铝对大鼠海马细胞内钙水平和钙通道蛋白基因表达的影响

目的 观察铝对大鼠海马细胞内游离钙([Ca2+]i)水平和钙通道蛋白表达的影响.方法 以64只健康Wistar大鼠为研究对象,按体质量将大鼠分为16组,经口灌胃给予氯化铝(AlCl3),按AlCl3剂量(mg/kg)分为O(对照)、37.3、74.7、248.7组;染铝时间为45、75、120 d,其中120 d的大鼠再正常饲养30 d;分别在实验45、75、120、150 d处死大鼠,取脑分离海马.应用荧光分光光度计测定海马[Ca2+]i,应用RT-PCR方法检测海马中Ryanodine受体2(RyR2)和L-型钙通道α1C亚基(L-Ca2+α1C)mRNA表达情况.结果 AlCl3剂量和实验时间均可增加大鼠海马[Ca2+],(F值分别为23.136、19.089,P<0.01),二者具有交互作用(F=2.270,P<0.05);实验120、150 d时,大鼠海马[Ca2+]i,37.3、74.7、248.7 mg/kg组[(299.3±48.7)、(342.7±35.3),(391.2±47.9)、(408.1±42.8),(397.9±55.8)、(405.2±22.7)nmol/L]均较对照组[(195.1±29.9)、(209.1±30.6)nmol/L]明显升高(P<0.01).AlCl3可使大鼠海马RyR2、L-Ca2+α1C mRNA表达增加(F值分别为23.301、60.812,P<0.01).实验时间对大鼠海马中RyR2 mRNA表达无影响(F=1.361,P>0.05),但可下调L-Ca2+α1CmRNA的表达(F=6.088,P<0.01);AlCl3剂量和实验时间对L-Ca2+α1C mRNA表达有交互作用(F=5.876,P<0.01).实验75、120、150 d时,大鼠海马L-Ca2+α1C mRNA表达水平74.7、248.7 mg/kg组(1.03±0.16、1.18±0.18、0.92±0.11,1.89±0.26、1.25±0.10、1.07±0.14)均较同时间对照组(0.63±0.09、0.78±0.16、0.69±0.11)明显增加(P<0.05或<0.01).实验45、75、120、150 d时,大鼠海马RyR2 mRNA表达水平,74.7、248.7ms/kg组(0.49±0.06、0.51±0.07、0.57±0.11、0.47±0.11,0.47±0.03、0.52±0.09、0.70±0.10、0.78±0.09)均较同时间对照组组(0.24±0.07、0.32±0.04、0.30±0.06、0.27±0.06)明显升高(P<0.05或<0.01).结论 铝可以通过上调RyR2 mRNA和L-Ca2+α1C mRNA的表达而增加海马[Ca2+]i,发挥不可恢复性的神经毒性作用.     

慢性铝暴露对大鼠海马钙离子稳态及CaMK Ⅱ和CREB活性的影响

目的通过研究慢性铝暴露对大鼠海马钙离子稳态及Ca MKⅡ和CREB活性的影响,探讨慢性铝暴露诱导神经损伤可能存在的机制。方法选用60只断乳后雄性Wistar大鼠,分为3组,每组20只;设定其中1组为对照组,用蒸馏水喂养;其他2组为铝暴露组,分别用含有0.2%和0.4%Al Cl3的蒸馏水喂养3个月。Morris水迷宫测定各组大鼠学习记忆能力;Fura-2/AM测定各组大鼠海马神经细胞自由Ca2+浓度变化;Western印迹方法检测各组大鼠海马中Ca MKⅡ、Ng和CREB的蛋白表达情况。结果大鼠的学习记忆能力明显降低;与对照组相比,铝暴露各组海马神经细胞中〔Ca2+〕i呈剂量依赖性升高;Ca MKⅡ和CREB总蛋白表达无显著变化,但p-Ca MKⅡ、Ng和p-CREB蛋白表达显著降低(P0.05)。结论慢性铝暴露损伤大鼠的学习记忆可能与上调Ca2+及抑制Ca MKⅡ和CREB活性相关。     

平滑肌胞内钙对L型钙通道的调控

平滑肌胞内钙对Ｌ型钙通道的调控桂培春（中国医学科学院基础医学研究所生理研究室，北京１００００５）ＭｏｄｕｌａｔｉｏｎｏｆＬＴｙｐｅＣａｌｃｉｕｍＣｈａｎｎｅｌｂｙ［Ｃａ２＋］ｉｉｎＳｍｏｏｔｈＭｕｓｃｌｅｃｅｌＧｕｉＰｅｉｃｈｕｎ（Ｄｅｐａｒｔｍｅｎ...     

铝对大鼠海马组织形态学及细胞凋亡相关基因表达的影响

目的 研究三氯化铝对大鼠海马组织形态学及细胞凋亡相关基因表达的影响.方法 选择雄性SD大鼠,按体重随机分为4组,在基础饲料中添加三氯化铝,Al3+剂量分别为0、11.2、55.9和111.9 mg/kg体重,连续染毒3个月.采用流式细胞仪检测大鼠海马Bcl-2 、Bax、p53表达,原子吸收法检测海马铝含量,并观察海马病理形态学变化.结果 与对照组相比,各染毒组大鼠海马Bcl-2表达明显减少(P＜0.05)、Bax、p53表达明显增加(P＜0.05),且随剂量增加渐趋明显.各染毒组大鼠海马铝含量均显著增高(P＜0.05),并与染毒剂量呈明显正相关(r=0.805,P＜0.01);病理检查发现111.9 mg/kg组大鼠海马颗粒空泡变性细胞明显增多,海马和皮质锥体细胞明显减少,并可见神经原纤维缠结样病理改变.结论 铝诱导大鼠出现AD典型的病理改变,其机制可能与Bcl-2表达下调及Bax、p53表达上调有关.     

异丙肾上腺素对心肌细胞内钙释放和肌浆网内钙容量的影响

目的 :心肌细胞膜上的 β-肾上腺素受体的激活对兴奋 -收缩耦联 （ECC）过程有重要的调节作用。本课题利用异丙肾上腺素 （ISO,1μmol/ L）激活 β-肾上腺素受体从而研究其对源自心肌细胞肌浆网的胞内钙释放 （ECC的重要环节 ）和肌浆网内钙容量的影响 ,进而分析钙释放与钙容量之间的关系。方法 :局部场刺激作用于成年大鼠心肌细胞 ,促使后者产生动作电位 ,进而诱发胞内钙瞬变 （ACT） ,由 ACT可估测胞内钙释放。肌浆网内钙容量则由咖啡因 （2 0 m mol/ L）诱发的钙瞬变 （CCT）估测。实验结果均由 Zeiss L SM- 5 10激光共聚焦显微镜系统记录。结果 :ISO作用下的 ACT峰值为 10 .2 9± 0 .35 （n=13）比正常情况下的 5 .74± 0 .2 7（n=18）高 （P<0 .0 1）。 ISO作用下的CCT峰值为 11.2 3± 0 .2 9（n=13）比正常情况下的 7.6 2± 0 .2 4 （n=18）高 （P<0 .0 1）。结论 :ISO可明显地提高心肌细胞内钙释放量和肌浆网内的钙容量。不管有无 ISO存在 ,胞内钙释放量总是只占肌浆网内钙容量的一部分。在正常情况下 ,心肌的钙释放量有较大的储备能力 ,且此储备可因β-肾上腺素受体的激活而动员。     

褪黑素对铝毒性痴呆小鼠海马组织病理变化的影响

有研究显示 ,褪黑素 (melatonin ,MLT)具有逆转阿尔茨海默病 (AD)动物学习记忆功能障碍的作用〔1,2〕。我们于2 0 0 1年 3～ 10月在铝毒性小鼠AD模型上 ,观察MLT对铝毒性痴呆小鼠海马组织病理变化 ,旨在探讨MLT对AD特征性病理变化是否具有改善作用。　　一、材料和方法　　 1.药品和试剂 :MLT为美国Sigma公司产品 ,使用前用无水乙醇溶解 ,再加生理盐水配制 ,乙醇终浓度为 5 %。氯化铝 (AlCl3 ·6H2 O ,分析纯 )为上海金山化工厂生产 ;其他试剂均为国产分析纯。　　 2 .动物 :雄性昆明种小鼠 ,体重 18～ 2 2 g ,福建医科大学实验动…     

增龄对大鼠海马N-Shc蛋白的影响

目的 研究不同月龄大鼠海马N-Shc蛋白的表达,以及经氧化应激后N-Shc磷酸化水平的改变随年龄的增加而变化的规律.方法 采用蛋白免疫印迹的方法,分别分析幼年组、成年组和老年组大鼠海马N-Shc蛋白的表达水平,以及经H2O2刺激后N-Shc磷酸化水平的改变.结果 幼年组、成年组、老年组海马N-Shc蛋白的含量依次减少(P＜0.05),但成年组和老年组之间没有显著性差异;H2O2处理后,幼年组、成年组、老年组海马N-Shc蛋白磷酸化水平也依次减少(P＜0.01),而成年组和老年组之间无显著差异.结论 随着年龄的增加,海马中N-Shc蛋白的表达逐渐减少并且N-Shc蛋白的活性逐渐降低.     

大鼠孕期染铝对子鼠海马星形胶质细胞的影响

目的 探讨母体孕期染铝对子代脑神经发育的毒性作用.方法 4～5月龄SD大鼠,雌雄各30只,1:1合笼喂养,每日早晨查雌鼠阴栓,发现阴栓即进入实验(EO),将孕鼠随机分为两组.染铝组ED～E18期间以AlCl3100mg/(ks·d)灌胃;正常对照组以等量蒸馏水灌胃.分别取孕鼠三个年龄段子代即18 d胚胎、生后继续喂养的1月子代及3月子代鼠海马组织,每只孕鼠任取其子代2只,每小组子代10只.常规石蜡切片,行胶质纤维酸性蛋白(GFAP)免疫组化染色,计数海马CAP区GFAP免疫组化染色阳性反应细胞,并用细胞形态学计量方法测量各阳性反应产物的平均光密度值.结果 铝中毒组子代GFAP阳性细胞明显多于对照组(P＜0.05),阳性反应产物平均光密度明显高于对照组(P＜0.05).结论 大鼠孕期染铝可导致子代海马CA2区星形胶质细胞增多;从而导致子代鼠神经发育障碍.     

氯沙坦对心力衰竭兔肌浆网钙调控蛋白的影响

目的探讨血管紧张素Ⅱ受体拮抗剂氯沙坦干预慢性心力衰竭对兔心肌肌浆网钙泵（SERCA2）、钙释放通道（RyR2）、受磷蛋白（PLB）基因表达的影响及意义。方法通过结扎兔冠状动脉前降支复制心肌梗死（心梗）模型,以氯沙坦进行干预。于心梗后8周比较观察左室结构、血流动力学的变化及SERCA2、RyR2、PLB基因的表达。结果与对照组相比,心梗组左室舒张末压（LVEDP）显著升高（P〈0．01）,左室压力上升和下降最大速度（＋dr,／dtmax、-dp／dtmax）显著降低（P〈0．01）;氯沙坦组LVEDP显著低于心梗组（P〈0．05）,＋dp／dtmax、-dp／dtmax显著高于心梗组（P〈0．05）。心梗组SERCA2、RyR2、PLBmRNA显著低于对照组（P〈0．01）,而氯沙坦组的上述三项显著高于心梗组（P〈0．05）。结论氯沙坦长期干预心力衰竭,能够改善心脏舒缩功能,可能与其上调肌浆网的钙调控蛋白SERCA2、RyR2、PLB的基因表达有关。     

钙通道拮抗剂对大鼠胰岛细胞胰岛素分泌的影响

目的　研究钙通道拮抗剂对大鼠胰岛细胞分泌胰岛素的影响和机制。方法　用放免和荧光方法分别检测不同治疗血浓度的硝苯地平 (NIF)、维拉帕米 (VER)、地尔硫 (DIL)等药物对低糖和高糖刺激状态下胰岛细胞Ca2 含量和胰岛素分泌量的影响。结果　低糖状态组 :NIF、VER、DIL在 2 5、50、10 0 μg/L药物浓度下未对胰岛细胞内Ca2 含量和胰岛素分泌量产生影响 ,其结果与对照组相比差异无统计学意义 (P >0 0 5)。在高糖状态组 ,NIF、VER、DIL在 2 5μg/L药物浓度下不抑制胰岛素分泌 ,NIF在 50、10 0 μg/L浓度时 ,胰岛细胞内Ca2 浓度和胰岛素分泌量明显减少 ,与对照组相比差异有显著性 (P <0 0 5)并呈剂量相关 (P <0 0 1)。VER在 50 μg/L时胰岛素分泌有减少趋势 ,10 0 μg/L时明显减少 (P <0 0 5)。DIL在 10 0 μg/L时胰岛素分泌量减少与对照组相比差异具有显著性 (P <0 0 5)。结论　高浓度药理治疗量的NIF、VER、DIL具有抑制高糖作用下的胰岛细胞外钙内流和使胰岛素分泌减少的作用     

玉郎伞多糖对Aβ25-35所致PC12细胞损伤细胞内钙离子浓度的影响

目的观察Aβ25-35诱导PC12细胞凋亡损伤模型胞内钙浓度的变化及YLSP的影响。方法采用Aβ25-35诱导PC12细胞凋亡损伤模型,石杉碱甲作为对照药,给予YLSP预处理后,加入Flu-3/AM,用流式细胞仪（FCM）测定各组细胞的平均荧光强度。结果各组PC12细胞内钙离子含量存在显著性差异（F=14.051,P〈0.01）,其中,Aβ25-35作用PC12细胞后,PC12细胞内钙离子含量显著升高（P〈0.01）,经YLSP处理后,PC12细胞内钙离子含量较模型组细胞显著降低（P〈0.01）,其中,YLSP高剂量组的PC12细胞内钙离子含量显著低于石杉碱甲组（P〈0.01）。结论 YLSP能对抗Aβ引起的细胞内钙离子浓度升高,防止钙超载的发生。     

Endothelin does not affect intracellular calcium ion concentration in the platelets of rats

We recently demonstrated that porcine endothelin increases intracellular calcium ion concentration ([Ca2+]i) in monolayers of cultured vascular smooth muscle cells isolated from rat renal arteries. The present study was designed to examine the effect of porcine endothelin on [Ca2+]i in the platelets of rats. [Ca2+]i was serially measured with the fluorescent dye, fura 2, and the photoprotein, aequorin. Endothelin (10(-9) mol/L, 10(-8) mol/L, and 10(-7) mol/L) did not induce any change in [Ca2+]i in the platelets of rats while thrombin (2 U/mL) dramatically increased [Ca2+]i as measured by these methods. Our results indicate that [Ca2+]i in rat platelets is not influenced by porcine endothelin.     

Effects of metabolic blockade on intracellular calcium concentration in isolated ferret ventricular muscle

Tension and intracellular free calcium concentration [( Ca2+]i) were measured in isolated ferret papillary muscles. When both anaerobic glycolysis and oxidative phosphorylation were prevented (metabolic blockade), there was a rapid decline of both developed tension and systolic [Ca2+]i signals. Subsequently, resting tension increased, and after a further delay, resting [Ca2+]i also rose. When oxidative metabolism was restarted after a period of metabolic blockade that was sufficient to elevate both resting tension and [Ca2+]i, a variable recovery of mechanical function occurred. In preparations that showed recovery, resting tension declined toward control level, and there was considerable recovery of developed tension. [Ca2+]i initially fell, but it then rose to a level similar to that at the end of the preceding period of metabolic blockade and exhibited large variations in amplitude with frequency components in the range 0.2-1 Hz. This elevated [Ca2+]i gradually declined. Arrhythmias were often present during this recovery period and appeared to be triggered by the spontaneous increases in [Ca2+]i. In preparations that failed to recover, resting tension remained elevated or increased, and developed tension showed little recovery. Such preparations showed larger rises in [Ca2+]i both during and after metabolic blockade, and [Ca2+]i continued to rise when oxidative metabolism was restarted. In experiments in which Na-Ca exchange was inhibited (by replacement of sodium by lithium or by the application of nickel), the rise of [Ca2+]i when oxidative metabolism was restarted was reduced, but recovery of mechanical function was improved. The correlation between elevated [Ca2+]i on reactivation of oxidative metabolism and failure of recovery of mechanical function suggests that elevated [Ca2+]i has a direct role in preventing the recovery of mechanical function.     

Mechanical stretch increases intracellular calcium concentration in cultured ventricular cells from neonatal rats

Summary We investigated the effects of mechanical stretch on intracellular calcium concentration ([Ca²⁺]_i) of cultured neonatal rat ventricular cells using micro-fluorometry with fura-2. Myocytes were cultured on laminin-coated silicon rubber and stretched by pulling the rubber with a manipulator. Myocytes were either mildly stretched (to less than 115% of control length), moderately so (to 115%–125% of control length), or extensively (to over 125% of the control length). “Quick stretches” (accomplished within 10s) of moderate to extensive intensities produced a large transient increase of [Ca²⁺]_i in the early phase of stretch (30s-2 min), followed by a small but sustained increase during the late phase of stretch (5–10 min). The initial transient increase in [Ca²⁺]_i after the “quick stretch” was preserved in the presence of gallopamil (10⁻⁷M) or ryanodine (10⁻⁵ M), but was absent in Ca²⁺-free medium or in the presence of gadolinium (10⁻⁷M). The late or steady state [Ca²⁺]_i increase was observed in the presence of gadolinium, gallopamil, or ryanodine but was abolished in Ca²⁺-free medium. A steady-state increase in [Ca²⁺]_i was also evoked by “slow stretch” in which cells were slowly pulled to the final length within 1–2min. As the presence of external Ca²⁺ was indispensable, increased trans-sarcolemmal Ca²⁺ influx appears to be involved in both initial and steady-state increases in [Ca²⁺]_i. The initial increase in [Ca²⁺]_i after the “quick stretch” can be attributed to the activation of gadolinium-sensitive, stretch-activated channels.     

Control of intracellular ionized calcium concentration by sarcolemmal and intracellular mechanisms

The regulation of the resting intracellular ionized calcium concentration [( Ca2+]i) has been studied in ferret papillary muscle using the photoprotein aequorin to measure [Ca2+]i. Elevating [Ca2+]o produced an initial rapid increase of [Ca2+]i and tension which then decayed to a steady level. This secondary fall of [Ca2+]i is attributed to a secondary decrease of Ca entry on Na-Ca exchange produced by the known fall of [Na+]i. Replacing external Na by K produced a large transient increase of both [Ca2+]i and tension which then decayed spontaneously to near the resting level. If Na was removed after metabolic inhibition with cyanide and deoxyglucose then neither tension nor [Ca2+]i recovered. The addition of the mitochondrial uncoupler FCCP to a muscle in Na-free solution produced a gradual rise of tension but only elevated [Ca2+]i after a delay of many minutes. Similarly caffeine did not elevate [Ca2+]i. These experiments do not support the hypothesis that the regulation of resting [Ca2+]i in Na-free solutions depends solely on intracellular sequestration of [Ca2+]i. The first twitch elicited in Na-containing solutions after exposure to Na-free solution was much larger than control and was associated with a large Ca transient attributed to increased loading of the sarcoplasmic reticulum with Ca in the Na-free solution. The elevation of [Ca2+]i in Na-free solutions was accompanied by spontaneous fluctuations of both [Ca2+]i and tension with a frequency of about 3 Hz. These fluctuations were abolished by drugs such as caffeine or ryanodine which interfere with sarcoplasmic reticulum function. These results provide direct evidence for the spontaneous release of Ca from the sarcoplasmic reticulum inferred from previous, less direct, work.     

Tissue-specific expression of calcium channels

The high-voltage-activated calcium channel is a multimeric protein complex containing α(1), α(2)/δ, β, and γ subunits. The α(1) subunit is the ion conduction channel and contains the binding sites for calcium channel blockers and toxins. Three genes code for distinct L-type, dihydropyridine-sensitive α(1) subunits; one gene codes for the neuronal P-type (Purkinje) α(1) subunit; and one gene codes for the neuronal N-type α(1) subunit. The smooth and cardiac muscle L-type calcium channel α(1) subunits are splice variants of the same gene. The α(1) subunits are coexpressed with a common α(2)/δ subunit and tissue-specific β subunits (at least three genes). The γ subunit apparently is expressed only in skeletal muscle. The properties of these cloned and expressed calcium channels are discussed here.     

丝胶对2型糖尿病大鼠海马Bad表达的影响

目的探讨丝胶(彩色蚕茧水提物)对2型糖尿病模型大鼠海马Bad表达的影响。方法取健康雄性SD大鼠,以25 mg/kg的链脲佐菌素连续(1次/d,注射3 d)腹腔注射的方法建立2型糖尿病大鼠模型,模型成功建立后给予模型大鼠丝胶(2.4 g·kg~(-1)·d~(-1))灌胃35 d,同时以二甲双胍(55.33 mg·kg~(-1)·d~(-1))作为阳性对照药物。分别采用蛋白印迹法和逆转录PCR法检测大鼠海马Bad蛋白和mRNA的表达情况。结果与糖尿病模型大鼠比较,丝胶组大鼠海马Bad蛋白和mRNA的表达明显降低(P<0.05)。结论丝胶可通过下调糖尿病模型大鼠海马Bad的表达,减弱Bad的促凋亡作用,这可能是丝胶保护糖尿病海马损伤的机制之一。     

COX-2抑制剂对慢性铝过负荷大鼠海马5-脂氧酶表达的影响

目的探讨COX-2抑制剂美洛昔康对慢性铝过负荷大鼠海马5-LO表达的影响。方法葡萄糖酸铝灌胃给予Wistar大鼠,1次/d,每周5 d,连续20 w,建立慢性铝过负荷致神经元退变大鼠模型。美洛昔康(1和3 mg/kg),在每次给予铝盐后30 min灌胃给予。RT-PCR检测大鼠海马5-LO mRNA的表达变化,Western印迹方法检测5-LO蛋白表达情况。结果慢性铝过负荷致大鼠海马5-LO mRNA和蛋白表达明显增加。美洛昔康能明显阻遏慢性铝过负荷诱导的大鼠海马5-LO mRNA和蛋白表达的增加。结论 COX-2抑制剂明显下调慢性铝过负荷大鼠海马5-LO表达。     

Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): Effects on intracellular calcium and platelet aggregation

Summary The goal of the present study was to evaluate if mibefradil, a novel nondihydropyridine Ca²⁺ antagonist, could block receptor-operated calcium channels (ROCC) present in human platelets and to determine the functional consequences of this blockade. Therefore, the effect of mibefradil on increases in intracellular Ca²⁺ concentrations and aggregation of human platelets induced by platelet activating factor (PAF) was examined. In order to differentiate effects on Ca²⁺ mobilization from intracellular stores from those on Ca²⁺ influx through ROCC, intracellular Ca²⁺ concentrations were measured either in fura-2-loaded platelets or in cells loaded with both BAPTA and fura-2. Mibefradil totally and dose dependently inhibited PAF-induced Ca²⁺ influx with a maximal effective concentration of 10 µM, but at this concentration only reduced Ca²⁺ mobilization from intracellular stores. A similar effect was observed when platelets were stimulated with ADP, suggesting that mibefradil was indeed interfering with ROCC and not specifically with PAF receptors. In the same range of concentrations, mibefradil inhibited Ca²⁺-dependent platelet aggregation induced by PAF. This effect was most likely due to the inhibition of ROCC, as Ca²⁺-independent aggregation induced by phorbolmyristyl-acetate (PMA) was insensitive to mibefradil. We conclude that mibefradil, which has previously been described to be an antagonist for L- and T-Type Ca²⁺ channels, also blocks receptor-operated Ca²⁺ channels. This blockade seems to be functionally relevant for platelet aggregation.