none of the ET increased [Ca2+]i in NCB-20 cells. Calcium channel blockers diltiazem or nicardipine had no effect on ET-induced increase in [CA2+]i, but extracellular Ca2(+)-depletion significantly reduced the response of NG108-15 cells to ETs. NG108-15 cells exhibited a homologous desensitization to sequential addition of ETs, but no heterologous desensitization among ET, bradykinin and PAF was observed. These data suggest that ET-induced receptor activation results in increased intracellular Ca2+ via a non voltage calcium channel mechanism and intracellular Ca2+ release.     

Variation in the pattern of [Ca2+]i change induced by acetylcholine in cultured hippocampal neurons

Acetylcholine (ACh) caused various patterns of change in the intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal neurons. We studied the underlying mechanisms of the [Ca2+]i changes with simultaneous recording of [Ca2+]i and membrane potential/current. In most cases, [Ca2+]i rise was accompanied by a membrane depolarization. The [Ca2+]i change was significantly reduced when the membrane was voltage clamped, which implies that most of the [Ca2+]i rise results from the Ca2+ influx through the voltage-gated Ca2+ channel activated by the membrane depolarization. The membrane depolarizations were classified into two types, one associated with membrane conductance decrease and the other associated with membrane conductance increase. The former results from potassium conductance ((gK+) decrease, and the latter may result from the activation of a Na(+)-permeable channel. However, [Ca2+]i elevation was also observed in some neurons showing membrane hyperpolarization in response to ACh. This seems to show that ACh liberates Ca2+ from the intracellular Ca2+ store, resulting in the activation of a calcium-dependent K+ channel (KCa). The variations of ACh response in the hippocampal neurons seem to result from a variety of muscarinic acetylcholine receptors and various species of ion channels governed by those receptors.     

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ to ouabain in carotid body glomus cells of adult rabbits.

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ ([Ca2+]i) to ouabain, an Na+/K+ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca2+]i (mean+/-S.E.M.; 38+/-5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca2+]i increase was abolished by the removal of extracellular Na+. D600 (50 microM), an L-type voltage-gated Ca2+ channel antagonist, inhibited the [Ca2+]i increase by 57+/-7% (n=4). Removal of extracellular Ca2+ eliminated the [Ca2+]i increase, but subsequent washing out of ouabain in Ca2+-free solution produced a rise in [Ca2+]i (62+/-8% increase, n=6, P<0.05), referred to as a [Ca2+]i rise after Ca2+-free/ouabain. The magnitude of the [Ca2+]i rise was larger than that of ouabain-induced [Ca2+]i increase. D600 (5 microM) inhibited the [Ca2+]i rise after Ca2+-free/ouabain by 83+/-10% (n=4). These results suggest that ouabain-induced [Ca2+]i increase was due to Ca2+ entry involving L-type Ca2+ channels which could be activated by cytosolic Na+ accumulation. Ca2+ removal might modify the [Ca2+]i response, resulting in the occurrence of a rise in [Ca2+]i after Ca2+-free/ouabain which mostly involved L-type Ca2+ channels.     

Antioxidants prevent elevation in [Ca(2+)](i) induced by low extracellular magnesium in cultured canine cerebral vascular smooth muscle cells: possible relationship to Mg(2+) deficiency-induced vasospasm and stroke

Low serum concentrations of Mg(2+) ions have been reported, recently, in patients with coronary disease, atherosclerosis and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [alpha-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Ca(2+) ([Ca(2+)](i)) overload associated with cerebral vascular injury induced by low extracellular free Mg(2+) ([Mg(2+)](o)). Exposure of cultured canine cerebral vascular smooth muscle cells to low [Mg(2+)](o) (0.15-0.6 mM) vs. normal [Mg(2+)](o) (1.2 mM) for either 10 min or 2 h induced concentration-dependent rises in [Ca(2+)](i). Treatment of the cultured cells with either PDTC (0.1 microM) or alpha-tocopherol (15 microM) for 24 h, alone, failed to interfere with basal [Ca(2+)](i) levels. However, preincubation of the cells with either alpha-tocopherol or PDTC for 24 h completely inhibited the elevation of [Ca(2+)](i) induced by exposure to low [Mg(2+)](o), not only for 10 min, but also for 2 h. These results indicate that alpha-tocopherol and PDTC prevent rises in [Ca(2+)](i) produced by low [Mg(2+)](o), which probably result from low [Mg(2+)](o)-induced lipid peroxidation of cerebral vascular smooth muscle cell membranes. Moreover, these new results suggest that such protective effects of alpha-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in cerebral vascular injury associated with low [Mg(2+)](o) and accumulation of [Ca(2+)](i).     

Low-[Mg2+]-induced Ca2+ fluctuations in organotypic hippocampal slice cultures

We show here by whole field monitoring of free intracellular Ca2+ ([Ca2+]i), locally recorded field potential (fp) and external [Ca2+], that low-[Mg2+] induces seizure like events (SLEs) accompanied by simultaneous fluctuations of [Ca2+]i and [Ca2+]e in cultured hippocampal slices. Within a SLE, complex [Ca2+]e fluctuations are seen throughout phases of Ca2+ depletion (tonic) and Ca2+ recovery (clonic) of the extracellular space. Information theory entropy-based analyses revealed strong asymmetric associations of [Ca2+]i and [Ca2+]e kinetics. By contrast, signal-associations between SLEs were found to be weak and of symmetric nature distinguishing seizure-like and interictal events by extensive coupling and decoupling of [Ca2+]i and [Ca2+]e fluctuations, respectively.