【作者】 李石；

【导师】 王永利；

【作者基本信息】 河北医科大学 ， 药理学， 2007， 博士

【摘要】 脑缺氧导致严重的细胞变性,因而脑功能丧失。当前普遍认为,缺氧损伤中谷氨酸大量释放,导致细胞内过量的Ca2+蓄积,从而引起神经元死亡。钠泵又称Na+, K+-ATP酶,是一种膜结合蛋白,可通过水解一分子ATP将三个Na+转运到细胞外同时摄取两个K+进入细胞,产生电化学梯度,维持细胞渗透压平衡和静息电位,因此钠泵是维持Na+动态平衡、体液和电解质稳态的关键。当哺乳动物脑组织的氧或血流供应低于临界值时就会出现能量障碍,仅仅缺氧5 min ATP下降90%,而丢失50～60%的ATP时钠泵活性下降,细胞膜去极化进而钠和水进入细胞内。去极化使电压门控性钙通道开放Ca2+内流,同时崩塌的Na+梯度引起Na+依赖性谷氨酸转运体排除谷氨酸到细胞间隙,激活谷氨酸受体导致大量Ca2+内流,继而出现Ca2+依赖性细胞损伤。由此可见,钠泵在脑缺血的发生、发展过程中起着重要的作用。但目前对其研究大多数集中在心肌细胞、肾脏、骨骼肌、血管平滑肌,而对脑组织钠泵的分布及功能特性研究较少,特别是其在脑缺血、缺氧状态下,功能和特性的改变及其可能机制的研究甚少,本研究旨在选取对缺血较敏感的皮层神经元(Ⅴ和Ⅵ层锥体细胞),研究钠泵电生理特性及其在缺氧中的改变,并探讨其可能机制。一、脑片皮层神经元钠泵的电生理特性目的:应用脑片皮层神经元研究钠泵电流的特性。方法:选取出生11~14天SD大鼠,深度麻醉后断头,快速取出脑组织放入冰冷的人工脑脊液(ACSF)中,取前脑切成300μm的薄片,储存在95%O2+5%CO2饱和的ACSF中。然后将制备好的皮层脑片放置于灌流槽中,持续灌流95% O2和5% CO2饱和的ASCF,流速为1～2ml/min,使脑片浸没于液面下。在红外微分干涉相差显微镜下,移动电极至细胞上方负压吸引形成封接,再以负压吸破细胞膜,使电极内液与细胞内液相通,并将细胞钳制在-60mV,当膜电流稳定时,先通过膜去极化程序(-60mV—0mV—-60mV)激活Na+电流来鉴别正在记录的细胞是神经元,然后换为含不同浓度哇巴因(Oua,10-12～10-3 mol/L)的ACSF,以全细胞膜片钳方式记录皮层神经元钠泵电流,并以10-3 mol/L Oua产生的泵电流幅度为100%,计算各浓度Oua产生的泵电流(Ip)的变化率(ΔIp),并以此为纵坐标,以Oua浓度([Oua])为横坐标,绘制ΔIp-[Oua]关系曲线。整个实验在室温(23±2℃)下进行结果:实验结果表明,当膜电压钳制在-60mV时,低浓度Oua灌流可引起膜电流的外向移动,但较高浓度的Oua灌流,则可剂量依赖性引起膜电流的内向移动,其中1 mmol/L Oua引起的膜电流内向移动幅度与切换无K细胞外液所致的膜电流内向移动幅度相同,证明该Oua敏感性电流为钠泵电流。本研究采用10-12~10-3 mol/L的Oua分别在96个神经元测定了泵电流,根据神经元钠泵对10-8 mol/L Oua的反应,可将钠泵电流对Oua(10-12～10-3 mol/L)的ΔIp-[Oua]关系曲线分为兴奋、抑制和无作用三种模式,。兴奋模式ΔIp-[Oua]关系曲线中10-12～10-3 mol/L Oua所对应的ΔIp值分别是0.027±0.114、0.032±0.082、0.050±0.213、0.172±0.226、0.125±0.152、-0.035±0.036、-0.146±0.124、-0.226±0.161、-0.421±0.076、-0.638±0.138和-1。其ΔIp-[Oua]关系曲线呈三位点结合特征,采用双亚基三位点结合模型可进行最优拟和,包括高亲和力兴奋性位点、高亲和力抑制性位点和低亲和力抑制性位点,其解离常数分别为0.31 nmol/L、41.27 nmol/L和152.48μmol/L。抑制模式ΔIp-[Oua]关系曲线中10-12～10-3 mol/LOua所对应的ΔIp值分别是-0.011±0.072、-0.028±0.071、-0.057±0.079、-0.093±0.063、-0.115±0.069、-0.124±0.067、-0.157±0.094、-0.281±0.144、-0.421±0.076、-0.690±0.112和-1.0。其ΔIp-[Oua]关系曲线呈两位点结合特征,采用双位点结合模型可进行最优拟和,包括高亲和力抑制性位点和低亲和力抑制性位点,其解离常数分别为71.12μmol/L和176.51μmol/L。无作用模式ΔIp-[Oua]关系曲线中10-12~10-7 mol/L Oua对钠泵电流无作用,10-6～10-3 mol/L Oua剂量依赖性抑制钠泵电流。其ΔIp-[Oua]关系曲线呈单位点结合特征,采用单位点结合模型可进行最优拟和,仅包括低亲和力抑制性位点,其解离常数为149μmol/L。综合以上三种模式,高亲和力钠泵电流占总泵电流的14.59%,可能经α2或α3亚基介导,而低亲和力钠泵电流占总泵电流的85.41%,可能经α1亚基介导。此外,钠泵电流还拥有电压依赖性,其I-V曲线从-90～+40 mV电压范围斜率为正值,翻转电位在-20 mV左右。结论:皮层的锥体神经元表达两种功能不同的钠泵,即高亲和力钠泵和低亲和力钠泵。钠泵电流不仅具有Oua敏感性还具有电压依赖性。二、缺氧对皮层神经元钠泵的影响目的:研究缺氧对皮层神经元钠泵活性和α亚基表达的影响。方法:通过缺氧培养皮层神经元以及用N2饱和的无糖ACSF灌流皮层脑片,建立皮层神经元的氧糖分离缺氧模型。通过检测缺氧0、2、4、8和12小时后培养皮层神经元培养基中乳酸脱氢酶(LDH)含量,评价缺氧对培养皮层神经元的损伤程度;应用激光共聚焦显微镜检测缺氧0、0.5、1、2、4和8小时后培养皮层神经元内Ca2+浓度([Ca2+]i)(fluo-3的荧光强度)的变化;采用TTC染色评价缺氧0、5、10、15、30、45和60min对皮层脑片神经元活性的影响。通过无机磷分光光度计法,检测缺氧培养皮层神经元在缺氧0、2、4和8小时钠泵活性的改变;通过双酶法,检测皮层脑片缺氧0、15、30和60min时钠泵活性的改变。采用脑片膜片钳装置观察缺氧0、2、4、6、8、10、15和20min对脑片皮层神经元钠泵电流的改变。采用RT-PCR和Westernblot方法,观察缺氧0、4、8和12小时对培养皮层神经元α1和α3亚基mRNA和蛋白表达的影响,以及缺氧0、5、10、15、30和60min对皮层脑片α1和α3亚基mRNA和蛋白表达的影响。结果:当培养的皮层神经元缺氧达4 h时,培养基中的LDH从正常状态下的324±16增加到417±50 U/L(p<0.05),随着缺氧时间的延长,培养基中的LDH显著增加,缺氧8 h时为584±98 U/L,缺氧12 h增加到791±87 U/L(p<0.05),提示缺氧可引起皮层神经元损伤。细胞内Ca2+超载是细胞受损另一具体表现,在无糖和缺氧培养条件下,培养的皮层神经元[Ca2+]i持续升高,在缺氧1 h时达到高峰(1.71±0.46),且在缺氧2、4和8 h时仍维持在高峰水平。用无糖低氧ACSF持续灌流皮层脑片仅5 min,活性即有明显降低(0.292±0.019,P<0.01),直至缺氧15min降低幅度并无明显改变,然而当缺氧时间继续延长时急剧下降(0.257±0.012,P<0.01)。随缺氧培养时间的延长,皮层神经元钠泵活性先升高后降低,正常状态下钠泵活性为1626±122μmol/NADH/mg·protein/h,缺氧培养2 h时其活性明显上升(4114±472μmol/NADH/mg·protein/h,p<0.01),然后钠泵活性随缺氧时间的延长逐渐下降,在缺氧4 h时显著减小到864±318μmol/NADH/mg·protein/h(p<0.01),继续缺氧到8 h时,其钠泵活性进一步大幅度下降,显著低于缺氧4 h时( 497±86μmol/NADH/mg·protein/h,p<0.05)。正常的皮层脑片钠泵活性是169±32μmol/mg protein/h,在缺氧30 min时其活性为572±28μmol/mg protein/h,显著高于正常皮层脑片(P<0.05),但在缺氧60 min后,脑片钠泵的活性则明显下降(243±72μmol/mg protein/h,P<0.05)。皮层脑片分别缺氧0、2、4、6、8、10、15和20 min后,钠泵电流先降低后升高。在缺氧4 min后钠泵电流即显著降低(从0.265±0.068 pA/pF降至0.160±0.046 pA/pF,P<0.01),但是当神经元缺氧20 min后泵电流却明显升高,为0.243±0.054 pA/pF(P<0.05)。用高浓度Oua(1mmol/L)完全抑制钠泵功能后,用95% O2和5% CO2饱和的ACSF冲洗,维持正常生理环境,则膜电流可恢复到未给Oua的初始水平,表明钠泵功能可以恢复。但若给予用95% N2和5% CO2饱和的ACSF冲洗,则膜电流不能恢复到未给Oua的初始水平,表明其钠泵功能在缺氧状态下不能恢复,提示缺氧可抑制钠泵功能。缺氧导致培养皮层神经元钠泵α1亚基mRNA表达增加,在缺氧0、2、4、8和12 h时mRNA水平分别为0.822±0.050、0.873±0.096、0.960±0.089、0.938±0.080和0.829±0.098,在缺氧4和8 h时有显著差别(p<0.01);α3亚基mRNA水平的改变与α1相反,出现下降的改变。在缺氧8 h时mRNA水平由0 h的1.173±0.156显著下降到0.540±0.076(p<0.01),到第12 h仍维持在较低水平0.536±0.100(p<0.01)。离体皮层脑片分别缺氧5、10、15、30和60 min后,α1和α3亚基mRNA表达随缺氧时间的延长变化不明显(P>0.05)。缺氧培养皮层神经元钠泵α1亚基的蛋白水平增加,同其mRNA的改变,在缺氧0、4和8 h时蛋白相对含量分别为0.471±0.0519、0.517±0.152和0.573±0.106 ,呈现增加的趋势但没有统计学意义( p>0.05),在缺氧12 h时蛋白含量显著增加到0.806±0.0776(p<0.01)。α3亚基的蛋白水平在缺氧后的变化也同其mRNA水平的改变,有明显的下降。在缺氧4 h时蛋白水平由0 h的0.730±0.065下降到0.501±0.097(p<0.05),以后随缺氧时间的延长而持续下降,8和12 h分别为0.446±0.077和0.448±0.109(p<0.01)。缺氧培养的皮层神经元上,α1亚基随缺氧时间的延长可见神经元荧光强度明显增强,而α3亚基明显减弱,支持蛋白的改变。离体皮层脑片分别缺氧15、30和60 min后,钠泵α1和α3亚基蛋白水平无明显改变(P<0.05)。结论:采用缺氧孵育脑片和缺氧培养神经元模型可成功模拟两种不同的缺氧性脑损伤。在缺氧早期阶段皮层神经元钠泵活性代偿性升高,但随缺氧时间的逐渐延长活性降低。缺氧培养调节皮层神经元mRNA和蛋白水平的表达,α1亚基表达升高,α3亚基表达降低三、低亲和力钠泵参与了皮层神经元缺氧损伤目的:比较皮层神经元高亲和力钠泵和低亲和力钠泵在缺氧损伤中的作用。方法:通过灌流不同时间无糖低氧ACSF,制备培养的皮层神经元和皮层脑片的氧糖分离模型。缺氧0、2、4、6、8或10min观察皮层脑片神经元膜电流的改变(?I/Cm),或者给予TTX(1μmol/L)后同时缺氧,或者缺氧6min时给予TTX继续缺氧观测膜电流的改变。钠泵对缺氧所致皮层脑片神经元膜电流变化的影响:采用Oua 10 nmol/L兴奋高亲和力钠泵、Oua 100 nmol/L抑制高亲和力钠泵或者Oua 10μmol/L部分抑制低亲和力钠泵后,检测缺氧0、2、4、6、8和10 min时?I/Cm。钠泵对缺氧所致皮层脑片神经元[Ca2+]i和[Na+]i变化的影响:采用可视化动缘探测系统检测培养的皮层神经元[Ca2+]i和[Na+]i荧光信号(Fura-2 10μmol/L和SBFI 10μmol/L),①无氧无糖的HBS灌流培养的皮层神经元10 min,分别于缺氧0、2、4、6、8和10 min时测定神经元[Ca2+]i荧光比值(?Ratio),比较不同时间点?Ratio。②连续灌流含Oua(10-9～10-3 mol/L)的正常HBS 2 min,测定[Na+]i ?Ratio。③以Oua 100 nmol/L、10μmol/L或1 mmol/L的正常HBS预先处理皮层神经元,然后换为含有同样浓度Oua的无氧无糖HBS,连续记录10 min,测量0、2、4、6、8和10 min时神经元[Ca2+]i ?Ratio。结果:缺氧增大神经元膜电流:无糖低氧ACSF灌流10 min,神经元膜电流则逐渐增大(P<0.01),在缺氧0、2、4、6、8和10 min时,其增加的膜电流密度分别为0、-0.014±0.006、-0.028±0.013、-0.035±0.016、-0.044±0.022、-0.058±0.026和-0.074±0.025 pA/pF,呈时间依赖性升高(r=0.98028,P<0.01),说明缺氧可引起皮层神经元膜上离子通道的异常开放。缺氧增大的膜电流为TTX敏感性Na通道电流:在阻断K+和Ca2+通道的液体环境中,所记录到的膜电流主要为钠电流,缺氧引起的脑片皮层神经元膜电流增加可以被TTX(1μmol/L)阻断。若先使神经元急性缺氧6 min后再给予TTX,则TTX可抑制缺氧增加的膜电流继续增大,仅使其维持在缺氧6 min时的电流水平而不能恢复至正常,在10 min时,膜电流的改变值为-0.033±0.013 pA/pF,与缺氧10 min时-0.058±0.028 pA/pF相比较有显著差异(P<0.01)。提示缺氧引起的膜电流增加可能与去极化所致的钠通道开放有关。钠泵对缺氧增大皮层神经元膜电流的影响:Oua 10μmol/L部分阻断低亲和力钠泵可促进缺氧所致膜电流的增大,在缺氧0、2、4、6、8和10 min时,?I/Cm分别为0、-0.031±0.010、-0.041±0.013、-0.053±0.007、-0.057±0.008和-0.069±0.012 pA/pF(P<0.05)。Oua 100 nmol/L的条件下急性缺氧10 min,?I/Cm呈降低趋势,分别为0、-0.021±0.003、-0.028±0.006、-0.038±0.016、-0.047±0.018和-0.055±0.020 pA/pF(P>0.05),但当同时给予Oua 10 nmol/L和缺氧, ?I/Cm较单纯缺氧明显减小,分别为0、-0.009±0.011、-0.011±0.015、-0.010±0.016、-0.009±0.018和-0.012±0.020 pA/pF ,(P<0.01),表明10 nmol/L Oua在一定程度可保护皮层神经元对抗缺氧性损伤。Oua对培养皮层神经元[Ca2+]i和[Na+]i的影响:有氧状态下,不同浓度的Oua抑制钠泵剂量依赖性地显著升高皮层神经元[Na+]i和[Ca2+]i。当Oua的浓度为10-5～10-3mol/L时,[Na+]i的ΔRatio值显著增加,分别为0.017±0.004、0.030±0.004和0.035±0.004(P<0.01);当Oua的浓度为10-7～10-3mol/L时, [Ca2+]i的ΔRatio值显著增加,分别为0.056±0.013、0.107±0.018、0.158±0.011、0.185±0.016和0.192±0.014(P<0.05或P<0.01),提示抑制钠泵可升高皮层神经元[Ca2+]i。缺氧使培养皮层神经元[Ca2+]i呈时间依赖性增加,在缺氧0、2、4、6、8和10 min时,[Ca2+]i的ΔRatio值分别为0、0.0220±0.0012、0.0693±0.0052、0.1047±0.0148、0.1397±0.0165和0.1637±0.0122,显著高于缺氧前(P<0.01)。钠泵对缺氧所致培养皮层神经元[Ca2+]i升高的影响:Oua 10 nmol/L可使缺氧所致的培养皮层神经元[Ca2+]i升高程度较单纯缺氧明显降低(P<0.05),缺氧后0、2、4、6、8和10 min时的ΔRatio值分别为0、0.0163±0.0009、0.0300±0.0050、0.0520±0.0141、0.0710±0.0155和0.0983±0.0183。提示兴奋高亲和力钠泵可部分代偿缺氧所致的培养皮层神经元[Ca2+]i升高。Oua 10μmol/L基础上缺氧,缺氧诱导的[Ca2+]i升高没有明显改变(P>0.05),主要是低亲和力钠泵参与缺氧损伤。Oua 1 mmol/L使正常培养皮层神经元[Ca2+]i值上升到相当高的水平后,缺氧可观察到4 min以内[Ca2+]i维持在缺氧之前的水平(P>0.05),ΔRatio值接近于0,与单纯缺氧相应时间点比较差异显著(P<0.01),提示完全阻断钠泵功能可对抗缺氧损伤最初的[Ca2+]i升高。然而随缺氧时间的延长,皮层神经元[Ca2+]i逐渐上升,在6、8和10 min时分别为0.0200±0.0144、0.0556±0.0141和0.0837±0.0128(P<0.05),与单纯缺氧相应时间点比较差异显著(P<0.01)。结论:缺氧使Na+进入皮层神经元以及[Ca2+]i升高,同时阻断钠泵功能可通过升高皮层神经元[Na+]i水平影响[Ca2+]i,以及完全抑制钠泵功能,可在4 min以内阻断缺氧介导的皮层神经元[Ca2+]i变化,提示钠泵通过间接调节[Ca2+]i参与皮层神经元的缺氧损伤,以及钠泵是缺氧损伤早期阶段的靶点之一,但是,还有其它机制参与了较长时间缺氧的损伤。兴奋高亲和力钠泵可能通过非转运功能对皮层神经元的缺氧损伤有保护作用。更多还原

【Abstract】 Cerebral ischemia results in severe cell degeneration and consequently loss of brain functions. Now, it is widely accepted that ischemic neuronal cell death results from excessive intracellular accumulation of Ca2+ caused by the massive release of glutamate during the insult.The Na pump, or Na+, K+-ATPase, is a membrane-bound protein. By utilizing the energy from the hydrolysis of one molecule of ATP, it translocates three Na+ out of the cell and two K+ into the cell. The electrochemical gradient the Na pump generates is critical in maintaining the osmotic balance of the cell and the resting membrane potential of most tissues. Thus the Na pump is essential in the maintenance of Na+, body fluid and electrolyte homeostasis.When the supply of oxygen or blood flow to the mammalian brain decreases to critical levels, energy failure occurs, with a decline in ATP by as much as 90% in within 5 min. When 50–65% of the ATP is lost, Na pump activity is inhibited and depolarization of the membrane voltage and subsequent uptake of sodium and water occurs. Depolarization causes Ca2+ influx through voltage-gated Ca2+ channels. The collapse of Na+ gradient causes the sodium-glutamate cotransporters to eject glutamate into the extracellular space. Glutamate triggers vigorous activation of glutamate receptors and the Ca2+-dependent cell injury.Obviously, Na pump plays an important role in the occurrence and development of neural damage after cerebral ischemia. Previous studies about the pump are focused on cadiomycyte, kidney, musculi skeleti, and vascular smooth muscle. Research on it in neurons, especially about its role in cerebral ischemic injury, has not yet been conducted extensively. The present study, carried out in cortical neurons (pyramidal neuron of layer V and VI) which are sensitive to hypoxia, is to explore the changes in the electrophysiological characteristics of Na pump in hypoxic injury of cortical neurons and underlying mechanism.I Electrophysiolofical characteristics of Na pump in neurons from cortical slices.Objectives: This part of investigation was undertaken to characterize the Na pump current in neurons from cortical slices.Methods: The young SD rats (11–14 d postnatal) were used to prepare the cortical slices. Animals were deeply anesthetized and decapitated. The brains were quickly removed and submerged in ice-cold ACSF. Sections (300μm thick) of frontal cortex were cut, immersed in ACSF aerated with a mixture of 95% O2/5% CO2. And then the slices were perfused with ACSF gassed with 95% O2/5% CO2 continually after putting them into filling trough and under the fluid. We moved the electrode above neuron by infrared differential interference contrast (DIC) optics, and then made a tight seal and broke the membrane by giving negative pressure. As pipette solution was dialyzing into the cell and membrane current reached a steady-state level at a holding potential of -60 mV, at first we identified that the recording cell was a neurons by membrane depolarization (-60mV—0mV—-60mV) evoking Na current, and then the bath solution was switched from normal ACSF to the ACSF contained Ouabain (Oua, 10-12~10-3 mol/L), which resulted in a inward shift of the membrane current. The Na pump current was determined as the difference in holding current levels in the absence and presence of Oua, and was identified as Oua-sensitive current by the way that the amplitude evoked by 1 mmol/L Oua equaled to that induced by free-K+ extracellular fluid. Then Oua-sensitive currents were measured as the concentration of Oua ([Oua]) ranged from 10-12 to 10-3 mol/L and a Oua dose-response curve was constructed. The Oua-sensitive currents (?Ip, stimulation, or inhibition) were normalized to the maximal value of ?Ip obtained in the same cell by total pump inhibition on application of 10-3 mol/L Oua and . Na pump current was also identified as the voltage-sensitive current and current-voltage (I-V) relationship was observed by square voltage steps to membrane potentials between +40 and -90 mV. The whole experiment was executed under the conditions of room temperature 23±2℃.Results: All cells in the experiments were pyramidal neurons because of Na+ current and larger volume and Oua-sensitive current represented Na pump current because the amplitude evoked by 1 mmol/L Oua equaled to that induced by free-K+ extracellular fluid.Perfusion of the ACSF contained different concentration of Oua evoked an inward shift of the holding current at -60mV in a concentration-dependent manner. The Na pump current was stimulated or inhibited by concentrations of Oua (10-12~10-3 mol/L), which were divided into three models, stimulation of Ip, inhibition of Ip, and no change of Ip, based on response of Na pump in neurons to low concentration Oua (10-8 mol/L).In the ?Ip-[Oua] relation curve for the stimulation of Ip (in 29 from 96 neurons), the ?Ip values produced by each concentration of Oua from 10-12 to 10-3 mol/L were 0.027±0.114, 0.032±0.082, 0.050±0.213, 0.172±0.226, 0.125±0.152, -0.035±0.036, -0.146±0.124, -0.226±0.161, -0.421±0.076, -0.638±0.138 and -1, respectively. Oua from 10-12 to 10-8 mol/L excited Na pump current and the excitory extend reached its peak at 10-9 mol/L, but concentration-dependent inhibition of Na pump current were recorded by using Oua from 10-7 to 10-3 mol/L. The curve was well fitted using a three-binding site model and the dissociation constants for high-affinity stimulatory binding site, high-affinity inhibitive binding site, and low-affinity inhibitive binding site were 0.31 nmol/L, 41.27 nmol/L, and 152.48μmol/L.In the ?Ip-[Oua] relation curve for the inhibition of Ip (in 61 from 96 neurons), all the ?Ip values on the curve (Oua from 10-12 to 10-3 mol/L) were -0.011±0.072, -0.028±0.071, -0.057±0.079, -0.093±0.063, -0.115±0.069, -0.124±0.067, -0.157±0.094, -0.281±0.144, -0.421±0.076, -0.690±0.112 and -1.0, respectively. Both Oua had inhibitory effects on Na pump current, but this curve displayed nearly plateau level at concentrations of 10-8 and 10-7 mol/L. The curve was well fitted using a two-binding site model and the dissociation constants for high-affinity inhibitive binding site and low-affinity inhibitive binding site were 71.12μmol/L and 176.51μmol/L.In the ?Ip-[Oua] relation curve for the no change of Ip (in 6 from 96 neurons), Oua from 10-12 to 10-7 mol/L had no effect on Na pump current and concentration-dependent inhibition of this current was recorded significantly by Oua from 10-6 to 10-3 mol/L. The curve was well fitted using one-binding site model with dissociation constant for low-inhibitive-affinity site of 149μmol/L.The high-affinity pumps generated 14.59% of the total Na pump current and very likely included theα2 andα3 isoform. The low-affinity pump corresponding to 85.41% of the total Na pump current attributed toα1 isoform. Additionally, the Na pump current exhibited a voltage-dependence; its I-V curve displayed a positive slope at potentials between -90 ~ +40 mV and the reversal potential was near -20 mV.Conclusion: Cortical pyramidal neurons express two functionally distinct pump as expected for high-affinity pump (α2 andα3 isoform) and low-affinity pump (α1 isoform). Na pump current is not only Oua-sensitive, but also voltage-dependent.II Effects of hypoxia on Na pump in cortical neuronsObjection: This part investigation was designed to examine how hypoxia affected Na pump activity and itsα-isoform expression in cortical neurons.Methods: Neurons were exposed to oxygen glucose deprivation by①neural culture under N2 and non-glycose conditions,②or cortical slices perfusion with free-glucose and low-oxygen ACSF. In order to determine whether the two hypoxic method were successfully, we examined the levels of lactate dehydrogenase (LDH) in culture medium at 0, 2, 4, 8 and 12 hour after hypoxia. The intracellular Ca2+ concentration ([Ca2+]i) (fluorescent intensity of fluo-3) was detected in cultured cortical neurons suffered from 0, 0.5, 1, 2, 4 and 8 hour hypoxia using confocal microscope, and TTC staining was employed to evaluate activity of cortical slices after perfusing free-glucose and low-oxygen ACSF at 0, 5, 10, 15, 30, 45 and 60 min. Change of Na pump activity induced by hypoxia in cultured cortical neurons was determined at 0, 2, 4 and 8 hours by inorganic phosphate spectrophotometry, and in cortical slices at 0, 15, 30 and 60 min by double enzymic method. Changes of Na pump current induced by hypoxia in pyramidal neurons from cortical slices were also examined at 0, 2, 4, 6, 8 or 10 min after hypoxia. After Oua of 1mmol/L perfusion, cortical slices were received a washout with normal ACSF and free-glucose and low-oxygen ACSF, to understand the recover velocity of Na pump function.And the expressions of mRNA and protein of Na pumpα1 andα3 isofrom were determined by RT-PCR and Western blot to evaluate effects of hypoxia on Na pump at 0, 4, 8 and 12 hour after hypoxia in cultured cortical neurons, and at 0, 5, 10, 15, 30 and 60 min after hypoxia in cortical slices.Results: LDH in medium of cultured cortical neurons increased from 324±16 to 417±50 U/L (p<0.05), and elevated further as prolonging hypoxic time, suggesting a hypoxic injury. Intracellular Ca2+ overload is another indicator to show cell damage, [Ca2+]i in cultured cortical neurons raised and reached its peak 1.71±0.46 within 1 hour, and maintained the maxiamal values for 2~8 hour hypoxia. TTC staining showed that the activity of cortical slices reduced to 0.292±0.019 (P<0.01) after perfusing free glucose and low oxygen ASCF for 5min, the lower level of the activity was kept until 15 min, and then dropped significantly (0.257±0.012, P<0.01).The activity of pump increased from 1626±122 to 4114±472μmol/NADH/mg·protein/h (p<0.01) after hypoxic culture for 2 hours, and then with prolonged hypoxic time, gradually decreased to 864±318μmol/NADH/mg·protein/h (p<0.01) at 4-hour after hypoxia and to 497±86μmol/NADH/mg·protein/h (p<0.05) at 8-hour after hypoxia. In additionally, this pump activity of cortical slices increased from 169±32 to 572±28μmol/mg protein/h (P<0.05) at 30 min after hypoxia, and then decreased at 60 min after hypoxia (243±72μmol/mg protein/h, P<0.05).Na pump current of cortical slices after hypoxic exposure increased and then decreased. The pump current was significantly lower at 4 min after hypoxia compared with control (0.265±0.068 vs 0.160±0.046 pA/pF,P<0.01), but it enhanced to 0.243±0.054 pA/pF (P<0.05) at 20 min after hypoxia. After holding current was inwardly shifted by 1 mmol/L Oua, the perfusion of free-glucose and low oxygen ACSF did not eliminate the inhibition of sodium pump function to recover the holding current to the original level before giving Oua, suggesting that hypoxia inhibited Na pump function.Hypoxic culture caused an increase in the expression ofα1 isoform mRNA in cultured cortical neurons, the mRNA levels were 0.822±0.050, 0.873±0.096, 0.960±0.089, 0.938±0.080 and 0.829±0.098, respectively, at 0, 2, 4, 8 and 12 hour after hypoxia, and there were significant differences (p<0.01) at 4 hour and 8 hour after hypoxic exposure. In contrast hypoxic culture caused a decrease in the expression ofα3 isoform mRNA, the mRNA levels declined from 1.173±0.156 to 0.540±0.076 (p<0.01), and kept the lower level until 12 hour (0.536±0.100, p<0.01). There were not obvious changes of mRNA level ofα1 andα3 isoform due to hypoxia in cortical slices.Hypoxic culture caused an increase in expressions ofα1 isoform protein in cultured cortical neurons, which is consent to its mRNA change. The levels of protein at 4 and 8 hour after hypoxia show a increasing trend which was not significant, but at 12 hour after hypoxia the level ofα1 isoform protein rose from 0.471±0.052 to 0.806±0.078 (p<0.01). The changes ofα3 isoform protein were similar to the decreases of its mRNA. The protein level at 4 hour after hypoxia decreased from 0.730±0.065 to 0.501±0.097 (p<0.05), and with prolonged hypoxic time it attenuated further from 0.446±0.077 at 8 hour after hypoxia to 0.448±0.109 at 12 hour after hypoxia (p<0.01), respectively. Changes in fluorescent intensity ofα1 andα3 isoform had the same trend as their protein expression. And there were not significant changes in protein levels ofα1 andα3 isoform in cortical slices at 15 min, 30 min and 60 min after hypoxia.Conclusion: Perfusing cortical slices with free-glucose and low oxygen and the hypoxic culture of cortical neurons simulate two different hypoxic injuries successfully. At the early stage of hypoxic exposure, the function of Na pump increases compensatoryly, but decreases step by step with prolonged hypoxic time. Hypoxic exposure downregulates the expressions of mRNA and protein of Na pumpα3 isoform and upregulates those ofα1 isoform. III Low-affinity Na pump involved in hypoxic injury in cortical neurons.Objective: This part of investigation was undertaken to understand the distinct functions of high- and low-affinity Na pump when suffering from hypoxia in cortical neuronsMethods: Hypoxic condition was simulated by perfusing free-glucose and low oxygen ACSF for different time.Changes of membrane current (?I/Cm) in neurons from cortical slices were determined at 0, 2, 4, 8 and 10 after hypoxia. We examined the membrane current through perfusion of both TTX (1μmol/L) and hypoxia or 6-min hypoxia before TTX in order to explore the relationship between ?I/Cm induced by hypoxia and activation of Na channel.We evaluated ?I/Cm by synchronously giving 10 min hypoxia and different concentrations of Oua (10 nmol/L, 100 nmol/L, and Oua 10μmol/L, which can excite high-affinity pump, inhibit high-affinity pump, and partially inhibit low-affinity pump respectively) and then calculated ?I/Cm at 0, 2, 4, 8, and 10 min after hypoxia, in order to understand the relationship between Na pump and hypoxia-mediated ?I/Cm.We examined fluorescent signals of [Ca2+]i and intracellular Na+ concentration ([Na+]i) in cultured cortical neurons using video based motion edge detection system (Fura-2 10μmol/L and SBFI 10μmol/L).①A change in ratio (?Ratio) of [Ca2+]i was determined by perfusing free-glucose and low oxygen HBS for 10 min, and calculated at 0, 2, 4, 6, 8 and 10 min after hypoxia.②?Ratio of [Na+]i and [Ca2+]i were determined by perfusing concentrations from 10-9 to 10-3 mol/L, and each concentration lasted for 2 min.③Cultured cortical neurons were pretreated by Oua of 100 nmol/L, 10μmol/L, or 1 mmol/L, and then suffered from hypoxia and Oua (the same concentration) synchronously.Results: Hypoxic exposure increased membrane current of neurons in cortical slices. Membrane current of neurons was enhanced time-dependently (r=0.98028,P<0.01) during the 10-min hypoxia exposure (P<0.01), and the increasing values were 0, -0.014±0.006, -0.028±0.013, -0.035±0.016, -0.044±0.022, -0.058±0.026 and -0.074±0.025 pA/pF, respectively, at 0, 2, 4, 6, 8 and 10 min after hypoxia, suggesting that the abnormal open of ionic channels was mediated by hypoxia.The increasing membrane current induced by hypoxia was completely blocked by TTX (1μmol/L) under condition without influences of Ca2+ and K+ channel. If cortical slices were pretreated by 6 min hypoxia and then suffered from both TTX and hypoxia, the increase of membrane current was not continued and kept the level at 6 min after hypoxia (-0.033±0.013 pA/pF), which was lower than that of alone hypoxia for 10 min (-0.058±0.028 pA/pF, P<0.01). These results indicate that increasing membrane current induced by hypoxia was TTX-sensitive Na current.Na pump affects the increase of membrane current induced by hypoxia. Oua 10μmol/L, partially inhibition of low-affinity pump, made membrane current increasing further, at 0, 2, 4, 6, 8 and 10 min after hypoxia the values of ?I/Cm were 0, -0.031±0.010, -0.041±0.013, -0.053±0.007, -0.057±0.008 and -0.069±0.012 pA/pF (P<0.05). By perfusing both Oua 100 nmol/L and 10 min oxygen-free A CSF, values of ?I/Cm displaying a decreasing extend were 0, -0.021±0.003, -0.028±0.006, -0.038±0.016, -0.047±0.018 and -0.055±0.020 pA/pF, respectively (P>0.05). However, values of ?I/Cm were obviously lower compared with each point of single hypoxia by perfusing both Oua 10 nmol/L and 10-min hypoxia, they were 0, -0.009±0.011, -0.011±0.015, -0.010±0.016, -0.009±0.018 and -0.012±0.020 pA/pF (P<0.01), which showed that 10 nmol/L Oua protected cortical neurons from hypoxic injury. Oua also affects the [Ca2+]i and [Na+]i in cultured cortical neurons.Inhibition of Na pump in cultured cortical neurons by Oua caused the increases in the [Ca2+]i and [Na+]i in a concentration-dependent manner in normal ACSF.ΔRatio of [Na+]i were 0.017±0.004, 0.030±0.004 and 0.035±0.004 (P<0.01) when perfusing Oua 10-5, 10-4 , and 10-3mol/L, which were higher compared with normal control.ΔRatio of [Ca2+]i also exhibited an significant elevate, which were 0.056±0.013, 0.107±0.018, 0.158±0.011, 0.185±0.016 and 0.192±0.014(P<0.05 or P<0.01) by perfusing Oua 10-7, 10-6, 10-5, 10-4, and 10-3 mol/L. These results suggest that inhibition of Na pump causes a raise in cortical neurons [Ca2+]i.Increase of [Ca2+]i mediated by hypoxia in cultured cortical neurons was time-dependent,ΔRatio of [Ca2+]i were 0, 0.0220±0.0012, 0.0693±0.0052, 0.1047±0.0148, 0.1397±0.0165 and 0.1637±0.0122, which were higher compared with control at 0, 2, 4, 6, 8 and 10 min point (P<0.01).Oua of 10 nmol/LdecreasedΔRatio of [Ca2+]i raised by hypoxia, and values ofΔRatio were 0, 0.0163±0.0009, 0.0300±0.0050, 0.0520±0.0141, 0.0710±0.0155 and 0.0983±0.0183, lower compared with control at 0, 2, 4, 6, 8 and 10 min point (P<0.05), suggesting that exciting high-affinity pump counterwork partially the increase of [Ca2+]i induced by hypoxia in cultured cortical neurons. Both hypoxia and Oua 10μmol/L perfusion did not change the increases in the [Ca2+]i induced by hypoxia, suggesting low-affinity pump involved mainly in hypoxic injury.When a higher level of the [Ca2+]i was caused by Oua of 1 mmol/L in cortical neurons, perfusion of hypoxic-ACSF contained Oua of 1 mmol/L could not make it further raising and kept the same level within 4-min hypoxia as them before hypoxia (P>0.05), this is, hypoxia-mediatedΔRatio of [Ca2+]i nearly equaled to 0, and much lower than these points of single hypoxia (P<0.01). However, with prolonged hypoxic time,ΔRatio of [Ca2+]i increased slightly, the values at 6, 8 and 10 min were 0.0200±0.0144, 0.0556±0.0141 and 0.0837±0.0128(P<0.05), which were lower than those in alone hypoxia.Conclusions: Hypoxia can cause Na+ entering cortical neurons and an increase of [Ca2+]i, the inhibition of Na pump also elevates the [Ca2+]i through increasing [Na+]i and the complete inhibition of Na pump can block hypoxia- mediated changes of [Ca2+]i in cortical neurons within 4 min hypoxic exposure, suggesting that Na pump involves in the effect of hypoxia on cortical neurons by regulating [Ca2+]i indirectly and that Na pump is one of targets at the early stage of hypoxic injury, but some other mechanisms involved in prolonged-time hypoxia. Exciting high-affinity pump protected cortical neurons from hypoxia injury.更多还原