缺氧/缺血诱导的离子平衡紊乱及Humanin的拮抗作用
|
摘要
Humanin(HN)是一个由24个氨基酸残基组成的神经保护肽,2001年首先由日本学者从阿尔采末病(Alzheimer’s disease, AD)患者未发病的枕叶cDNA文库中筛查发现。最初的研究显示,HN的神经保护作用主要针对AD相关毒性的损伤,因此发现者将其定义为AD相关的神经保护肽。随后的研究提示,HN可能具有更为广范的神经保护作用。我们研究室以往的工作也证实,HN对缺氧损伤具有神经保护作用。然而HN在缺氧早期事件中的作用尚不清楚,其神经保护作用的机制有待进一步阐明,尤其是缺乏电生理机制方面的研究报道。
一般认为缺氧或缺血造成的神经细胞损伤可大致分为三个阶段:(1)急性缺氧或缺血早期,其特征表现为膜通道的功能活动及膜电位的改变,细胞内环境的改变,如细胞内Na~+、Ca~(2+)、和细胞外K~+离子的显著增加等;(2)损伤相关的酶激活(蛋白酶、磷脂酶等);(3)细胞功能和结构改变,最终导致细胞死亡。由于损伤性质和细胞类型的不同,从急性缺氧缺血到延迟的细胞死亡发生所经历的时间可有很大差异(从数分钟到数小时或数周),其病理变化也有很大区别,包括最终引起的神经细胞的死亡也有不同方式,如坏死、凋亡等。而早期发生的离子稳态失衡在缺氧缺血所致的神经损伤过程发挥重要作用,至少包括以下三个方面:(1)缺氧早期引起的Na~+和Ca~(2+)在神经细胞中的积聚是缺氧造成损伤的重要诱因;(2)缺氧或缺血早期对神经元的影响几乎只与离子稳态的改变相关;(3)这些早期改变不仅决定细胞短期(即时)的命运(例如细胞损伤和坏死),而且也将引起细胞的长期改变(例如代谢改变、酶的活化、基因激活及引起凋亡等),即神经元的很多损伤性改变都继发于离子稳态失调。因此,探讨缺氧早期引起的离子稳态失衡的机制对于理解缺氧导致的病理生理改变及早期、晚期的病理损伤具有极其重要的意义。
在生理条件下,离子跨膜移动由多种机制调控,从而维持各种离子在胞内外分布的相对恒定;维持静息电位稳定及在此基础上产生的动作电位;维持神经组织正常的兴奋性。然而,急性缺氧会造成离子通道活动的改变及膜电位改变如去极化等,并继而引起细胞结构、功能改变直至细胞死亡。为探讨缺氧引起的早期改变,探讨HN对于早期缺氧/缺血的神经保护作用及其离子机制。我们以缺氧和谷氨酸处理神经元模拟体内的缺血损伤,采用膜片钳技术和钙离子成像技术,观察急性缺氧引起的离子稳态失衡,包括K+、Na~+离子电流改变(通道活动改变)和细胞内钙信号改变(胞内Ca~(2+)升高)以及HN的保护作用。
实验中采用急性分离的海马CA1细胞和膜片钳技术以及离体培养的原代神经元和离子测定技术,观察了急性缺氧诱导的电压门控性钾电流(全钾电流、瞬间钾电流I_A、延迟整流钾电流I_K)、电压门控性钠电流(持续性钠电流I_(NaP)、瞬间钠电流I_(NaT))的改变及HN对急性缺氧诱导产生的通道功能改变的影响。考虑到兴奋性神经毒和钙离子超载在包括缺血/缺氧在内的各种神经病理中的重要作用,观察了HN对谷氨酸诱导的胞内Ca~(2+)的影响,并初步探讨了其机制。主要结果显示:(1)急性缺氧造成电压门控I_K、I_(Na)电流的异常改变(电流幅度增加);(2)HN(5μM)与缺氧同时作用于细胞,HN能够拮抗缺氧诱导的I_K、I_(NaP)电流的异常改变;(3)谷氨酸造成胞内Ca~(2+)明显升高,HN(5μM)预处理抑制了谷氨酸诱导的胞内Ca~(2+)升高,其机制可能是HN通过抑制经谷氨酸受体的Ca~(2+)内流和保护线粒体的钙缓冲能力而发挥作用。
上述结果显示,离子稳态失衡,包括缺氧引起的I_K增加、I_(NaP)增加及[Ca~(2+)]i增加,是缺氧引起的早期改变。这些改变可能通过影响膜电位(导致去极化)、影响兴奋性、影响胞内Ca~(2+)(导致钙超载),及由此继发性地引起其他病理改变,;在缺氧诱导的病理损伤过程发挥重要作用。HN通过抑制离子通道异常活动和稳定线粒体的功能而维持离子稳态,从而保护神经元对抗缺氧/缺血和兴奋毒造成的损伤,即HN可对急性缺氧损伤提供有效保护。这些结果也支持HN可能具有广谱的神经保护作用的假说。
第一部分: HN对缺氧诱导的海马CA1神经元电压门控性钾电流异常改变的影响
通道的异常活动造成的离子失稳态被认为是缺氧/缺血诱导的神经元损伤的最初和关键的改变,包括缺氧缺血早期引起的细胞外钾浓度增高。研究显示,细胞内钾的外流与缺氧诱导的去极化密切相关,升高细胞外钾浓度能够模拟缺氧去极化,而缺氧去极化被认为是导致神经细胞死亡的重要因素;此外钾外流造成的细胞内钾的丢失引起神经细胞皱缩、解除对caspase和核内切酶的抑制作用从而介导凋亡过程。资料证实,持续性的细胞外高钾可引起神经细胞死亡,而阻断钾外流可拮抗缺氧和缺血诱导的神经元死亡。这些结果提示,维持钾通道功能稳定及钾在细胞内外正常分布在缺血损伤及保护中发挥重要意义。神经元上的钾通道包括五类,目前针对缺氧对钾通道调制的研究大多数集中在K_(ATP)通道和K_(Ca)通道,而对电压门控性钾通道包括延迟整流钾电流(I_K)、瞬间钾电流(I_A)在缺血、缺氧状态下功能特性的改变研究甚少。
HN是一个内源性神经肽,最初被认为只针对AD相关毒性的特异神经保护作用。然而最近的研究提示,HN可能具有更为广谱的保护作用。本实验室前期工作也显示,HN对缺氧诱导的神经损伤具有保护作用,其作用机制包括抗凋亡和稳定线粒体功能。
为了探讨电压门控性钾通道在缺氧导致钾离子稳态失衡中的作用,以及HN急性处理对缺氧损伤可能发挥的影响,我们采用急性分离的海马CA1细胞和膜片钳技术,观察了急性缺氧对电压门控性钾通道(全钾电流、瞬间钾电流I_A、延迟整流钾电流I_K)的作用及HN在常氧状态(单独作用)于缺氧状态(与缺氧同时作用)下对钾通道的影响。
主要结果如下:1)急性缺氧(3min)显著增强I_K电流,+70mV去极化脉冲下I_K电流幅度增加为对照组的196±15% (P<0.05, n=6);而急性缺氧对I_A没有明显影响,+70mV去极化脉冲下I_A电流幅度与对照组相比无统计学差异(P=0.871, n=6);2)HN(5μM)单独作用(即在常氧状态下)使I_A电流幅度增加,I_A电流幅度从对照组的2250.33±608.77pA增加为3051.47±781.62 pA (P<0.05, n=6); HN单独作用不影响I_K电流幅度,HN作用前后I_K电流幅度分别为1240.96±171.71 pA和1609.63±353.49 pA (P>0.05, n=6);3)HN与缺氧同时作用时,取消了缺氧对I_K电流的影响。在有HN存在的情况下,缺氧不再引起I_K电流幅度的增加,记录到的I_K电流为对照组的0.89±0.23,与对照组相比无显著性差异(P>0.05, n=11)。
这些结果显示:1)缺氧早期引起延迟整流钾电流I_K的增强,I_K电流的增强是介导缺氧早期K~+外流的主要途径,并可能由此造成缺氧的损伤性改变及导致其他病理改变;2)缺氧对瞬间钾电流I_A未产生明显作用;3)HN抑制缺氧引起的I_K增强,这一作用可能通过减轻K~+分布的异常从而对缺氧/缺血损伤发挥神经保护作用; 4)HN在常氧和缺氧条件下都能增强I_A电流幅度,其生理即病理生理意义尚不明确。以上结果提示,HN可能通过拮抗缺氧对电压依赖性钾通道的作用、维持钾离子稳态而对抗缺氧缺血引起的早期改变,从而对缺氧性脑损伤发挥有效保护作用。
第二部分:HN对缺氧诱导的海马CA1神经元电压门控性钠电流异常改变的影响
缺氧早期发生快速的细胞内环境的改变,包括细胞内先于胞浆Ca~(2+)的增高而发生的Na~+增高。大量研究提示了钠通道功能改变以及胞浆Na~+增高在缺氧/缺血性损伤中发挥的作用。细胞外Na~+内流促进缺氧去极化的发生;胞浆Na~+增高不仅刺激钠钾泵的活动加快能量耗竭,而且通过逆转Na~+/Ca~(2+)交换体的转运方向从而引起细胞内Ca~(2+)增高,加剧随之发生的细胞损伤。钠通道阻断剂和细胞外低Na~+溶液可减少缺血或缺氧性细胞损伤和延迟的缺氧去极化,证明了钠通道以及胞浆Na~+增高在缺氧性损伤中所发挥的作用。
HN是最近发现的内源性神经肽,初始被认为特异拮抗AD相关的神经毒,然而新近的研究提示HN可能具有更为广谱的神经保护作用。本实验室的前期工作显示HN预处理能够拮抗缺氧诱导的神经损伤。
为探讨HN对缺氧性损伤可能具有的保护作用及其作用的电生理机制,本研究中我们采用了全细胞膜片钳技术、急性分离海马神经元,观察HN在常氧和缺氧状态下对电压门控性钠通道(持续性钠电流I_(NaP)、瞬间钠电流I_(NaT))的作用。
主要结果如下:1)急性缺氧2 min后,I_(NaP)电流幅度显著增大为对照组的265±40% (P < 0·01, n = 5),而缺氧引起了I_(NaT)电流幅度的降低为对照组的65.4±7% (P<0.05, n= 7);2)HN (5μmol/l)单独作用2 min后I_(NaP)电流幅度不受影响;3)HN与缺氧同时作用2min,HN抑制了缺氧诱导的I_(NaP)电流幅度的增加,在HN存在的情况下I_(NaP)电流幅度为对照组的144±24% (P > 0.05, n=5),即HN拮抗了缺氧诱导的I_(NaP)增强。这些结果显示:1)缺氧早期,不失活的电压门控钠电流即持续性钠电流I_(NaP)的活动增强是引起[Na~+]i升高的主要途径,并可能继而造成Na~+、Ca~(2+)稳态失调、持续膜去极化和细胞损伤; 2)急性缺氧抑制瞬间钠电流I_(NaT);3)HN急性处理能够拮抗缺氧诱导的I_(NaP)增强,从而可能通过减轻Na~+分布异常、及其继发损伤而在缺氧早期发挥神经保护作用。瞬间钠电流I_(NaT)改变的生理及病理生理意义尚不明确。
第三部分:HN对谷氨酸诱导的钙离子稳态失衡的影响及机制
本实验室以往的工作显示,HN对缺氧和兴奋毒损伤具有神经保护作用。考虑到谷氨酸受体过度激活和钙超载在缺氧缺血性脑损伤中的关键作用,本研究观察在培养的皮层神经元,HN能否拮抗谷氨酸(500μM)所导致的细胞内钙离子稳态失衡,并初步探讨可能的机制包括线粒体机制。
本研究以谷氨酸处理神经元模拟缺血的神经损伤,采用培养皮层神经元Ca~(2+)荧光成像技术观察神经元的[Ca~(2+)]i;在急性分离神经元采用全细胞膜片钳技术观察HN对谷氨酸受体的作用。为探讨HN对谷氨酸诱导钙反应的拮抗作用是否有线粒体钙缓冲机制的参与,使用了线粒体解耦联剂FCCP。FCCP可使线粒体质子梯度、线粒体膜电位消失,从而导致将胞浆内Ca~(2+)摄取进入线粒体内的动力消失,因而抑制线粒体摄取Ca~(2+)、并可促进其释放Ca~(2+)。因此,在谷氨酸预处理引起钙反应的稳态期应用FCCP,由FCCP诱导的细胞内Ca~(2+)
增加可反应谷氨酸暴露期间线粒体摄取Ca~(2+)的量。
主要结果如下:1)谷氨酸(500μM)导致培养皮层神经元显著的[Ca~(2+)]i增高,平台期fluo-3标准化荧光增加为基线水平的2.29±0.38;2)HN预处理显著抑制兴奋毒浓度谷氨酸暴露所导致的细胞内Ca~(2+)增加;在HN预处理的细胞,谷氨酸导致的[Ca~(2+)]i增加为1.38±0.19,与对照组相比抑制率接近40% (P<0.01);3)在对照细胞和HN预处理细胞,FCCP诱导的[Ca~(2+)]i增加分别为1.64±0.15和2.83±0.29,HN预处理细胞FCCP诱导释放的Ca~(2+)量显著增加(P<0.05),说明HN预处理导致了谷氨酸暴露期间线粒体摄取Ca~(2+)增加;4)在谷氨酸之前预先给予FCCP,通过破坏线粒体质子梯度、导致线粒体膜电位丧失,从而抑制线粒体的摄取Ca~(2+)功能。在FCCP存在情况下,HN处理组和对照组谷氨酸诱导的[Ca~(2+)]i增加分别为1.29±0.14和1.09±0.35,HN仍能抑制兴奋毒浓度谷氨酸诱导的[Ca~(2+)]i反应(P<0.05),提示HN的拮抗作用除线粒体外还有其他机制;4)电生理结果显示,HN显著抑制谷氨酸(200μM)和NMDA(100μM)所诱导的全细胞膜电流。HN预处理2min使谷氨酸、NMDA诱导的峰电流分别降低为基线水平的53.1±7.4% (P<0.01, n=13)、67.1±3.6%(P<0.01, n=12)。
这些结果显示:(1)HN(5μM)能够拮抗兴奋毒浓度谷氨酸所导致的细胞内钙升高;(2)这种拮抗作用部分通过稳定线粒体功能以及恢复钙缓冲能力而实现;(3)部分通过抑制经谷氨酸受体的细胞外Ca~(2+)内流而实现。
综上结果,可得出如下结论:
1)缺氧诱导电压门控性钠、钾通道电流的改变,提示电压门控性钠、钾通道在缺氧早期发生的离子稳态失衡中起重要作用。
2)缺氧早期引起延迟整流钾电流IK的增强,这可能是介导缺氧早期K~+外流、K~+稳态失衡的主要途径及损伤机制;缺氧对瞬间钾电流I_A未产生明显作用,I_A可能由于其快速失活的特性而在缺氧早期K~+稳态失衡中不起重要作用。
3)缺氧早期引起不失活的电压门控钠电流即持续性钠电流I_(NaP)的活动增强,Na~+经I_(NaP)内流是缺氧早期造成细胞内Na~+升高的主要途径,并继而引起Na~+、Ca~(2+)稳态失衡、持续的膜去极化和细胞损伤;急性缺氧抑制瞬间钠电流INaT。
4)HN预处理可拮抗缺氧引起的I_K、I_(NaP)增强。这一作用通过阻止K~+、Na~+稳态失衡及其继发损伤从而在缺氧/缺血早期发挥神经保护作用。HN对I_A产生了影响,但生理及病理生理意义尚不明确。
5)谷氨酸能够诱发细胞内钙超载而对神经元发挥损伤作用。HN能够拮抗谷氨酸导致的钙升高,从而通过减轻缺血缺氧和兴奋毒损伤期间发生的细胞内Ca~+超载。
6)HN拮抗谷氨酸诱导的钙超载的机制,包括HN可保护线粒体的钙缓冲能力以及抑制经谷氨酸受体的Ca~(2+)内流。
Humanin (HN) is a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal brain region from patients with Alzheimer’s disease (AD) in 2001. From those initial observations it seemed that HN was a selectively neuroprotective factor rescuing neurons from Alzheimer’s disease-related insults, hence it is defined as AD-related neuroprotive peptide. However, later efforts suggested a broader spectrum of its survival-promoting activity. Several lines of studies provided evidence for the argument. Prior works in our laboratory proved that HN treatment provided long-term neuroprotection against hypoxia insults in primary cortical neurons. Yet effects of HN when given acutely on early neuronal damage during hypoxia/ischemia remain unclear. And the mechanisms of HN neuroprotection remain to be fully illustrated, especially because of a paucity of electrophysiological studies.
It is generally accepted that that neuronal cell death can be described as three stages: (1) early intracellular ionic and chemical changes; early onset of acute hypoxia or ischemia is characterized by changes of ion channel activity and membrane potential, and major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na~+, Ca~(2+), and extracellular K~+ (2) activation of damaging enzymes and (3) changes in cellular functions and structures, eventually leading to cell death. The delayed before cell death occurs varies greatly (from minutes to hours or weeks), depending on the nature of the insults and the cell types. A large number of the damaging changes occurring in the neurons are secondary to loss of ion homeostasis (Lipton 1999), the pathological changes vary greatly, including the mode of final cell death. These early ionic alterations play important roles in hypoxia/ischemia induced neuronal damages: (a) the accumulation of Ca~(2+) and Na~+ in nerve cells during hypoxia has been shown by many investigators to be deleterious; (b) the main changes in neuronal function early in brain hypoxia or ischemia are almost solely related to changes in ionic homeostasis and (c) it is likely that these early changes dictate not only cell fate in the short term, such as cell injury and necrosis, but also long term changes such as activation of damaging enzymes and programmed cell death. A large number of the damaging changes occurring later in the neurons are secondary to disturbed ion homeostasis. Therefore, it is of great importance to investigate mechanisms of ionic imbalance during early hypoxia/ischemia insults.
Ion transmembrane flux is controlled by multiple different mechanisms under physiology conditions. However, changes in ionic channel activities by hypoxia are the major pathway for redistribution of ions across the membrane and the membrane depolarization, contributing to succeeding cell death. To examine the effects of HN on early hypoxia/ischemia induced disturbance of ionic homeostasis, using hypoxia model and glutamate as simulated-ischemia conditions, we investigate the effects of HN on ischemia-related insults induced disturbance of membrane ionic currents and the intracellular calcium ([Ca~(2+)]i) deregulation using whole-cell patch clamp technique and calcium imaging.
Using freshly isolated hippocampal CA1 neurons and patch clamp techniques, primary cultured neurons and ion imaging techniques, we investigate effects of hypoxia-induced disturbance of sodium and potassium currents and effects of HN. Given the crucial roles of excitotoxicity and dysfunction of calcium homeostasis in various neuropathologic lesions including hypoxia/ischemia, we investigated the effects of HN on Ca~(2+) deregulation induced by toxic concentration of glutamate exposure and its possible mechanisms. The results showed that:1) acute hypoxia induced disturbance of voltage-gated potassium and sodium currents (IK、INa); 2) HN (5μM) co-application with hypoxia solution attenuated hypoxia–induced disturbance of potassium current (IK) and persistent potassium current (INaP); and 3) HN (5μM) pretreatment attenuated Ca~(2+) deregulation during glutamate exposure through attenuated calcium entry via glutamate receptor channels and preserved mitochondrial buffering capacity.
Our results showed that disturbance of ionic homeostasis, including enhancement of IK, INaP and [Ca~(2+)]i, were the early changes induced by hypoxia and played important roles in hypoxic pathology through actions on membrane potential, excitability. HN was able to maintain ionic homeostasis via attenuating disturbance of ion channels and stabilizing mitochondrial function, and thus protect neurons against early hypoxia/ischemia insults. Furthermore, HN provided neuronal protection by ionic mechanisms acting at the very beginning of insults, i.e., that it is protective against acute hypoxia injury. These results provide further evidence for the broader spectrum of neuroprotective effects of HN against insults other than AD.
PartⅠ: Effects of HN on Hypoxia-Induced Disturbance of Voltage-Dependent Potassium Currents in Rat Hippocampal CA1 Neurons
Disruption of ionic homeostasis induced by disturbance of ion channel activity has been generally regarded as an initial and key alterations in anoxia/ischemia-induced neuronal injury. Enhanced ef?ux of K~+ has been shown to be closely associated with anoxia-induced depolarization, which is believed to be a crucial factor leading to neuronal death. The anxia depolarization is mimicked by elevation of the extracellular potassium concentration. The loss of intracellular K~+ causes cell shrinkage (apoptotic volume decrease) and creates a permissible environment for apoptosis by relieving the inhibition of endogenous caspases and nucleases. Sustained exposure to elevated extracellular K~+ causes signi?cant neuronal death even under conditions of normoxia and abundant glucose supply, whereas blockade of K~+ ef?ux has been shown to attenuate hypoxia and ischemia-induced neuronal death. These results suggest that maintaining K~+ channels functions and K~+ distribution across plasmamembrane may be of therapeutic bene?t in the treatment of ischemia-related insults. Among the five types of plasmalemma potassium channels, a great deal of researches focus on KATP and KCa channels, while voltage-gated potassium channels get less attention.
Mammalian neurons react rapidly to a lack of oxygen with alterations of ion channels activities, being either adaptive or deleterious responses. Of particular interest is the effects that hypoxia has on potassium channels since these channels are one of the fundamental factors that regulate membrane potential and neuronal excitability, in addition there are major alterations in K~+ ions homeostasis during early hypoxia. Thus K~+ channels and K~+ homeostasis may play important roles in the initiation and development of hypoxia/ischemia. Among the five classes of K~+ channels identified in excitable cells, penetrating researches have been focused on roles of KATP and KCa channels; while changes of voltage-gated potassium channels during hypoxia/ischemia got much less attentions.
HN was originally identified as an endogenous peptide that protects neuronal cells from death caused by Alzheimer’s disease (AD)-related genes and amyloid-β(Aβ). While recent studies suggested that HN might have a broader spectrum of protections. And the mechanisms underlying the protections of HN remain to be illustrated. Preliminary works in our laboratory showed that HN protected neurons from hypoxia-insults with mechanisms including anti-apoptosis and preserving mitochondrial functions.
To illustrate the effects of HN during early hypoxia/ischemia insults and illuminate underlying electrophysiological mechanisms for its potential neuroprotections, we investigated the response of voltage-gated K~+ (KV) channels (total potassium current, transient potassium current, and delayed rectifier potassium current) to hypoxia and the effects of HN in freshly isolated hippocampal CA1 neurons from rats aged 10～12 days.
Major results as following: 1) acute exposure to hypoxia (3 min) significantly enhanced IK currents. In five neurons exposed to hypoxia, the amplitude of IK at +70 mV was 1.96±0.15 times the amplitude of control group (P<0.05, n=6). While the amplitude of IA at +70 mV was 1.05±0.05 times of controls group, i.e. not significantly different from control (P>0.05, n=6); 2) HN (5μM) alone had no effect on IK at constant normoxic PO2, the amplitude of IK were 1240.96±171.71 pA and 1609.63±353.49 pA before and after HN application, respectively (P>0.05, n=6); HN, however, significantly increased IA amplitude under both hypoxia and nomoxia, the amplitude of IA increased from 2250.33±608.77 pA to 3051.47±781.62 pA after HN treatment (P<0.05, n=6); 3) HN co-application abolished the effect of hypoxia on IK. In the presence of HN, hypoxia no longer increased IK current amplitude. The IK current amplitude in the presence of HN was 0.89±0.23 times of control (P<0.05, n=11).
These results indicated that: 1) acute hypoxia induced an enhancement of the IK channel in central neurons, which may be the major pathway for K~+ efflux and results in hypoxia-induced changes and other pathological changes;2) transient IA currents were not influenced by acute hypoxia; 3) HN attenuated hypoxia-induced disturbance of IK, this effect might be neuroprotective during ischemia by attenuated the disturbance of potassium ionic distribution that occured. 4) HN increases IA current amplitude; the physiological and pathological significance of this fact remain unclear. These results suggested that HN may attenuate hypoxia-induced changes of IK currents and thus maintain K~+ homeostasis, protect neurons against hypoxia-induced damages.
PartⅡ: Effects of HN on Hypoxia-Induced Disturbance of Voltage-Dependent Sodium Currents in Rat Hippocampal CA1 Neurons
During hypoxia, cells undergo alterations in membrane potential and detrimental changes in their intracellular environment, including an early increase in intracellular Na~+ that contributes to the pathophysiology of neuronal death. There are mounting studies illustrating the relation between Na~+ channels and hypoxia. A large number of the damaging changes occurring in the neurons are secondary to an increase in intracellular Na~+ levels (Lipton 1999). In dissociated cells and cell cultures, hypoxia raised intracellular Na~+ concentration ([Na~+]i). Increased [Na~+]i may aggravate neuronal lessons by imposing an energy demand on cells due to stimulation of the plasmalemmal Na~+, K~+-ATPase. In addition, it contributes to an impairment of Ca~(2+) homeostasis by driving Ca~(2+) into the cells via the Na~+-Ca~(2+) exchanger, and to the development of other acute dysfunctions. Drugs that selectively block this current might reduce damage to cells during ischemia or hypoxia.
HN is a recently described endogenous peptide that antagonizes neurotoxicity caused by Alzheimer’s disease (AD) relevant insults, while recent studies suggest that HN may have a broader spectrum of protection. To illustrate the electrophysiological mechanism of HN on neurons and the possible neuroprotection against hypoxia insults, whole-cell patch clamp techniques were applied on isolated hippocampal neurons to study the possible effects of HN (5μM) on persistent and transient sodium current under normoxia and hypoxia.
The major results as following: 1) After 2 min hypoxia stimulation, persistent sodium current (INaP) increased significantly to 2.65±0.40 times control group (P < 0·01, n = 5), transient sodium current (INaT) decreased significantly to 65.4±7% of baseline (P<0.05, n= 7); 2) After application of HN (5μmol/l) alone, the current amplitude of INaP remained unchanged; 3)After co-application of HN and hypoxia, current density of INaP increased to 1.44±0.24 times control group, significantly different compared with the value of hypoxia group (P<0.05).
These results indicated that: (1) During early hypoxia, enhanced INaP amplitude constitutes the primary pathway of Na~+ influx, and hence resulted in Na~+ and Ca~(2+) imbalance, delayed membrane depolarization and cell death; (2)Acute hypoxia inhibited transient INaT; (3) HN could attenuate hypoxia-induced increased of INaP and thus mitigate Na~+ imbalance and secondary lessons. The physiological and pathological significance of INaT changes remain unclear.
PartⅢ: Effects of HN on Ca~(2+) Dysregulation during Excitotoxic Glutamate Exposure and its Mechanisms
Lines of evidence suggested that HN provided neuroprotection against hypoxia insults and glutamate excitotoxicity. Due to the central involvement of the activation of glutamate receptors and calcium overload in ischemic brain injury, the aim of this work was to investigate whether and how HN can prevent calcium homeostasis deregulation induced by glutamate (500μM) in cultured cortical neurons, and the potential involvements of membrane receptors and mitochondrial mechanisms.
In the present study, intracellular Fluo-3 Ca~(2+)-imaging was conducted with laser confocal microscopy in cultured cortical neurons. Mitochondrial uncoupler FCCP was used as a tool to collapse the mitochondrial membrane potential and thus block mitochondrial Ca~(2+) uptake, in addition release Ca~(2+) stored within,. Direct estimates of effects of HN on glutamate receptor channels were conducted with whole-cell path clamp in acutely isolated neurons.
The major results as following: 1) glutamate (500μM) induced marked increase in intracellular calcium ions in cultured cortical neurons. The mean changes in [Ca~(2+)]i at the steady-state level stimulated by 500μM glutamate were 2.29±0.38. 2) HN pretreatment significantly attenuated the increase of intracellular calcium responses upon excitotoxic concentration of glutamate exposure. The mean changes in [Ca~(2+)]i stimulated by glutamate were 1.38±0.19 in HN-treated neurons, nearly 40% inhibition compared with control neurons (P<0.01). 3) to investigate the potential involvement of mitochondrial mechanisms in the antagonisms of dysregulation of HN, we used uncouplers FCCP to semi-quantify the amount of Ca~(2+) taken up by mitochondria. Cells were exposed to glutamate until the steady-state phase (plateau) at which time point 1μM FCCP was applied to rapidly dump any Ca~(2+) stored in the mitochondrion back into the cytosol. FCCP application resulted in a significantly greater [Ca~(2+)]i increase in HN-treated cells. The changes in Fluo-3 fluorescence were 1.64±0.15 and 2.83±0.29 for control neurons and HN-treated ones, respectively (P< 0.05), indicating an HN-induced increase in the mitochondrial calcium ([Ca~(2+)]m) load during glutamate exposure; 4) when FCCP was applied before glutamate exposure, the glutamate-induced [Ca~(2+)]i increase was 1.29±0.14 and 1.09±0.35 in control group and HN-pretreated group, respectively (P<0.05). Effects of HN were not abolished in the presence of FCCP, indicating involvement of other mitochondrial-independent mechanisms in antagonism of dysregulation of Ca~(2+); 5) electrophysiological experiments showed that HN significantly inhibited glutamate receptor-mediated currents. The mean peak currents were significantly decreased to 53.1±7.4% (P <0.01, n=13) and 67.1±3.6% (P <0.01, n=12) of initial value for glutamate (200μM)- and NMDA (100μM)-induced currents in neurons treated with HN for 2 min.
These results indicated that: 1) HN (5μM) attenuated intracellular calcium dysregulation during excitotoxic glutamate exposure; 2) this attenuation was resulted from preserved mitochondrial functions and calcium buffering capacity and 3) decreased Ca~(2+) entry via glutamate receptor channels.
Taken together, we can draw conclusions blow:
(1) Acute hypoxia induced disturbance of voltage-gated sodium and potassium channels, suggesting important implications of these channels in early hypoxia.
(2) Acute hypoxia induced an enhancement of the delayed rectifier voltage-dependent K~+ channel in central neurons, which might be a deleterious response that constitutes the pathway for K~+ efflux and resulted in K~+ homeostasis deregulation during early hypoxia;transient IA currents were not influenced by acute hypoxia.
(3) During early hypoxia, enhanced INaP amplitude constituted the primary pathway of Na~+ influx during early hypoxia, and hence resulted in Na~+ and Ca~(2+) imbalance, delayed membrane depolarization and cell death;
(4) HN pre-application could attenuated hypoxia-induced increased of IK and INaP and thus mitigate K~+, Na~+ imbalance and secondary lessons. The physiological and pathological functional implication of actions of HN on IA remains unclear.
(5) Glutamate induced intracellular calcium overload and damage neurons; humanin attenuated intracellular calcium dysregulation during excitotoxic glutamate exposure and thus might mitigate intracellular calcium overload during hypoxia./ischemia.
(6) HN attenuated delayed calcium deregulation via preserved mitochondrial buffering capacity and inhibited Ca~(2+) influx through glutamate receptor channels.
一般认为缺氧或缺血造成的神经细胞损伤可大致分为三个阶段:(1)急性缺氧或缺血早期,其特征表现为膜通道的功能活动及膜电位的改变,细胞内环境的改变,如细胞内Na~+、Ca~(2+)、和细胞外K~+离子的显著增加等;(2)损伤相关的酶激活(蛋白酶、磷脂酶等);(3)细胞功能和结构改变,最终导致细胞死亡。由于损伤性质和细胞类型的不同,从急性缺氧缺血到延迟的细胞死亡发生所经历的时间可有很大差异(从数分钟到数小时或数周),其病理变化也有很大区别,包括最终引起的神经细胞的死亡也有不同方式,如坏死、凋亡等。而早期发生的离子稳态失衡在缺氧缺血所致的神经损伤过程发挥重要作用,至少包括以下三个方面:(1)缺氧早期引起的Na~+和Ca~(2+)在神经细胞中的积聚是缺氧造成损伤的重要诱因;(2)缺氧或缺血早期对神经元的影响几乎只与离子稳态的改变相关;(3)这些早期改变不仅决定细胞短期(即时)的命运(例如细胞损伤和坏死),而且也将引起细胞的长期改变(例如代谢改变、酶的活化、基因激活及引起凋亡等),即神经元的很多损伤性改变都继发于离子稳态失调。因此,探讨缺氧早期引起的离子稳态失衡的机制对于理解缺氧导致的病理生理改变及早期、晚期的病理损伤具有极其重要的意义。
在生理条件下,离子跨膜移动由多种机制调控,从而维持各种离子在胞内外分布的相对恒定;维持静息电位稳定及在此基础上产生的动作电位;维持神经组织正常的兴奋性。然而,急性缺氧会造成离子通道活动的改变及膜电位改变如去极化等,并继而引起细胞结构、功能改变直至细胞死亡。为探讨缺氧引起的早期改变,探讨HN对于早期缺氧/缺血的神经保护作用及其离子机制。我们以缺氧和谷氨酸处理神经元模拟体内的缺血损伤,采用膜片钳技术和钙离子成像技术,观察急性缺氧引起的离子稳态失衡,包括K+、Na~+离子电流改变(通道活动改变)和细胞内钙信号改变(胞内Ca~(2+)升高)以及HN的保护作用。
实验中采用急性分离的海马CA1细胞和膜片钳技术以及离体培养的原代神经元和离子测定技术,观察了急性缺氧诱导的电压门控性钾电流(全钾电流、瞬间钾电流I_A、延迟整流钾电流I_K)、电压门控性钠电流(持续性钠电流I_(NaP)、瞬间钠电流I_(NaT))的改变及HN对急性缺氧诱导产生的通道功能改变的影响。考虑到兴奋性神经毒和钙离子超载在包括缺血/缺氧在内的各种神经病理中的重要作用,观察了HN对谷氨酸诱导的胞内Ca~(2+)的影响,并初步探讨了其机制。主要结果显示:(1)急性缺氧造成电压门控I_K、I_(Na)电流的异常改变(电流幅度增加);(2)HN(5μM)与缺氧同时作用于细胞,HN能够拮抗缺氧诱导的I_K、I_(NaP)电流的异常改变;(3)谷氨酸造成胞内Ca~(2+)明显升高,HN(5μM)预处理抑制了谷氨酸诱导的胞内Ca~(2+)升高,其机制可能是HN通过抑制经谷氨酸受体的Ca~(2+)内流和保护线粒体的钙缓冲能力而发挥作用。
上述结果显示,离子稳态失衡,包括缺氧引起的I_K增加、I_(NaP)增加及[Ca~(2+)]i增加,是缺氧引起的早期改变。这些改变可能通过影响膜电位(导致去极化)、影响兴奋性、影响胞内Ca~(2+)(导致钙超载),及由此继发性地引起其他病理改变,;在缺氧诱导的病理损伤过程发挥重要作用。HN通过抑制离子通道异常活动和稳定线粒体的功能而维持离子稳态,从而保护神经元对抗缺氧/缺血和兴奋毒造成的损伤,即HN可对急性缺氧损伤提供有效保护。这些结果也支持HN可能具有广谱的神经保护作用的假说。
第一部分: HN对缺氧诱导的海马CA1神经元电压门控性钾电流异常改变的影响
通道的异常活动造成的离子失稳态被认为是缺氧/缺血诱导的神经元损伤的最初和关键的改变,包括缺氧缺血早期引起的细胞外钾浓度增高。研究显示,细胞内钾的外流与缺氧诱导的去极化密切相关,升高细胞外钾浓度能够模拟缺氧去极化,而缺氧去极化被认为是导致神经细胞死亡的重要因素;此外钾外流造成的细胞内钾的丢失引起神经细胞皱缩、解除对caspase和核内切酶的抑制作用从而介导凋亡过程。资料证实,持续性的细胞外高钾可引起神经细胞死亡,而阻断钾外流可拮抗缺氧和缺血诱导的神经元死亡。这些结果提示,维持钾通道功能稳定及钾在细胞内外正常分布在缺血损伤及保护中发挥重要意义。神经元上的钾通道包括五类,目前针对缺氧对钾通道调制的研究大多数集中在K_(ATP)通道和K_(Ca)通道,而对电压门控性钾通道包括延迟整流钾电流(I_K)、瞬间钾电流(I_A)在缺血、缺氧状态下功能特性的改变研究甚少。
HN是一个内源性神经肽,最初被认为只针对AD相关毒性的特异神经保护作用。然而最近的研究提示,HN可能具有更为广谱的保护作用。本实验室前期工作也显示,HN对缺氧诱导的神经损伤具有保护作用,其作用机制包括抗凋亡和稳定线粒体功能。
为了探讨电压门控性钾通道在缺氧导致钾离子稳态失衡中的作用,以及HN急性处理对缺氧损伤可能发挥的影响,我们采用急性分离的海马CA1细胞和膜片钳技术,观察了急性缺氧对电压门控性钾通道(全钾电流、瞬间钾电流I_A、延迟整流钾电流I_K)的作用及HN在常氧状态(单独作用)于缺氧状态(与缺氧同时作用)下对钾通道的影响。
主要结果如下:1)急性缺氧(3min)显著增强I_K电流,+70mV去极化脉冲下I_K电流幅度增加为对照组的196±15% (P<0.05, n=6);而急性缺氧对I_A没有明显影响,+70mV去极化脉冲下I_A电流幅度与对照组相比无统计学差异(P=0.871, n=6);2)HN(5μM)单独作用(即在常氧状态下)使I_A电流幅度增加,I_A电流幅度从对照组的2250.33±608.77pA增加为3051.47±781.62 pA (P<0.05, n=6); HN单独作用不影响I_K电流幅度,HN作用前后I_K电流幅度分别为1240.96±171.71 pA和1609.63±353.49 pA (P>0.05, n=6);3)HN与缺氧同时作用时,取消了缺氧对I_K电流的影响。在有HN存在的情况下,缺氧不再引起I_K电流幅度的增加,记录到的I_K电流为对照组的0.89±0.23,与对照组相比无显著性差异(P>0.05, n=11)。
这些结果显示:1)缺氧早期引起延迟整流钾电流I_K的增强,I_K电流的增强是介导缺氧早期K~+外流的主要途径,并可能由此造成缺氧的损伤性改变及导致其他病理改变;2)缺氧对瞬间钾电流I_A未产生明显作用;3)HN抑制缺氧引起的I_K增强,这一作用可能通过减轻K~+分布的异常从而对缺氧/缺血损伤发挥神经保护作用; 4)HN在常氧和缺氧条件下都能增强I_A电流幅度,其生理即病理生理意义尚不明确。以上结果提示,HN可能通过拮抗缺氧对电压依赖性钾通道的作用、维持钾离子稳态而对抗缺氧缺血引起的早期改变,从而对缺氧性脑损伤发挥有效保护作用。
第二部分:HN对缺氧诱导的海马CA1神经元电压门控性钠电流异常改变的影响
缺氧早期发生快速的细胞内环境的改变,包括细胞内先于胞浆Ca~(2+)的增高而发生的Na~+增高。大量研究提示了钠通道功能改变以及胞浆Na~+增高在缺氧/缺血性损伤中发挥的作用。细胞外Na~+内流促进缺氧去极化的发生;胞浆Na~+增高不仅刺激钠钾泵的活动加快能量耗竭,而且通过逆转Na~+/Ca~(2+)交换体的转运方向从而引起细胞内Ca~(2+)增高,加剧随之发生的细胞损伤。钠通道阻断剂和细胞外低Na~+溶液可减少缺血或缺氧性细胞损伤和延迟的缺氧去极化,证明了钠通道以及胞浆Na~+增高在缺氧性损伤中所发挥的作用。
HN是最近发现的内源性神经肽,初始被认为特异拮抗AD相关的神经毒,然而新近的研究提示HN可能具有更为广谱的神经保护作用。本实验室的前期工作显示HN预处理能够拮抗缺氧诱导的神经损伤。
为探讨HN对缺氧性损伤可能具有的保护作用及其作用的电生理机制,本研究中我们采用了全细胞膜片钳技术、急性分离海马神经元,观察HN在常氧和缺氧状态下对电压门控性钠通道(持续性钠电流I_(NaP)、瞬间钠电流I_(NaT))的作用。
主要结果如下:1)急性缺氧2 min后,I_(NaP)电流幅度显著增大为对照组的265±40% (P < 0·01, n = 5),而缺氧引起了I_(NaT)电流幅度的降低为对照组的65.4±7% (P<0.05, n= 7);2)HN (5μmol/l)单独作用2 min后I_(NaP)电流幅度不受影响;3)HN与缺氧同时作用2min,HN抑制了缺氧诱导的I_(NaP)电流幅度的增加,在HN存在的情况下I_(NaP)电流幅度为对照组的144±24% (P > 0.05, n=5),即HN拮抗了缺氧诱导的I_(NaP)增强。这些结果显示:1)缺氧早期,不失活的电压门控钠电流即持续性钠电流I_(NaP)的活动增强是引起[Na~+]i升高的主要途径,并可能继而造成Na~+、Ca~(2+)稳态失调、持续膜去极化和细胞损伤; 2)急性缺氧抑制瞬间钠电流I_(NaT);3)HN急性处理能够拮抗缺氧诱导的I_(NaP)增强,从而可能通过减轻Na~+分布异常、及其继发损伤而在缺氧早期发挥神经保护作用。瞬间钠电流I_(NaT)改变的生理及病理生理意义尚不明确。
第三部分:HN对谷氨酸诱导的钙离子稳态失衡的影响及机制
本实验室以往的工作显示,HN对缺氧和兴奋毒损伤具有神经保护作用。考虑到谷氨酸受体过度激活和钙超载在缺氧缺血性脑损伤中的关键作用,本研究观察在培养的皮层神经元,HN能否拮抗谷氨酸(500μM)所导致的细胞内钙离子稳态失衡,并初步探讨可能的机制包括线粒体机制。
本研究以谷氨酸处理神经元模拟缺血的神经损伤,采用培养皮层神经元Ca~(2+)荧光成像技术观察神经元的[Ca~(2+)]i;在急性分离神经元采用全细胞膜片钳技术观察HN对谷氨酸受体的作用。为探讨HN对谷氨酸诱导钙反应的拮抗作用是否有线粒体钙缓冲机制的参与,使用了线粒体解耦联剂FCCP。FCCP可使线粒体质子梯度、线粒体膜电位消失,从而导致将胞浆内Ca~(2+)摄取进入线粒体内的动力消失,因而抑制线粒体摄取Ca~(2+)、并可促进其释放Ca~(2+)。因此,在谷氨酸预处理引起钙反应的稳态期应用FCCP,由FCCP诱导的细胞内Ca~(2+)
增加可反应谷氨酸暴露期间线粒体摄取Ca~(2+)的量。
主要结果如下:1)谷氨酸(500μM)导致培养皮层神经元显著的[Ca~(2+)]i增高,平台期fluo-3标准化荧光增加为基线水平的2.29±0.38;2)HN预处理显著抑制兴奋毒浓度谷氨酸暴露所导致的细胞内Ca~(2+)增加;在HN预处理的细胞,谷氨酸导致的[Ca~(2+)]i增加为1.38±0.19,与对照组相比抑制率接近40% (P<0.01);3)在对照细胞和HN预处理细胞,FCCP诱导的[Ca~(2+)]i增加分别为1.64±0.15和2.83±0.29,HN预处理细胞FCCP诱导释放的Ca~(2+)量显著增加(P<0.05),说明HN预处理导致了谷氨酸暴露期间线粒体摄取Ca~(2+)增加;4)在谷氨酸之前预先给予FCCP,通过破坏线粒体质子梯度、导致线粒体膜电位丧失,从而抑制线粒体的摄取Ca~(2+)功能。在FCCP存在情况下,HN处理组和对照组谷氨酸诱导的[Ca~(2+)]i增加分别为1.29±0.14和1.09±0.35,HN仍能抑制兴奋毒浓度谷氨酸诱导的[Ca~(2+)]i反应(P<0.05),提示HN的拮抗作用除线粒体外还有其他机制;4)电生理结果显示,HN显著抑制谷氨酸(200μM)和NMDA(100μM)所诱导的全细胞膜电流。HN预处理2min使谷氨酸、NMDA诱导的峰电流分别降低为基线水平的53.1±7.4% (P<0.01, n=13)、67.1±3.6%(P<0.01, n=12)。
这些结果显示:(1)HN(5μM)能够拮抗兴奋毒浓度谷氨酸所导致的细胞内钙升高;(2)这种拮抗作用部分通过稳定线粒体功能以及恢复钙缓冲能力而实现;(3)部分通过抑制经谷氨酸受体的细胞外Ca~(2+)内流而实现。
综上结果,可得出如下结论:
1)缺氧诱导电压门控性钠、钾通道电流的改变,提示电压门控性钠、钾通道在缺氧早期发生的离子稳态失衡中起重要作用。
2)缺氧早期引起延迟整流钾电流IK的增强,这可能是介导缺氧早期K~+外流、K~+稳态失衡的主要途径及损伤机制;缺氧对瞬间钾电流I_A未产生明显作用,I_A可能由于其快速失活的特性而在缺氧早期K~+稳态失衡中不起重要作用。
3)缺氧早期引起不失活的电压门控钠电流即持续性钠电流I_(NaP)的活动增强,Na~+经I_(NaP)内流是缺氧早期造成细胞内Na~+升高的主要途径,并继而引起Na~+、Ca~(2+)稳态失衡、持续的膜去极化和细胞损伤;急性缺氧抑制瞬间钠电流INaT。
4)HN预处理可拮抗缺氧引起的I_K、I_(NaP)增强。这一作用通过阻止K~+、Na~+稳态失衡及其继发损伤从而在缺氧/缺血早期发挥神经保护作用。HN对I_A产生了影响,但生理及病理生理意义尚不明确。
5)谷氨酸能够诱发细胞内钙超载而对神经元发挥损伤作用。HN能够拮抗谷氨酸导致的钙升高,从而通过减轻缺血缺氧和兴奋毒损伤期间发生的细胞内Ca~+超载。
6)HN拮抗谷氨酸诱导的钙超载的机制,包括HN可保护线粒体的钙缓冲能力以及抑制经谷氨酸受体的Ca~(2+)内流。
Humanin (HN) is a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal brain region from patients with Alzheimer’s disease (AD) in 2001. From those initial observations it seemed that HN was a selectively neuroprotective factor rescuing neurons from Alzheimer’s disease-related insults, hence it is defined as AD-related neuroprotive peptide. However, later efforts suggested a broader spectrum of its survival-promoting activity. Several lines of studies provided evidence for the argument. Prior works in our laboratory proved that HN treatment provided long-term neuroprotection against hypoxia insults in primary cortical neurons. Yet effects of HN when given acutely on early neuronal damage during hypoxia/ischemia remain unclear. And the mechanisms of HN neuroprotection remain to be fully illustrated, especially because of a paucity of electrophysiological studies.
It is generally accepted that that neuronal cell death can be described as three stages: (1) early intracellular ionic and chemical changes; early onset of acute hypoxia or ischemia is characterized by changes of ion channel activity and membrane potential, and major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na~+, Ca~(2+), and extracellular K~+ (2) activation of damaging enzymes and (3) changes in cellular functions and structures, eventually leading to cell death. The delayed before cell death occurs varies greatly (from minutes to hours or weeks), depending on the nature of the insults and the cell types. A large number of the damaging changes occurring in the neurons are secondary to loss of ion homeostasis (Lipton 1999), the pathological changes vary greatly, including the mode of final cell death. These early ionic alterations play important roles in hypoxia/ischemia induced neuronal damages: (a) the accumulation of Ca~(2+) and Na~+ in nerve cells during hypoxia has been shown by many investigators to be deleterious; (b) the main changes in neuronal function early in brain hypoxia or ischemia are almost solely related to changes in ionic homeostasis and (c) it is likely that these early changes dictate not only cell fate in the short term, such as cell injury and necrosis, but also long term changes such as activation of damaging enzymes and programmed cell death. A large number of the damaging changes occurring later in the neurons are secondary to disturbed ion homeostasis. Therefore, it is of great importance to investigate mechanisms of ionic imbalance during early hypoxia/ischemia insults.
Ion transmembrane flux is controlled by multiple different mechanisms under physiology conditions. However, changes in ionic channel activities by hypoxia are the major pathway for redistribution of ions across the membrane and the membrane depolarization, contributing to succeeding cell death. To examine the effects of HN on early hypoxia/ischemia induced disturbance of ionic homeostasis, using hypoxia model and glutamate as simulated-ischemia conditions, we investigate the effects of HN on ischemia-related insults induced disturbance of membrane ionic currents and the intracellular calcium ([Ca~(2+)]i) deregulation using whole-cell patch clamp technique and calcium imaging.
Using freshly isolated hippocampal CA1 neurons and patch clamp techniques, primary cultured neurons and ion imaging techniques, we investigate effects of hypoxia-induced disturbance of sodium and potassium currents and effects of HN. Given the crucial roles of excitotoxicity and dysfunction of calcium homeostasis in various neuropathologic lesions including hypoxia/ischemia, we investigated the effects of HN on Ca~(2+) deregulation induced by toxic concentration of glutamate exposure and its possible mechanisms. The results showed that:1) acute hypoxia induced disturbance of voltage-gated potassium and sodium currents (IK、INa); 2) HN (5μM) co-application with hypoxia solution attenuated hypoxia–induced disturbance of potassium current (IK) and persistent potassium current (INaP); and 3) HN (5μM) pretreatment attenuated Ca~(2+) deregulation during glutamate exposure through attenuated calcium entry via glutamate receptor channels and preserved mitochondrial buffering capacity.
Our results showed that disturbance of ionic homeostasis, including enhancement of IK, INaP and [Ca~(2+)]i, were the early changes induced by hypoxia and played important roles in hypoxic pathology through actions on membrane potential, excitability. HN was able to maintain ionic homeostasis via attenuating disturbance of ion channels and stabilizing mitochondrial function, and thus protect neurons against early hypoxia/ischemia insults. Furthermore, HN provided neuronal protection by ionic mechanisms acting at the very beginning of insults, i.e., that it is protective against acute hypoxia injury. These results provide further evidence for the broader spectrum of neuroprotective effects of HN against insults other than AD.
PartⅠ: Effects of HN on Hypoxia-Induced Disturbance of Voltage-Dependent Potassium Currents in Rat Hippocampal CA1 Neurons
Disruption of ionic homeostasis induced by disturbance of ion channel activity has been generally regarded as an initial and key alterations in anoxia/ischemia-induced neuronal injury. Enhanced ef?ux of K~+ has been shown to be closely associated with anoxia-induced depolarization, which is believed to be a crucial factor leading to neuronal death. The anxia depolarization is mimicked by elevation of the extracellular potassium concentration. The loss of intracellular K~+ causes cell shrinkage (apoptotic volume decrease) and creates a permissible environment for apoptosis by relieving the inhibition of endogenous caspases and nucleases. Sustained exposure to elevated extracellular K~+ causes signi?cant neuronal death even under conditions of normoxia and abundant glucose supply, whereas blockade of K~+ ef?ux has been shown to attenuate hypoxia and ischemia-induced neuronal death. These results suggest that maintaining K~+ channels functions and K~+ distribution across plasmamembrane may be of therapeutic bene?t in the treatment of ischemia-related insults. Among the five types of plasmalemma potassium channels, a great deal of researches focus on KATP and KCa channels, while voltage-gated potassium channels get less attention.
Mammalian neurons react rapidly to a lack of oxygen with alterations of ion channels activities, being either adaptive or deleterious responses. Of particular interest is the effects that hypoxia has on potassium channels since these channels are one of the fundamental factors that regulate membrane potential and neuronal excitability, in addition there are major alterations in K~+ ions homeostasis during early hypoxia. Thus K~+ channels and K~+ homeostasis may play important roles in the initiation and development of hypoxia/ischemia. Among the five classes of K~+ channels identified in excitable cells, penetrating researches have been focused on roles of KATP and KCa channels; while changes of voltage-gated potassium channels during hypoxia/ischemia got much less attentions.
HN was originally identified as an endogenous peptide that protects neuronal cells from death caused by Alzheimer’s disease (AD)-related genes and amyloid-β(Aβ). While recent studies suggested that HN might have a broader spectrum of protections. And the mechanisms underlying the protections of HN remain to be illustrated. Preliminary works in our laboratory showed that HN protected neurons from hypoxia-insults with mechanisms including anti-apoptosis and preserving mitochondrial functions.
To illustrate the effects of HN during early hypoxia/ischemia insults and illuminate underlying electrophysiological mechanisms for its potential neuroprotections, we investigated the response of voltage-gated K~+ (KV) channels (total potassium current, transient potassium current, and delayed rectifier potassium current) to hypoxia and the effects of HN in freshly isolated hippocampal CA1 neurons from rats aged 10～12 days.
Major results as following: 1) acute exposure to hypoxia (3 min) significantly enhanced IK currents. In five neurons exposed to hypoxia, the amplitude of IK at +70 mV was 1.96±0.15 times the amplitude of control group (P<0.05, n=6). While the amplitude of IA at +70 mV was 1.05±0.05 times of controls group, i.e. not significantly different from control (P>0.05, n=6); 2) HN (5μM) alone had no effect on IK at constant normoxic PO2, the amplitude of IK were 1240.96±171.71 pA and 1609.63±353.49 pA before and after HN application, respectively (P>0.05, n=6); HN, however, significantly increased IA amplitude under both hypoxia and nomoxia, the amplitude of IA increased from 2250.33±608.77 pA to 3051.47±781.62 pA after HN treatment (P<0.05, n=6); 3) HN co-application abolished the effect of hypoxia on IK. In the presence of HN, hypoxia no longer increased IK current amplitude. The IK current amplitude in the presence of HN was 0.89±0.23 times of control (P<0.05, n=11).
These results indicated that: 1) acute hypoxia induced an enhancement of the IK channel in central neurons, which may be the major pathway for K~+ efflux and results in hypoxia-induced changes and other pathological changes;2) transient IA currents were not influenced by acute hypoxia; 3) HN attenuated hypoxia-induced disturbance of IK, this effect might be neuroprotective during ischemia by attenuated the disturbance of potassium ionic distribution that occured. 4) HN increases IA current amplitude; the physiological and pathological significance of this fact remain unclear. These results suggested that HN may attenuate hypoxia-induced changes of IK currents and thus maintain K~+ homeostasis, protect neurons against hypoxia-induced damages.
PartⅡ: Effects of HN on Hypoxia-Induced Disturbance of Voltage-Dependent Sodium Currents in Rat Hippocampal CA1 Neurons
During hypoxia, cells undergo alterations in membrane potential and detrimental changes in their intracellular environment, including an early increase in intracellular Na~+ that contributes to the pathophysiology of neuronal death. There are mounting studies illustrating the relation between Na~+ channels and hypoxia. A large number of the damaging changes occurring in the neurons are secondary to an increase in intracellular Na~+ levels (Lipton 1999). In dissociated cells and cell cultures, hypoxia raised intracellular Na~+ concentration ([Na~+]i). Increased [Na~+]i may aggravate neuronal lessons by imposing an energy demand on cells due to stimulation of the plasmalemmal Na~+, K~+-ATPase. In addition, it contributes to an impairment of Ca~(2+) homeostasis by driving Ca~(2+) into the cells via the Na~+-Ca~(2+) exchanger, and to the development of other acute dysfunctions. Drugs that selectively block this current might reduce damage to cells during ischemia or hypoxia.
HN is a recently described endogenous peptide that antagonizes neurotoxicity caused by Alzheimer’s disease (AD) relevant insults, while recent studies suggest that HN may have a broader spectrum of protection. To illustrate the electrophysiological mechanism of HN on neurons and the possible neuroprotection against hypoxia insults, whole-cell patch clamp techniques were applied on isolated hippocampal neurons to study the possible effects of HN (5μM) on persistent and transient sodium current under normoxia and hypoxia.
The major results as following: 1) After 2 min hypoxia stimulation, persistent sodium current (INaP) increased significantly to 2.65±0.40 times control group (P < 0·01, n = 5), transient sodium current (INaT) decreased significantly to 65.4±7% of baseline (P<0.05, n= 7); 2) After application of HN (5μmol/l) alone, the current amplitude of INaP remained unchanged; 3)After co-application of HN and hypoxia, current density of INaP increased to 1.44±0.24 times control group, significantly different compared with the value of hypoxia group (P<0.05).
These results indicated that: (1) During early hypoxia, enhanced INaP amplitude constitutes the primary pathway of Na~+ influx, and hence resulted in Na~+ and Ca~(2+) imbalance, delayed membrane depolarization and cell death; (2)Acute hypoxia inhibited transient INaT; (3) HN could attenuate hypoxia-induced increased of INaP and thus mitigate Na~+ imbalance and secondary lessons. The physiological and pathological significance of INaT changes remain unclear.
PartⅢ: Effects of HN on Ca~(2+) Dysregulation during Excitotoxic Glutamate Exposure and its Mechanisms
Lines of evidence suggested that HN provided neuroprotection against hypoxia insults and glutamate excitotoxicity. Due to the central involvement of the activation of glutamate receptors and calcium overload in ischemic brain injury, the aim of this work was to investigate whether and how HN can prevent calcium homeostasis deregulation induced by glutamate (500μM) in cultured cortical neurons, and the potential involvements of membrane receptors and mitochondrial mechanisms.
In the present study, intracellular Fluo-3 Ca~(2+)-imaging was conducted with laser confocal microscopy in cultured cortical neurons. Mitochondrial uncoupler FCCP was used as a tool to collapse the mitochondrial membrane potential and thus block mitochondrial Ca~(2+) uptake, in addition release Ca~(2+) stored within,. Direct estimates of effects of HN on glutamate receptor channels were conducted with whole-cell path clamp in acutely isolated neurons.
The major results as following: 1) glutamate (500μM) induced marked increase in intracellular calcium ions in cultured cortical neurons. The mean changes in [Ca~(2+)]i at the steady-state level stimulated by 500μM glutamate were 2.29±0.38. 2) HN pretreatment significantly attenuated the increase of intracellular calcium responses upon excitotoxic concentration of glutamate exposure. The mean changes in [Ca~(2+)]i stimulated by glutamate were 1.38±0.19 in HN-treated neurons, nearly 40% inhibition compared with control neurons (P<0.01). 3) to investigate the potential involvement of mitochondrial mechanisms in the antagonisms of dysregulation of HN, we used uncouplers FCCP to semi-quantify the amount of Ca~(2+) taken up by mitochondria. Cells were exposed to glutamate until the steady-state phase (plateau) at which time point 1μM FCCP was applied to rapidly dump any Ca~(2+) stored in the mitochondrion back into the cytosol. FCCP application resulted in a significantly greater [Ca~(2+)]i increase in HN-treated cells. The changes in Fluo-3 fluorescence were 1.64±0.15 and 2.83±0.29 for control neurons and HN-treated ones, respectively (P< 0.05), indicating an HN-induced increase in the mitochondrial calcium ([Ca~(2+)]m) load during glutamate exposure; 4) when FCCP was applied before glutamate exposure, the glutamate-induced [Ca~(2+)]i increase was 1.29±0.14 and 1.09±0.35 in control group and HN-pretreated group, respectively (P<0.05). Effects of HN were not abolished in the presence of FCCP, indicating involvement of other mitochondrial-independent mechanisms in antagonism of dysregulation of Ca~(2+); 5) electrophysiological experiments showed that HN significantly inhibited glutamate receptor-mediated currents. The mean peak currents were significantly decreased to 53.1±7.4% (P <0.01, n=13) and 67.1±3.6% (P <0.01, n=12) of initial value for glutamate (200μM)- and NMDA (100μM)-induced currents in neurons treated with HN for 2 min.
These results indicated that: 1) HN (5μM) attenuated intracellular calcium dysregulation during excitotoxic glutamate exposure; 2) this attenuation was resulted from preserved mitochondrial functions and calcium buffering capacity and 3) decreased Ca~(2+) entry via glutamate receptor channels.
Taken together, we can draw conclusions blow:
(1) Acute hypoxia induced disturbance of voltage-gated sodium and potassium channels, suggesting important implications of these channels in early hypoxia.
(2) Acute hypoxia induced an enhancement of the delayed rectifier voltage-dependent K~+ channel in central neurons, which might be a deleterious response that constitutes the pathway for K~+ efflux and resulted in K~+ homeostasis deregulation during early hypoxia;transient IA currents were not influenced by acute hypoxia.
(3) During early hypoxia, enhanced INaP amplitude constituted the primary pathway of Na~+ influx during early hypoxia, and hence resulted in Na~+ and Ca~(2+) imbalance, delayed membrane depolarization and cell death;
(4) HN pre-application could attenuated hypoxia-induced increased of IK and INaP and thus mitigate K~+, Na~+ imbalance and secondary lessons. The physiological and pathological functional implication of actions of HN on IA remains unclear.
(5) Glutamate induced intracellular calcium overload and damage neurons; humanin attenuated intracellular calcium dysregulation during excitotoxic glutamate exposure and thus might mitigate intracellular calcium overload during hypoxia./ischemia.
(6) HN attenuated delayed calcium deregulation via preserved mitochondrial buffering capacity and inhibited Ca~(2+) influx through glutamate receptor channels.
引文
Alex V. Nowicky and Michael R. Duchen. Changes in [Ca~(2+)]i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons. JPhysiol. 1998; 507:131—145.
Ashcroft FM. Adenosine 5’-triphosphate-sensitive potassium channels. Annu Rev Neurosci 1988; 11: 97–118.
Ballanyi K. Protective role of neuronal KATP channels in brain hypoxia. J Experimental Biology. 2004; 207(pt 18):3201-3212.
Bortner CD, Hughes FM Jr, Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem. 1997; 272(51):32436-32442.
Brevnova, Elena E., Oleksandr Platoshyn, Shen Zhang, and Jason X.-J. Yuan. Overexpression of human KCNA5 increases IK(V) and enhances apoptosis. Am J Physiol Cell Physiol 2004; 287: C715–C722.
Carmelle V. Remillard and Jason X.-J. Yuan. Activation of K+ channels: an essential pathway in programmed cell death. Am J Physiol Lung Cell Mol Physiol 2004; 286:49-67.
Chen XS, SP Chi, MN Liu, W Yang, TT Wei, Z Qi, and FY Yang. Inhibitory effect of ganglioside GD1b on K+ current in hippocampal neurons and its involvement in apoptosis suppression. J Lipid Res. 2005; 46(12):2580-2585.
Chen XX., LL Yuan, CP Zhao, et al. Deletion of Kv4.2 Gene Eliminates Dendritic A-Type K+ Current and Enhances Induction of Long-Term Potentiation in Hippocampal CA1 Pyramidal Neurons. J Neurosci., 2006; 26:12143–12151.
Chi XX and Xu ZC. Differential changes of potassium currents in CA1 pyramidal neurons after transient forebrain ischemia. J Neurophysiol. 2000; 84: 2834-2843.
Cummins TR, Donnelly DF, and Haddad GG. Effect of metabolic inhibition on the excitability of isolated hippocampal CA1 neurons: developmental aspects. J Neurophysiol. 1991; 66: 1471–1482.
Dallaporta B, Hirsch T, Susin SA, Zamzami N, Larochette N, Brenner C, Marzo I, Kroemer G. Potassium leakage during the apoptotic degradation phase. J Immunol. 1998;160(11):5605-5615.
Deng P, Pang ZP, Zhang Y, Xu ZC.Increase of delayed rectifier potassium currents in large aspiny neurons in the neostriatum following transient forebrain ischemia. Neuroscience. 2005;131(1):135-146.
Ekhterae D, Platoshyn O, Krick S, Yu Y, McDaniel SS, and Yuan JX. Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells. Am J Physiol Cell Physiol 2001; 281: C157–C165.
Ekhterae D, Platoshyn O, Zhang S, Remillard CV, and Yuan JX. Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents. Am J Physiol Cell Physiol 2003; 284: C1405–C1410.
Erdemli G, Xu YZ, and Krnjevic K. Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia. J Neurophysiol. 1998; 80: 2378–2390.
Ficker E and Heinemann U. Slow and fast transient potassium currents in cultured rat hippocampal cells. J Physiol (Lond) 1992; 445: 431–455.
Fujita H, Takizawa S, Nanri K, Matsushima K, Ogawa S, Shinohara Y. Potassium channel opener reduces extracellular glutamate concentration in rat focal cerebral ischemia. Brain Res Bull. 1997;43:365–368
Fujiwara N, Higashi H , Shimojik, and Yoshimura M. Effects of hypoxia on rat hippocampal neurons in vitro. J Physiol (Lond) 1987; 384: 131–151.
Fujiwura N, Tanaka E, Yamamoto S, Shigemori M, and Higashi H. Contribution of ATP-sensitive potassium channels to hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro. J. Neurophysiol. 1997; 77: 378–385.
Gebhardt C and Heinemann U. Anoxic decrease in potassium outward currents of hippocampal cultured neurons in absence and presence of dithionite. Brain Res. 1999; 837: 270–276.
Griesemer D, Zawar C, Neumcke B. Cell-type specific depression of neuronal excitability in rat hippocampus by activation of ATP-sensitive potassium channels. Eur Biophys J. 2002; 31(6):467-477.
Guo B, Zhai DY, Cabezas E, Welsh K, Nouraini S, Satterthwait AC, Reed JC. Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature. 2003; 423: 456–461. Hansen AJ, Hounsgaard J, and Jahnsen H. Anoxia increases potassium conductance in hippocampal nerve cells. Acta Physiol Scand. 1982; 115: 301–310.
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981; 391(2):85-100.
Hashimoto Y, Ito Y, Niikura T, Shao Z, Hata M., Oyama F, Nishimoto I. Mechanisms of neuroprotection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein. Biochem. Biophys. Res. Commun. 2001a , 283: 460–468.
Hashimoto Y., Niikura T., Tajima H., Yasukawa T., Sudo H., Ito Y., Kita Y., Kawasumi M., Kouyama K., Doyu M., Sobue G., Koide T., Tsuji S., Lang J., Kurokawai K., Nishimoto I. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Abeta. Proc. Natl. Acad. Sci. 2001b , USA 98: 6336–6341.
Hashimoto Y, Niikura T, Ito Y, Sudo H, Hata M, Arakawa E, Abe Y, Kita Y, Nishimoto I. Detailed Characterization of Neuroprotection by a Rescue Factor Humanin against Various Alzheimer’s Disease-Relevant Insults. J. Neurosci. 2001c; 21:9235–9245.
Hashimoto Y., Suzuki H., Aiso S., Niikura T., Nishimoto I., Matsuoka M. Involvement of tyrosine kinases and STAT3 in Humanin-mediated neuroprotection. Life Sciences 2005; 77: 3092–3104.
Huang L, Li W, Li B, Zou F. Activation of ATP-sensitive K channels protects hippocampal CA1 neurons from hypoxia by suppressing p53 expression. Neurosci Lett. 2006; 398:34-38.
Hughes FM Jr, Bortner CD, Purdy GD, Cidlowski JA. Intracellular K+ suppresses the activation of apoptosis in lymphocytes. J Biol Chem. 1997; 272(48):30567-30576.
Hyllienmark L and Brismar T. Effect of metabolic inhibition on K+ channels in pyramidal cells of the hippocampal CA1 region in rat brain slices. J Physiol (Lond) 1996; 491: 155–164.
Jiang C and Haddad GG. Effect of anoxia on intracellular and extracellular potassium activity in hypoglossal neurons in vitro. J Neurophysiol 1991, 66: 103–111.
Jiang C, Xia Y, Haddad GG. Role of ATP-sensitive K+ channels during anoxia: major differences between rat (newborn and adult) and turtle neurons. J Physiol. 1992; 448:599-612.
Kariya S, Takahashi N, Ooba N, Kawahara M, Nakayama H, Ueno S. Humanin inhibits cell death of serum-deprived PC12 cells. Neuroreport 2002; 13: 903–907.
Kariya S, Takahashi N, Hirano M, Ueno S. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes. Mol. Cell. Biochem. 2003, 254: 83–89.
Kariya S, Hirano M, Furiya Y, Ueno S. Effect of humanin on cellular ATP production: a possible therapeutic candidate for mitochondrial disorders. Ann. Neurol. 2004, 56 (Suppl. 8): s65.
Kariya S, Hirano M, Furiya Y, Ueno S. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides 2005; 39: 97–101.
Kariya S, Hirano M, Furiya Y, Sugie K, Ueno S. Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent. Acta Neuropathol 2005; 109: 367–372.
Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982; 239(1):57-69.
Klee R, Ficker E, Heinemann U. Comparison of voltage-dependent potassium current in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3. J. Neurphysiol. 1995; 75: 1982–1995.
Krick S, Platoshyn O, Sweeney M, Kim H, and Jason Yuan XJ. Activation of K+ channels induces apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol 2001; 280: C970–C979.
Krick S., Platoshyn O, McDaniel SS, Rubin LJ, and Yuan JX. Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis. Am J PhysiolLung Cell Mol Physiol. 2001; 281: L887–L894.
Lipton P. Ischemic Cell Death in Brain Neurons. Physiol Rev 1999; 79:1431-1568. Mironov SL, Langohr K, Haller M and Richter DW. Hypoxia activates ATP-dependent
potassium channels in inspiratory neurones of neonatal mice. Journal of Physiology 1998; 509: 755-766.
Murai Y, Ishibashi H, Koyama S, Akaike N. Ca~(2+)-activated K~+ currents in rat locus coeruleus neurons induced by experimental ischemia, anoxia, and hypoglycemia. J Neurophysiol. 1997; 78: 2674–2681.
Nakao K, Tokuyama S, Takahashi M, Kanet H and Ueda H. Antiamnesic Action of Cromakalim, a Potassium Channel Opener, in Mice Treated with Hypoxia- and Cerebral Ischemia-Type Stress Stimuli Cellular and Molecular Neurobiology. 1998; 18:4.
Niikura T, Hashimoto Y, Tajima H, Nishimoto I. Death and survival of neuronal cells exposed to Alzheimer’s insults. J Neurosci. Res. 2002; 70: 380–391.
Nishimoto I., Matsuoka M. and Niikura T. Unravelling the role of Humanin. TRENDS in Molecular Medicine. 2004; 10: 102～105.
Pal S, Hartnett KA, Nerbonne JM, Levitan ES, and Aizenman E. Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels. J Neurosci 2003; 23: 4798–4802.
Pek-Scott Marta and Lutz PL. ATP-sensitive K+ channel activation provides transient protection to the anoxic turtle brain. Am. J. Physiol. 275 (Regulatory Integrative Comp. Physiol. 1998; 44: R2023–R2027.
Sebastian H, Gerich FJ, M?ller M. Sulfhydryl oxidation reduces hippocampal susceptibility to hypoxia-induced spreading depression by activating BK channels. J Neurophysiol 2005; 94: 1091–1103.
Sun HS, Feng ZP, Miki T, Seino S, French RJ. Enhanced neuronal damage after ischemic insults in mice lacking Kir6.2-containing ATP-sensitive K+ channels. J Neurophysiol 2006; 95: 2590–2601.
Takaba H, Nagao T, Yao H, Kitazono T, Ibayashi S, Fujishima M. An ATP-sensitive potassium channel activator reduces infarct volume in focal cerebral ischemia in rats. Am J Physiol. 1997; 273:R583–R586.
Tajima H, Niikura T, Hashimoto Y, Ito Y, Kita Y, Terashita K, Yamazaki K, Koto A, Aiso S, Nishimoto I. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer’s disease-related insults. Neuroscience Letters 2002, 324: 227–231.
Tanaka T, Yoshida M, Yokoo H, Mizoguchi K, and Tanaka M. The role of ATP-sensitive potassium channels in striatal dopamine release: an in vivo microdialysis study. Pharmacol Biochem. Behav. 1995; 52: 831–835.
Tanaka E, Yamamoto S, Kudo Y, Mihara S, Higashi H.. Mechanisms underlying the rapid depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J. Neurophysiol. 1997; 78: 891–902.
Wei L., SP Yu; F Gottron; B. Joy Snider; Gregory J. Zipfel and Dennis W. Choi. Potassium Channel Blockers Attenuate Hypoxia- and Ischemia-Induced Neuronal Death In Vitro and In Vivo. Stroke 2003; 34: 1281-1286.
Yamamoto S, Tanaka E and Higashi H. Mediation by intracellular calcium-dependent signals of hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro. J Neurophysiol. 1997; 77: 386-392.
Yu SP, Yeh CH, Sensi SL, Gwag BJ, Canzoniere LMT, Farhangrazi ZS, Ying HS, Tian M, Dugan LL, and Choi DW. Mediation of neuronal apoptosis by enhancement of outward potassium current. Science. 1997; 278: 114–117.
Yu SP, Farhangraxi ZS, Ying HS, Yeh CH, and Choi DW. Enhancement of outward potassium current may participate inβ-amyloid peptide-induced cortical neuronal death. Neurobiol Dis. 1998; 5: 81–88.
Yu SP, Yeh CH, Strasser U, Tian M, and Choi DW. NMDA receptor-mediated K+ efflux and neuronal apoptosis. Science. 1999b; 284: 336–339.
Zawar C, Neumcke B. Differential activation of ATP-sensitive potassium channels during energy depletion in CA1 pyramidal cells and interneurones of rat hippocampus. J Physiol 2000;439:256–262.
Zhai DY, F Luciano, XW Zhu, B Guo, Arnold C. Satterthwait, and John C. Reed. Humanin Binds and Nullifies Bid Activity by Blocking Its Activation of Bax and Bak. J Biological Chemistry 2005; 280: 15815–15824.
Zou B, Li Y, Deng P,Xu Zc. Alterations of potassium currents in ischemia-vulnerable and ischemia-resistant neurons in the hippocampus after ischemia.Brain Res. 2005, 1033:78-89.
Alzheimer C, Schwindt PC, and Grill WE. Modal Gating of Na~+ Channels as a Mechanism of Persistent Na~+ Current in Pyramidal Neurons from Rat and Cat Sensorimotor Cortex. J Neurosci, 1993, 73: 660-673.
Boening JA, Kass IS, Cottrell JE, et al.The effect of blocking sodium influx on anoxic damage in the rat hippocampal slice. Neuroscience. 1989, 3:263-8.
Boehmer G, Greffrath W, Martin E, et al. Subthreshold oscillation of the membrane potential in magnocellular neurones of the rat supraoptic nucleus. J Physiol. 2000, 526:115-28.
Brown AM, Schwindt PC, Crill WE. Different voltage dependence of transient and persistent Na~+ currents is compatible with modal-gating hypothesis for sodium channels. J Neurophysiol. 1994, 71:2562-5.
Chandler WK and Meves H. Evidence For Two Types Of Sodium Conductance In Axons Perfused With Sodium Fluoride Solution. J. Physiol. 1970, 211: 653-678.
Cummins TR, Donnelly DF, Haddad GG. Effect of metabolic inhibition on the excitability of isolated hippocampal CA1 neurons: developmental aspects. J Neurophysiol. 1991, 66:1471-82. Cummins TR, Jiang C and Haddad GG. Human Neocortical Excitability Is Decreased during Anoxia Via Sodium Channel Modulation. J. Clin. Invest. 1993, 91:608-615.
Davis RE and Stuart AE. A Persistent, TTX-Sensitive Sodium Current in an Invertebrate Neuron with Neurosecretory Ultrastructure. J Neurosci. 1988, 8: 3978～3991.
Do MT, Bean BP.Subthreshold sodium currents and pacemaking of subthalamic neurons: modulation by slow inactivation. Neuron. 2003, 39:109-20.
French CR, Sah P, K. J. Buckett KJ, et al. A Voltage-dependent Persistent Sodium Current in Mammalian Hippocampal Neurons. J. Gen. Physiol. 1990, 95:1139-1157.
Fried PA, Chambers G, Cottrell JE, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices: mechanisms of protection by lidocaine E. J of Physiol. 1995, 489: 557-565.
Friedman JE, Haddad GG.Removal of extracellular sodium prevents anoxia-induced injury in freshly dissociated rat CA1 hippocampal neurons. Brain Res. 1994, 641:57-64.
Friedman JE, Haddad GG.Anoxia induces an increase in intracellular sodium in rat central neurons in vitro. Brain Res. 1994, 663:329-34.
Fung ML, Croning MD, Haddad GG. Sodium homeostasis in rat hippocampal slices during oxygen and glucose deprivation: role of voltage-sensitive sodium channels. Neurosci Lett. 1999, 275:41-4.
Fung ML, Haddad GG.Anoxia-induced depolarization in CA1 hippocampal neurons: role of Na~+-dependent mechanisms. Brain Res. 1997,762:97-102.
Golomb D, Yue C, and Yaari Y. Contribution of persistent Na~+ current and M-type K+ current to somatic bursting in CA1 pyramidal cells: combined experimental and modeling Study. J Neurophysiol. 2006, 96: 1912–1926.
Haigney MC, Lakatta EG, Stern MD, et al. Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading. Circulation. 1994, 90:391-9.
Hammarstr(o|¨)m AKM and Gage PW. Inhibition of oxidative metabolism increases persistent sodium current in rat CA1 hippocampal neurons. J of Physiol. 1998, 510:735~741.
Hammarstr(o|¨)m AKM and Gage PW. Oxygen-sensing persistent sodium channels in rat hippocampus. J of Physiol. 2000, 529: 107–118.
Hammarstr(o|¨)m AKM and Gage PW. Hypoxia and persistent sodium current. Eur Biophys J. 2002, 31: 323–330.
Hashimoto Y., Niikura T., Tajuma H,, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Aβ. Proc. Natl. Acad. Sci. 2001, 98: 6336~6341.
Herzog RI, Cummins TR and Waxman SG. Persistent TTX-Resistant Na1 Current Affects Resting Potential and Response to Depolarization in Simulated Spinal Sensory Neurons. J Neurophysiol 2001, 86:1351-1364.
Horn EM. and Waldrop TG. Hypoxic augmentation of fast-inactivating and persistent sodium currents in rat caudal hypothalamic neurons. J Neurophysiol.2000, 84: 2572–2581.
Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived pc12 cells. Neuroreport 2002; 13: 903-7.
Kariya S., Takahashi N., Hirano M., et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deproved human hymphocytes. Mol Cell Biochem., 2003, 254):83～89.
Kariya S., Hirano M., Furiya Y., et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides, 2005, 39: 97～101.
Kariya S., Hirono M., Furiya Y., et al. Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent. Acta Neuropathol (Berl). 2005, 109: 367～372.
Keynes RD. Bimodal gating of the Na~+ channel. Trends Neurosci. 1994 ;17:58-61.
Li Y and Bennett DJ. Persistent Sodium and Calcium Currents Cause Plateau Potentials in Motoneurons of Chronic Spinal Rats. J Neurophysiol 2003,90: 857–869.
Lipton P. Ischemic cell death in brain neurons. Physiol Rev 1999, 79:1431–1568.
Liu Z, Song W, Dong K. Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel. PNAS 2004, 101: 11862–11867.
Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280:15815～15924.
Magistretti J and Alonso A. Fine Gating Properties of Channels Responsible for Persistent Sodium Current Generation in Entorhinal Cortex Neurons. J Gen Physiol. 2002, 120:855-73.
Magistretti J, Castelli L, Forti L, et al. Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study. J Physiol.2006, 573: 83～106.
Martinez-Sánchez M, Striggow F, Schr(o|¨)der UH, et al. Na~+ and Ca~(2+) homeostasis pathways, cell death and protection after oxygen-glucose deprivation in organotypic hippocampal slice cultures.Neuroscience 2004, 128:729–740.
Niikura T., Hashimoto Y, Tajima H, et al. A tripartite motif protein TRIMI1 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer’s disease-relevant insults. Eur J Neurosci., 2003, 17 :1150～1158.
Niikura T, Chiba T, Aiso S, et al. Humanin : after the discovery. Mol Neurobiol., 2004, 30:327～340.
O'Reilly JP, Cummins TR, Haddad GG. Oxygen deprivation inhibits Na~+ current in rat hippocampal neurones via protein kinase C. J Physiol. 1997, 503:479-88.
Park Choon-Ok, Xiao XH, and Allen DG. Changes in intracellular Na1 and pH in rat heart during ischemia: role of Na1/H1 exchanger. Am J Physiol Heart Circ Physiol 1999, 276:1581-1590.
Raley-Susman KM, Kass IS, Cottrell JE, et al.Sodium influx blockade and hypoxic damage to CA1 pyramidal neurons in rat hippocampal slices. J Neurophysiol. 2001, 86:2715-26
Sponne I, Fifre A, Koziel V, et al. Humanin rescues cortical neurons from prion-peptide-induced apoptosis. Mol Cell Neurosci. 2004, 25:95-102.
Stys PK, Waxman SG, and Ransom BR. Ionic Mechanisms of Anoxic Injury in Mammalian CNS White Matter: Role of Na~+ Channels and Na~+-Ca~(2+) Exchanger. J Neurosci. 1992, 12: 430-439.
Su H, Alroy G, Kirson ED, et al. Extracellular Calcium Modulates Persistent Sodium Current-Dependent Burst-Firing in Hippocampal Pyramidal Neurons. J Neurosci. 2001, 27: 4173～4182.
Taddese A and Bean BP. Subthreshold Sodium Current from Rapidly Inactivating Sodium Channels Drives Spontaneous Firing of Tuberomammillary Neurons. Neuron. 2002, 33: 587–600.
Tajima H., Niikura T., Hashimoto Y., et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer’s disease-related insults. Neurosci. Lett. 2002, 324: 227～231.
Taylor CP. Na~+ currents that fail to inactivate. Trends Neurosci. 1993, 16:455-60.
Toner CC, Stamford JA. Sodium channel blockade unmasks two temporally distinct mechanisms of striatal dopamine release during hypoxia/hypoglycaemia in vitro. Neuroscience. 1997, 81:999-1007.
Urenjak J, Obrenovitch TP.Neuroprotection--rationale for pharmacological modulation of Na~+-channels. Amino Acids. 1998,14:151-8.
Van Drongelen W, Koch H, Elsen F, et al. Role of Persistent Sodium Current in Bursting Activity of Mouse Neocortical Networks In Vitro. J Neurophysiol. 2006, 96:2564-2577.
Weber ML, Taylor CP. Damage from oxygen and glucose deprivation in hippocampal slices is prevented by tetrodotoxin, lidocaine and phenytoin without blockade of action potentials. Brain Res. 1994 , 664:167-77.
Wu N, Enomoto A, Tanaka S, et al. Persistent sodium currents in mesencephalic V neurons participate in burst generation and control of membrane excitability. J Neurophysiol. 2005, 93: 2710–2722.
Xiao XH, Allen DG. Role of Na~+/H+ exchanger during inchemia and preconditioning in the isolated rat heart. Circ Res. 1999; 85: 723-30.
Xu X, Chua CC, Gao J, et al. Humanin is a novel neuroprotective agent against stroke. Stroke 2006, 37:2613-9.
Yamasaki Y, Kogure K, Hara H, et al.The possible involvement of tetrodotoxin-sensitive ion channels in ischemic neuronal damage in the rat hippocampus. Neurosci Lett. 1991, 121:251-4. Links
Zeng J, Powers RK, Newkirk G, et al. Contribution of Persistent Sodium Currents to Spike-Frequency Adaptation in Rat Hypoglossal Motoneurons. J Neurophysiol 2005, 93: 1035–1041.
Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem., 2005, 280: 15815～15824.
Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neuronsby altering fibrogenesis of Aβ1-40. Peptides. 2003, 24:679～685.
Andrey Y. Abramov, Michael R. Duchen. Mechanisms underlying the loss of mitochondrial membrane potential in glutamate excitotoxicity. Biochimica et Biophysica Acta. 2008; 1777: 953–964.
Benveniste H, Drejer J, Schousboe A, Diemer NH. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem. 1984; 43(5):1369-74.
Benveniste H. Glutamate, microdialysis, and cerebral ischemia: lost in translation? Anesthesiology. 2009;110(2):422-5.
Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity. FASEB J. 2002; 16(10): 1331～1333.
Choi DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1988; 1: 623～634.
Du C, Koretsky AP, Izrailtyan I, Benveniste H. Simultaneous detection of blood volume, oxygenation, and intracellular calcium changes during cerebral ischemia and reperfusion in vivo using diffuse reflectance and fluorescence. J Cereb Blood Flow Metab. 2005;25(8):1078-92.
Bolshakov AP, Mikhailova MM, Szabadkai G, Pinelis VG, Brustovetsky N, Rizzuto R, Khodorov BI. Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca~(2+) deregulation. Cell Calcium. 2008; 43(6):602-14.
Ganitkevich V.Y. The role of mitochondria in cytoplasmic Ca~(2+) cycling. Experimental Physiology 2003,88(1): 91–97.
Green D, Kroemer G. The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol. 1998;8(7):267-71.
Hajnóczky G., Csordás G., Madesh M. and Pacher P. The machinery of local Ca$ signalling between sarco-endoplasmic reticulum and mitochondria. J Physiology. 2000, 529(1): 69-81.
Hwang IK, Yoo KY, Kim DW, Choi SY, Kang TC, Kim YS, Won MH. Ionized calcium-binding adapter molecule 1 immunoreactive cells change in the gerbil hippocampal CA1 region after ischemia/reperfusion. Neurochem Res. 2006, 1(7):957-65.
Ikonen M, Liu B, Hashimoto Y, et al. Interaction between the Alzheimer’s survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. Proc Natl Acad Sci USA. (PNAS). 2003; 100(22): 13042–13047.
Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deproved human hymphocytes. Mol Cell Biochem. 2003; 254(1-2):83～89.
Kariya S, Hirano M, Furiya Y, et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides. 2005; 39(): 97～101.
Khodorov B, Pinelis V, Storozhevykh T, Yuravichus A, Khaspekhov L. Blockade of mitochondrial Ca~(2+) uptake by mitochondrial inhibitors amplifies the glutamate-induced calcium response in cultured cerebellar granule cells. FEBS Lett. 1999; 458(2):162-6.
Kirichok Y, Krapivinsky G, Clapham DE. The mitochondrial calcium uniporter is a highly selective ion channel. Nature. 2004; 427(6972):360-4.
Li LM, Qiao JT, Zhang C. Effects of humanin on elevation of intracellular calcium concentration induced by beta-amyloid peptide(31-35) in cultured cortical neurons. Sheng Li Xue Bao. 2009; 61(2):127-31.
Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999 ; 79(4):1431-568.
Lipton SA: pathologically-activated therapeutics for neuroprotection: mechanism of NMDA receptor block by memantine and s-nitrosylation. Current Drug Targets, 2007; 8(5):621～632.
Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem. 2005; 280(16):15815～15924.
Merritt, J. E., Jacob, R. & Hallam, T. J. J. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J Biol Chem. 1989; 264(3):1522-7.
Omann GM, Harter JM. Pertussis toxin effects on chemoattractant-induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry. Cytometry. 1991;12(3):252-9.
Palmer GC. Neuroprotection by NMDA Receptor Antagonists in a Variety of Neuropathologies. Current Drug Targets, 2001; 2(3):241～271.
Parekh. Anant B. Mitochondrial Regulation of Intracellular Ca~(2+) Signaling: More Than Just Simple Ca~(2+) Buffers. News Physiol Sci. 2003; 18: 252-256.
Potier M. and Trebak M. New developments in the signaling mechanisms of the store-operated calcium entry pathway. Pflugers Arch. 2008; 457(2): 405–415.
Shishkin V, Potapenko E, Kostyuk E, Girnyk O, Voitenko N, Kostyuk P. Role of mitochondria in intracellular calcium signaling in primary and secondary sensory neurones of rats. Cell Calcium. 2002;32(3):121-30.
Stout A.K., Reynolds I.J. High-af?nity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations, Neuroscience 1999; 89: 91–100.
Thayer SA, Miller RJ. Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro. J Physiol. 1990 Jun;425:85-115.
Tsujimoto Y. Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria? Genes to Cells. 1998; 3: 697–707
Vergun O., Keelan J., Khodorov B.I., Duchen M.R. Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones, J. Physiol. (London). 1999; 519: 451–466.
Verkhratsky A., Fernyhough P. Mitochondrial malfunction and Ca~(2+) dyshomeostasis drive neuronal pathology in diabetes. Cell Calcium. 2008; 44: 112—122.
Xu, X., Chua, C.C., Gao, J., Hamdy, R.C., Chua, B.H., 2006. Humanin is a novel neuroprotective agent against stroke. Stroke, 2006; 37, 2613–2619.
Xu, X., Chua, C.C., Gao, J., Kao-Wei Chua, Wang, H., Hamdy, R.C., Chua, B.H., Neuroprotective effect of humanin on cerebral ischemia/reperfusion injury is mediated by a PI3K/Akt pathway. Brain Research, 2008, 1227:12-18.
Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem. 2005; 280(16): 15815～15824.
Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ1-40. Peptides. 2003; 24(5):679～685.
1. Barhwal K, Hota SK, Baitharu I, Prasad D, Singh SB, Ilavazhagan G. Isradipine antagonizes hypobaric hypoxia induced CA1 damage and memory impairment: Complementary roles of L-type calcium channel and NMDA receptors. Neurobiol Dis. 2009; 34(2):230-44.
2. Barks JD. Current controversies in hypothermic neuroprotection. Semin Fetal Neonatal Med. 2008;13(1):30-4.
3. Ben-Ari Z, Pappo O, Yitzhaki S, Cheporko Y, Shainberg A, Zinman T, Ravid A, Zemel R, Bachmatov L, Kurtzwald E, Mor E, Hochhauser E. Uridine-5'-triphosphate protects against hepatic- ischemic/reperfusion injury in mice. Transplantation. 2009; 87(8):1155-62.
4. Biagas K. Hypoxic-ischemic brain injury: advancements in the understanding of mechanisms and potential avenues for therapy. Curr Opin Pediatr. 1999; 11: 223-228.
5. Bickler PE. Clinical perspectives: neuroprotection lessons from hypoxia-tolerant organisms. J. Exp Biol. 2004; 207: 3243-3249.
6. Bickler PE, Fahlman CS. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons. Neuroscience. 2004;127(3):673-83.
7. Bona E, Andersson AL, Blomgren K, et al. Chemokine and inflammatory cell response to hypoxia-ischemia in immature rats. Pediatr Res. 1999;45:500-509.
8. Bruce AJ, Boling W, Kindy MS, et al. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat Med. 1996; 2:788-794.
9. Chen X, Kintner DB, Luo J, Baba A, Matsuda T, Sun D. Endoplasmic reticulum Ca~(2+) dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter. J Neurochem. 2008; 106(4):1563-76.
10. Djali S and and Dawson LA. Characterization of endogenous Amino acid efflux from hippocampal slices during chemically-induced ischemia. Neurochemical Research. 200126(2):135–143.
11. Fujimoto S, Katsuki H, Kume T, Kaneko S, Akaike A. Mechanisms of oxygen glucose deprivation-induced glutamate release from cerebrocortical slice cultures. Neurosci Res. 2004; 50(2):179-87.
12. Hagberg H, Gilland E, Bona E, et al. Enhanced expression ofinterleukin (IL)-I and IL-6 messenger RNA and bioactive protein after hypoxia-ischemia in neonatal rats. Pediatr Res. 1996; 40:603-609.
13. Haile M, Limson F, Gingrich K, Li YS, Quartermain D, Blanck T, Bekker A. Nimodipine prevents transient cognitive dysfunction after moderate hypoxia in adult mice. J Neurosurg Anesthesiol. 2009; 21(2):140-4.
14. Henrich M, Buckler KJ. Effects of anoxia and aglycemia on cytosolic calcium regulation in rat sensory neurons. J Neurophysiol. 2008; 100(1):456-73.
15. Hossain MA. Molecular mediators of hypoxic–ischemic injury and implications for epilepsy in the developing brain. Epilepsy & Behavior 2005; 7: 204–213.
16. Ichiba H, Tamai H, Negishi H, et al, for the Kansai Magnesium Study Group. Randomized controlled trial of magnesium sulfate infusion for severe birth asphyxia. Pediatr Int. 2002;44:505-509.
17. Grow J, Barks JD. Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: Current concepts. Clin Perinatol. 2002; 29:585-602.
18. Koinig H, Vornik V, Rueda C, Zornow MH. Lubeluzole inhibits accumulation of extracellular glutamate in the hippocampus during transient global cerebral ischemia. Brain Res. 2001; 898(2):297-302.
19. Kulik A, Trapp S, Ballanyi K. Ischemia but not anoxia evokes vesicular and Ca2+-independent glutamate release in the dorsal vagal complex in vitro. J Neurophysiol. 2000; 83(5):2905-15.
20. Lipton P. Ischemic cell death in brain neurons. Physiol Rev.1999; 79:1431-1568.
21. Lipton SA. Pathologically-activated therapeutics for neuroprotection: mechanism of NMDA receptor block by memantine and s-nitrosylation. Curr. Drug Targets, 2007, 8: 621-632.
22. Manning SM, Talos DM, Zhou C, Selip DB, Park HK, Park CJ, Volpe JJ, Jensen FE. NMDA receptor blockade with memantine attenuates white matter injury in a rat model of periventricular leukomalacia. J Neurosci. 2008;28(26):6670-8.
23. Mark LP, Prost RW, Ulmer JL, et al. Pictorial review of glutamate excitotoxicity: fundamental concepts for neuroimaging. Am J Neuroradiol 2001; 22:1813–1824.
24. McManus T, Sadgrove M, Pringle AK, Chad JE, Sundstrom LE. Intraischaemic hypothermia reduces free radical production and protects against ischaemic insults in cultured hippocampal slices. J Neurochem. 2004 91(2):327-36.
25. Mueller RN, Deyo DJ, Brantley DR, Disterhoft JF, Zornow MH. Lubeluzole and conditioned learning after cerebral ischemia. Exp Brain Res. 2003;152(3):329-34.
26. Muir KW, Lees KR. Excitatory amino acid antagonists for acute stroke. Cochrane Database Syst Rev. 2003;(3):CD001244. Review
27. Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies. Curr.Drug Targets, 2001, 2:241-271.
28. Palmer C. Hypoxic-ischemic encephalopathy. Therapeutic approaches against microvascular injury, and role of neutrophils, PAF, and free radicals. Clin Perinatol. 1995;22: 481-517.
29. Papazisis G, Kallaras K, Kaiki-Astara A, Pourzitaki C, Tzachanis D, Dagklis T, Kouvelas D. Neuroprotection by lamotrigine in a rat model of neonatal ypoxic-ischaemic encephalopathy.Int J Neuropsychopharmacol. 2008;11(3):321-9.
30. Perlman JM, MB, ChB. Intervention strategies for neonatal hypoxic-ischemic cerebral injury. Clin Ther. 2006; 28: 1353-1365.
31. Sahuquillo J and Vilalta A. Cooling the injured brain: how does moderate hypothermia influence the pathophysiology of traumatic brain injury. Curr Pharm Des., 2007; 13: 2310-2322.
32. Vannucci SJ, Hagberg H. Hypoxia–ischemia in the immature brain. J. Exp Biol. 2004; 207: 3149-3154.
33. Vidwans AS, Hewett SJ. Enhanced release of synaptic glutamate underlies the potentiation of oxygen-glucose deprivation-induced neuronal injury after induction of NOS-2. Exp Neurol. 2004; 190(1):91-101.
34. Volbracht C, van Beek J, Zhu C, Blomgren K, Leist M. Neuroprotective properties of memantine in different in vitro and in vivo models of excitotoxicity. Eur J Neurosci. 2006;23(10):2611-22.
35. Wardas J. Neuroprotective role of adenosine in the CNS. J. Pharmacol. 2002; 54: 313–326.
36. Waters KA, Machaalani R. NMDA receptors in the developing brain and effects of noxious insults. Neurosignals. 2004;13:162–174.
37. Yamaguchi S, Endo K, Kitajima T, Ogata H, Hori Y. Involvement of the glutamate transporter and the sodium-calcium exchanger in the hypoxia-induced increase in intracellular Ca2+ in rat hippocampal slices. Brain Res. 1998; 813(2):351-8.
Ashcroft FM. Adenosine 5’-triphosphate-sensitive potassium channels. Annu Rev Neurosci 1988; 11: 97–118.
Ballanyi K. Protective role of neuronal KATP channels in brain hypoxia. J Experimental Biology. 2004; 207(pt 18):3201-3212.
Bortner CD, Hughes FM Jr, Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem. 1997; 272(51):32436-32442.
Brevnova, Elena E., Oleksandr Platoshyn, Shen Zhang, and Jason X.-J. Yuan. Overexpression of human KCNA5 increases IK(V) and enhances apoptosis. Am J Physiol Cell Physiol 2004; 287: C715–C722.
Carmelle V. Remillard and Jason X.-J. Yuan. Activation of K+ channels: an essential pathway in programmed cell death. Am J Physiol Lung Cell Mol Physiol 2004; 286:49-67.
Chen XS, SP Chi, MN Liu, W Yang, TT Wei, Z Qi, and FY Yang. Inhibitory effect of ganglioside GD1b on K+ current in hippocampal neurons and its involvement in apoptosis suppression. J Lipid Res. 2005; 46(12):2580-2585.
Chen XX., LL Yuan, CP Zhao, et al. Deletion of Kv4.2 Gene Eliminates Dendritic A-Type K+ Current and Enhances Induction of Long-Term Potentiation in Hippocampal CA1 Pyramidal Neurons. J Neurosci., 2006; 26:12143–12151.
Chi XX and Xu ZC. Differential changes of potassium currents in CA1 pyramidal neurons after transient forebrain ischemia. J Neurophysiol. 2000; 84: 2834-2843.
Cummins TR, Donnelly DF, and Haddad GG. Effect of metabolic inhibition on the excitability of isolated hippocampal CA1 neurons: developmental aspects. J Neurophysiol. 1991; 66: 1471–1482.
Dallaporta B, Hirsch T, Susin SA, Zamzami N, Larochette N, Brenner C, Marzo I, Kroemer G. Potassium leakage during the apoptotic degradation phase. J Immunol. 1998;160(11):5605-5615.
Deng P, Pang ZP, Zhang Y, Xu ZC.Increase of delayed rectifier potassium currents in large aspiny neurons in the neostriatum following transient forebrain ischemia. Neuroscience. 2005;131(1):135-146.
Ekhterae D, Platoshyn O, Krick S, Yu Y, McDaniel SS, and Yuan JX. Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells. Am J Physiol Cell Physiol 2001; 281: C157–C165.
Ekhterae D, Platoshyn O, Zhang S, Remillard CV, and Yuan JX. Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents. Am J Physiol Cell Physiol 2003; 284: C1405–C1410.
Erdemli G, Xu YZ, and Krnjevic K. Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia. J Neurophysiol. 1998; 80: 2378–2390.
Ficker E and Heinemann U. Slow and fast transient potassium currents in cultured rat hippocampal cells. J Physiol (Lond) 1992; 445: 431–455.
Fujita H, Takizawa S, Nanri K, Matsushima K, Ogawa S, Shinohara Y. Potassium channel opener reduces extracellular glutamate concentration in rat focal cerebral ischemia. Brain Res Bull. 1997;43:365–368
Fujiwara N, Higashi H , Shimojik, and Yoshimura M. Effects of hypoxia on rat hippocampal neurons in vitro. J Physiol (Lond) 1987; 384: 131–151.
Fujiwura N, Tanaka E, Yamamoto S, Shigemori M, and Higashi H. Contribution of ATP-sensitive potassium channels to hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro. J. Neurophysiol. 1997; 77: 378–385.
Gebhardt C and Heinemann U. Anoxic decrease in potassium outward currents of hippocampal cultured neurons in absence and presence of dithionite. Brain Res. 1999; 837: 270–276.
Griesemer D, Zawar C, Neumcke B. Cell-type specific depression of neuronal excitability in rat hippocampus by activation of ATP-sensitive potassium channels. Eur Biophys J. 2002; 31(6):467-477.
Guo B, Zhai DY, Cabezas E, Welsh K, Nouraini S, Satterthwait AC, Reed JC. Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature. 2003; 423: 456–461. Hansen AJ, Hounsgaard J, and Jahnsen H. Anoxia increases potassium conductance in hippocampal nerve cells. Acta Physiol Scand. 1982; 115: 301–310.
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981; 391(2):85-100.
Hashimoto Y, Ito Y, Niikura T, Shao Z, Hata M., Oyama F, Nishimoto I. Mechanisms of neuroprotection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein. Biochem. Biophys. Res. Commun. 2001a , 283: 460–468.
Hashimoto Y., Niikura T., Tajima H., Yasukawa T., Sudo H., Ito Y., Kita Y., Kawasumi M., Kouyama K., Doyu M., Sobue G., Koide T., Tsuji S., Lang J., Kurokawai K., Nishimoto I. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Abeta. Proc. Natl. Acad. Sci. 2001b , USA 98: 6336–6341.
Hashimoto Y, Niikura T, Ito Y, Sudo H, Hata M, Arakawa E, Abe Y, Kita Y, Nishimoto I. Detailed Characterization of Neuroprotection by a Rescue Factor Humanin against Various Alzheimer’s Disease-Relevant Insults. J. Neurosci. 2001c; 21:9235–9245.
Hashimoto Y., Suzuki H., Aiso S., Niikura T., Nishimoto I., Matsuoka M. Involvement of tyrosine kinases and STAT3 in Humanin-mediated neuroprotection. Life Sciences 2005; 77: 3092–3104.
Huang L, Li W, Li B, Zou F. Activation of ATP-sensitive K channels protects hippocampal CA1 neurons from hypoxia by suppressing p53 expression. Neurosci Lett. 2006; 398:34-38.
Hughes FM Jr, Bortner CD, Purdy GD, Cidlowski JA. Intracellular K+ suppresses the activation of apoptosis in lymphocytes. J Biol Chem. 1997; 272(48):30567-30576.
Hyllienmark L and Brismar T. Effect of metabolic inhibition on K+ channels in pyramidal cells of the hippocampal CA1 region in rat brain slices. J Physiol (Lond) 1996; 491: 155–164.
Jiang C and Haddad GG. Effect of anoxia on intracellular and extracellular potassium activity in hypoglossal neurons in vitro. J Neurophysiol 1991, 66: 103–111.
Jiang C, Xia Y, Haddad GG. Role of ATP-sensitive K+ channels during anoxia: major differences between rat (newborn and adult) and turtle neurons. J Physiol. 1992; 448:599-612.
Kariya S, Takahashi N, Ooba N, Kawahara M, Nakayama H, Ueno S. Humanin inhibits cell death of serum-deprived PC12 cells. Neuroreport 2002; 13: 903–907.
Kariya S, Takahashi N, Hirano M, Ueno S. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes. Mol. Cell. Biochem. 2003, 254: 83–89.
Kariya S, Hirano M, Furiya Y, Ueno S. Effect of humanin on cellular ATP production: a possible therapeutic candidate for mitochondrial disorders. Ann. Neurol. 2004, 56 (Suppl. 8): s65.
Kariya S, Hirano M, Furiya Y, Ueno S. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides 2005; 39: 97–101.
Kariya S, Hirano M, Furiya Y, Sugie K, Ueno S. Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent. Acta Neuropathol 2005; 109: 367–372.
Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982; 239(1):57-69.
Klee R, Ficker E, Heinemann U. Comparison of voltage-dependent potassium current in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3. J. Neurphysiol. 1995; 75: 1982–1995.
Krick S, Platoshyn O, Sweeney M, Kim H, and Jason Yuan XJ. Activation of K+ channels induces apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol 2001; 280: C970–C979.
Krick S., Platoshyn O, McDaniel SS, Rubin LJ, and Yuan JX. Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis. Am J PhysiolLung Cell Mol Physiol. 2001; 281: L887–L894.
Lipton P. Ischemic Cell Death in Brain Neurons. Physiol Rev 1999; 79:1431-1568. Mironov SL, Langohr K, Haller M and Richter DW. Hypoxia activates ATP-dependent
potassium channels in inspiratory neurones of neonatal mice. Journal of Physiology 1998; 509: 755-766.
Murai Y, Ishibashi H, Koyama S, Akaike N. Ca~(2+)-activated K~+ currents in rat locus coeruleus neurons induced by experimental ischemia, anoxia, and hypoglycemia. J Neurophysiol. 1997; 78: 2674–2681.
Nakao K, Tokuyama S, Takahashi M, Kanet H and Ueda H. Antiamnesic Action of Cromakalim, a Potassium Channel Opener, in Mice Treated with Hypoxia- and Cerebral Ischemia-Type Stress Stimuli Cellular and Molecular Neurobiology. 1998; 18:4.
Niikura T, Hashimoto Y, Tajima H, Nishimoto I. Death and survival of neuronal cells exposed to Alzheimer’s insults. J Neurosci. Res. 2002; 70: 380–391.
Nishimoto I., Matsuoka M. and Niikura T. Unravelling the role of Humanin. TRENDS in Molecular Medicine. 2004; 10: 102～105.
Pal S, Hartnett KA, Nerbonne JM, Levitan ES, and Aizenman E. Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels. J Neurosci 2003; 23: 4798–4802.
Pek-Scott Marta and Lutz PL. ATP-sensitive K+ channel activation provides transient protection to the anoxic turtle brain. Am. J. Physiol. 275 (Regulatory Integrative Comp. Physiol. 1998; 44: R2023–R2027.
Sebastian H, Gerich FJ, M?ller M. Sulfhydryl oxidation reduces hippocampal susceptibility to hypoxia-induced spreading depression by activating BK channels. J Neurophysiol 2005; 94: 1091–1103.
Sun HS, Feng ZP, Miki T, Seino S, French RJ. Enhanced neuronal damage after ischemic insults in mice lacking Kir6.2-containing ATP-sensitive K+ channels. J Neurophysiol 2006; 95: 2590–2601.
Takaba H, Nagao T, Yao H, Kitazono T, Ibayashi S, Fujishima M. An ATP-sensitive potassium channel activator reduces infarct volume in focal cerebral ischemia in rats. Am J Physiol. 1997; 273:R583–R586.
Tajima H, Niikura T, Hashimoto Y, Ito Y, Kita Y, Terashita K, Yamazaki K, Koto A, Aiso S, Nishimoto I. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer’s disease-related insults. Neuroscience Letters 2002, 324: 227–231.
Tanaka T, Yoshida M, Yokoo H, Mizoguchi K, and Tanaka M. The role of ATP-sensitive potassium channels in striatal dopamine release: an in vivo microdialysis study. Pharmacol Biochem. Behav. 1995; 52: 831–835.
Tanaka E, Yamamoto S, Kudo Y, Mihara S, Higashi H.. Mechanisms underlying the rapid depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J. Neurophysiol. 1997; 78: 891–902.
Wei L., SP Yu; F Gottron; B. Joy Snider; Gregory J. Zipfel and Dennis W. Choi. Potassium Channel Blockers Attenuate Hypoxia- and Ischemia-Induced Neuronal Death In Vitro and In Vivo. Stroke 2003; 34: 1281-1286.
Yamamoto S, Tanaka E and Higashi H. Mediation by intracellular calcium-dependent signals of hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro. J Neurophysiol. 1997; 77: 386-392.
Yu SP, Yeh CH, Sensi SL, Gwag BJ, Canzoniere LMT, Farhangrazi ZS, Ying HS, Tian M, Dugan LL, and Choi DW. Mediation of neuronal apoptosis by enhancement of outward potassium current. Science. 1997; 278: 114–117.
Yu SP, Farhangraxi ZS, Ying HS, Yeh CH, and Choi DW. Enhancement of outward potassium current may participate inβ-amyloid peptide-induced cortical neuronal death. Neurobiol Dis. 1998; 5: 81–88.
Yu SP, Yeh CH, Strasser U, Tian M, and Choi DW. NMDA receptor-mediated K+ efflux and neuronal apoptosis. Science. 1999b; 284: 336–339.
Zawar C, Neumcke B. Differential activation of ATP-sensitive potassium channels during energy depletion in CA1 pyramidal cells and interneurones of rat hippocampus. J Physiol 2000;439:256–262.
Zhai DY, F Luciano, XW Zhu, B Guo, Arnold C. Satterthwait, and John C. Reed. Humanin Binds and Nullifies Bid Activity by Blocking Its Activation of Bax and Bak. J Biological Chemistry 2005; 280: 15815–15824.
Zou B, Li Y, Deng P,Xu Zc. Alterations of potassium currents in ischemia-vulnerable and ischemia-resistant neurons in the hippocampus after ischemia.Brain Res. 2005, 1033:78-89.
Alzheimer C, Schwindt PC, and Grill WE. Modal Gating of Na~+ Channels as a Mechanism of Persistent Na~+ Current in Pyramidal Neurons from Rat and Cat Sensorimotor Cortex. J Neurosci, 1993, 73: 660-673.
Boening JA, Kass IS, Cottrell JE, et al.The effect of blocking sodium influx on anoxic damage in the rat hippocampal slice. Neuroscience. 1989, 3:263-8.
Boehmer G, Greffrath W, Martin E, et al. Subthreshold oscillation of the membrane potential in magnocellular neurones of the rat supraoptic nucleus. J Physiol. 2000, 526:115-28.
Brown AM, Schwindt PC, Crill WE. Different voltage dependence of transient and persistent Na~+ currents is compatible with modal-gating hypothesis for sodium channels. J Neurophysiol. 1994, 71:2562-5.
Chandler WK and Meves H. Evidence For Two Types Of Sodium Conductance In Axons Perfused With Sodium Fluoride Solution. J. Physiol. 1970, 211: 653-678.
Cummins TR, Donnelly DF, Haddad GG. Effect of metabolic inhibition on the excitability of isolated hippocampal CA1 neurons: developmental aspects. J Neurophysiol. 1991, 66:1471-82. Cummins TR, Jiang C and Haddad GG. Human Neocortical Excitability Is Decreased during Anoxia Via Sodium Channel Modulation. J. Clin. Invest. 1993, 91:608-615.
Davis RE and Stuart AE. A Persistent, TTX-Sensitive Sodium Current in an Invertebrate Neuron with Neurosecretory Ultrastructure. J Neurosci. 1988, 8: 3978～3991.
Do MT, Bean BP.Subthreshold sodium currents and pacemaking of subthalamic neurons: modulation by slow inactivation. Neuron. 2003, 39:109-20.
French CR, Sah P, K. J. Buckett KJ, et al. A Voltage-dependent Persistent Sodium Current in Mammalian Hippocampal Neurons. J. Gen. Physiol. 1990, 95:1139-1157.
Fried PA, Chambers G, Cottrell JE, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices: mechanisms of protection by lidocaine E. J of Physiol. 1995, 489: 557-565.
Friedman JE, Haddad GG.Removal of extracellular sodium prevents anoxia-induced injury in freshly dissociated rat CA1 hippocampal neurons. Brain Res. 1994, 641:57-64.
Friedman JE, Haddad GG.Anoxia induces an increase in intracellular sodium in rat central neurons in vitro. Brain Res. 1994, 663:329-34.
Fung ML, Croning MD, Haddad GG. Sodium homeostasis in rat hippocampal slices during oxygen and glucose deprivation: role of voltage-sensitive sodium channels. Neurosci Lett. 1999, 275:41-4.
Fung ML, Haddad GG.Anoxia-induced depolarization in CA1 hippocampal neurons: role of Na~+-dependent mechanisms. Brain Res. 1997,762:97-102.
Golomb D, Yue C, and Yaari Y. Contribution of persistent Na~+ current and M-type K+ current to somatic bursting in CA1 pyramidal cells: combined experimental and modeling Study. J Neurophysiol. 2006, 96: 1912–1926.
Haigney MC, Lakatta EG, Stern MD, et al. Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading. Circulation. 1994, 90:391-9.
Hammarstr(o|¨)m AKM and Gage PW. Inhibition of oxidative metabolism increases persistent sodium current in rat CA1 hippocampal neurons. J of Physiol. 1998, 510:735~741.
Hammarstr(o|¨)m AKM and Gage PW. Oxygen-sensing persistent sodium channels in rat hippocampus. J of Physiol. 2000, 529: 107–118.
Hammarstr(o|¨)m AKM and Gage PW. Hypoxia and persistent sodium current. Eur Biophys J. 2002, 31: 323–330.
Hashimoto Y., Niikura T., Tajuma H,, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Aβ. Proc. Natl. Acad. Sci. 2001, 98: 6336~6341.
Herzog RI, Cummins TR and Waxman SG. Persistent TTX-Resistant Na1 Current Affects Resting Potential and Response to Depolarization in Simulated Spinal Sensory Neurons. J Neurophysiol 2001, 86:1351-1364.
Horn EM. and Waldrop TG. Hypoxic augmentation of fast-inactivating and persistent sodium currents in rat caudal hypothalamic neurons. J Neurophysiol.2000, 84: 2572–2581.
Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived pc12 cells. Neuroreport 2002; 13: 903-7.
Kariya S., Takahashi N., Hirano M., et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deproved human hymphocytes. Mol Cell Biochem., 2003, 254):83～89.
Kariya S., Hirano M., Furiya Y., et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides, 2005, 39: 97～101.
Kariya S., Hirono M., Furiya Y., et al. Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent. Acta Neuropathol (Berl). 2005, 109: 367～372.
Keynes RD. Bimodal gating of the Na~+ channel. Trends Neurosci. 1994 ;17:58-61.
Li Y and Bennett DJ. Persistent Sodium and Calcium Currents Cause Plateau Potentials in Motoneurons of Chronic Spinal Rats. J Neurophysiol 2003,90: 857–869.
Lipton P. Ischemic cell death in brain neurons. Physiol Rev 1999, 79:1431–1568.
Liu Z, Song W, Dong K. Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel. PNAS 2004, 101: 11862–11867.
Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280:15815～15924.
Magistretti J and Alonso A. Fine Gating Properties of Channels Responsible for Persistent Sodium Current Generation in Entorhinal Cortex Neurons. J Gen Physiol. 2002, 120:855-73.
Magistretti J, Castelli L, Forti L, et al. Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study. J Physiol.2006, 573: 83～106.
Martinez-Sánchez M, Striggow F, Schr(o|¨)der UH, et al. Na~+ and Ca~(2+) homeostasis pathways, cell death and protection after oxygen-glucose deprivation in organotypic hippocampal slice cultures.Neuroscience 2004, 128:729–740.
Niikura T., Hashimoto Y, Tajima H, et al. A tripartite motif protein TRIMI1 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer’s disease-relevant insults. Eur J Neurosci., 2003, 17 :1150～1158.
Niikura T, Chiba T, Aiso S, et al. Humanin : after the discovery. Mol Neurobiol., 2004, 30:327～340.
O'Reilly JP, Cummins TR, Haddad GG. Oxygen deprivation inhibits Na~+ current in rat hippocampal neurones via protein kinase C. J Physiol. 1997, 503:479-88.
Park Choon-Ok, Xiao XH, and Allen DG. Changes in intracellular Na1 and pH in rat heart during ischemia: role of Na1/H1 exchanger. Am J Physiol Heart Circ Physiol 1999, 276:1581-1590.
Raley-Susman KM, Kass IS, Cottrell JE, et al.Sodium influx blockade and hypoxic damage to CA1 pyramidal neurons in rat hippocampal slices. J Neurophysiol. 2001, 86:2715-26
Sponne I, Fifre A, Koziel V, et al. Humanin rescues cortical neurons from prion-peptide-induced apoptosis. Mol Cell Neurosci. 2004, 25:95-102.
Stys PK, Waxman SG, and Ransom BR. Ionic Mechanisms of Anoxic Injury in Mammalian CNS White Matter: Role of Na~+ Channels and Na~+-Ca~(2+) Exchanger. J Neurosci. 1992, 12: 430-439.
Su H, Alroy G, Kirson ED, et al. Extracellular Calcium Modulates Persistent Sodium Current-Dependent Burst-Firing in Hippocampal Pyramidal Neurons. J Neurosci. 2001, 27: 4173～4182.
Taddese A and Bean BP. Subthreshold Sodium Current from Rapidly Inactivating Sodium Channels Drives Spontaneous Firing of Tuberomammillary Neurons. Neuron. 2002, 33: 587–600.
Tajima H., Niikura T., Hashimoto Y., et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer’s disease-related insults. Neurosci. Lett. 2002, 324: 227～231.
Taylor CP. Na~+ currents that fail to inactivate. Trends Neurosci. 1993, 16:455-60.
Toner CC, Stamford JA. Sodium channel blockade unmasks two temporally distinct mechanisms of striatal dopamine release during hypoxia/hypoglycaemia in vitro. Neuroscience. 1997, 81:999-1007.
Urenjak J, Obrenovitch TP.Neuroprotection--rationale for pharmacological modulation of Na~+-channels. Amino Acids. 1998,14:151-8.
Van Drongelen W, Koch H, Elsen F, et al. Role of Persistent Sodium Current in Bursting Activity of Mouse Neocortical Networks In Vitro. J Neurophysiol. 2006, 96:2564-2577.
Weber ML, Taylor CP. Damage from oxygen and glucose deprivation in hippocampal slices is prevented by tetrodotoxin, lidocaine and phenytoin without blockade of action potentials. Brain Res. 1994 , 664:167-77.
Wu N, Enomoto A, Tanaka S, et al. Persistent sodium currents in mesencephalic V neurons participate in burst generation and control of membrane excitability. J Neurophysiol. 2005, 93: 2710–2722.
Xiao XH, Allen DG. Role of Na~+/H+ exchanger during inchemia and preconditioning in the isolated rat heart. Circ Res. 1999; 85: 723-30.
Xu X, Chua CC, Gao J, et al. Humanin is a novel neuroprotective agent against stroke. Stroke 2006, 37:2613-9.
Yamasaki Y, Kogure K, Hara H, et al.The possible involvement of tetrodotoxin-sensitive ion channels in ischemic neuronal damage in the rat hippocampus. Neurosci Lett. 1991, 121:251-4. Links
Zeng J, Powers RK, Newkirk G, et al. Contribution of Persistent Sodium Currents to Spike-Frequency Adaptation in Rat Hypoglossal Motoneurons. J Neurophysiol 2005, 93: 1035–1041.
Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem., 2005, 280: 15815～15824.
Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neuronsby altering fibrogenesis of Aβ1-40. Peptides. 2003, 24:679～685.
Andrey Y. Abramov, Michael R. Duchen. Mechanisms underlying the loss of mitochondrial membrane potential in glutamate excitotoxicity. Biochimica et Biophysica Acta. 2008; 1777: 953–964.
Benveniste H, Drejer J, Schousboe A, Diemer NH. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem. 1984; 43(5):1369-74.
Benveniste H. Glutamate, microdialysis, and cerebral ischemia: lost in translation? Anesthesiology. 2009;110(2):422-5.
Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity. FASEB J. 2002; 16(10): 1331～1333.
Choi DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1988; 1: 623～634.
Du C, Koretsky AP, Izrailtyan I, Benveniste H. Simultaneous detection of blood volume, oxygenation, and intracellular calcium changes during cerebral ischemia and reperfusion in vivo using diffuse reflectance and fluorescence. J Cereb Blood Flow Metab. 2005;25(8):1078-92.
Bolshakov AP, Mikhailova MM, Szabadkai G, Pinelis VG, Brustovetsky N, Rizzuto R, Khodorov BI. Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca~(2+) deregulation. Cell Calcium. 2008; 43(6):602-14.
Ganitkevich V.Y. The role of mitochondria in cytoplasmic Ca~(2+) cycling. Experimental Physiology 2003,88(1): 91–97.
Green D, Kroemer G. The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol. 1998;8(7):267-71.
Hajnóczky G., Csordás G., Madesh M. and Pacher P. The machinery of local Ca$ signalling between sarco-endoplasmic reticulum and mitochondria. J Physiology. 2000, 529(1): 69-81.
Hwang IK, Yoo KY, Kim DW, Choi SY, Kang TC, Kim YS, Won MH. Ionized calcium-binding adapter molecule 1 immunoreactive cells change in the gerbil hippocampal CA1 region after ischemia/reperfusion. Neurochem Res. 2006, 1(7):957-65.
Ikonen M, Liu B, Hashimoto Y, et al. Interaction between the Alzheimer’s survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. Proc Natl Acad Sci USA. (PNAS). 2003; 100(22): 13042–13047.
Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deproved human hymphocytes. Mol Cell Biochem. 2003; 254(1-2):83～89.
Kariya S, Hirano M, Furiya Y, et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides. 2005; 39(): 97～101.
Khodorov B, Pinelis V, Storozhevykh T, Yuravichus A, Khaspekhov L. Blockade of mitochondrial Ca~(2+) uptake by mitochondrial inhibitors amplifies the glutamate-induced calcium response in cultured cerebellar granule cells. FEBS Lett. 1999; 458(2):162-6.
Kirichok Y, Krapivinsky G, Clapham DE. The mitochondrial calcium uniporter is a highly selective ion channel. Nature. 2004; 427(6972):360-4.
Li LM, Qiao JT, Zhang C. Effects of humanin on elevation of intracellular calcium concentration induced by beta-amyloid peptide(31-35) in cultured cortical neurons. Sheng Li Xue Bao. 2009; 61(2):127-31.
Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999 ; 79(4):1431-568.
Lipton SA: pathologically-activated therapeutics for neuroprotection: mechanism of NMDA receptor block by memantine and s-nitrosylation. Current Drug Targets, 2007; 8(5):621～632.
Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem. 2005; 280(16):15815～15924.
Merritt, J. E., Jacob, R. & Hallam, T. J. J. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J Biol Chem. 1989; 264(3):1522-7.
Omann GM, Harter JM. Pertussis toxin effects on chemoattractant-induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry. Cytometry. 1991;12(3):252-9.
Palmer GC. Neuroprotection by NMDA Receptor Antagonists in a Variety of Neuropathologies. Current Drug Targets, 2001; 2(3):241～271.
Parekh. Anant B. Mitochondrial Regulation of Intracellular Ca~(2+) Signaling: More Than Just Simple Ca~(2+) Buffers. News Physiol Sci. 2003; 18: 252-256.
Potier M. and Trebak M. New developments in the signaling mechanisms of the store-operated calcium entry pathway. Pflugers Arch. 2008; 457(2): 405–415.
Shishkin V, Potapenko E, Kostyuk E, Girnyk O, Voitenko N, Kostyuk P. Role of mitochondria in intracellular calcium signaling in primary and secondary sensory neurones of rats. Cell Calcium. 2002;32(3):121-30.
Stout A.K., Reynolds I.J. High-af?nity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations, Neuroscience 1999; 89: 91–100.
Thayer SA, Miller RJ. Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro. J Physiol. 1990 Jun;425:85-115.
Tsujimoto Y. Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria? Genes to Cells. 1998; 3: 697–707
Vergun O., Keelan J., Khodorov B.I., Duchen M.R. Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones, J. Physiol. (London). 1999; 519: 451–466.
Verkhratsky A., Fernyhough P. Mitochondrial malfunction and Ca~(2+) dyshomeostasis drive neuronal pathology in diabetes. Cell Calcium. 2008; 44: 112—122.
Xu, X., Chua, C.C., Gao, J., Hamdy, R.C., Chua, B.H., 2006. Humanin is a novel neuroprotective agent against stroke. Stroke, 2006; 37, 2613–2619.
Xu, X., Chua, C.C., Gao, J., Kao-Wei Chua, Wang, H., Hamdy, R.C., Chua, B.H., Neuroprotective effect of humanin on cerebral ischemia/reperfusion injury is mediated by a PI3K/Akt pathway. Brain Research, 2008, 1227:12-18.
Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem. 2005; 280(16): 15815～15824.
Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ1-40. Peptides. 2003; 24(5):679～685.
1. Barhwal K, Hota SK, Baitharu I, Prasad D, Singh SB, Ilavazhagan G. Isradipine antagonizes hypobaric hypoxia induced CA1 damage and memory impairment: Complementary roles of L-type calcium channel and NMDA receptors. Neurobiol Dis. 2009; 34(2):230-44.
2. Barks JD. Current controversies in hypothermic neuroprotection. Semin Fetal Neonatal Med. 2008;13(1):30-4.
3. Ben-Ari Z, Pappo O, Yitzhaki S, Cheporko Y, Shainberg A, Zinman T, Ravid A, Zemel R, Bachmatov L, Kurtzwald E, Mor E, Hochhauser E. Uridine-5'-triphosphate protects against hepatic- ischemic/reperfusion injury in mice. Transplantation. 2009; 87(8):1155-62.
4. Biagas K. Hypoxic-ischemic brain injury: advancements in the understanding of mechanisms and potential avenues for therapy. Curr Opin Pediatr. 1999; 11: 223-228.
5. Bickler PE. Clinical perspectives: neuroprotection lessons from hypoxia-tolerant organisms. J. Exp Biol. 2004; 207: 3243-3249.
6. Bickler PE, Fahlman CS. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons. Neuroscience. 2004;127(3):673-83.
7. Bona E, Andersson AL, Blomgren K, et al. Chemokine and inflammatory cell response to hypoxia-ischemia in immature rats. Pediatr Res. 1999;45:500-509.
8. Bruce AJ, Boling W, Kindy MS, et al. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat Med. 1996; 2:788-794.
9. Chen X, Kintner DB, Luo J, Baba A, Matsuda T, Sun D. Endoplasmic reticulum Ca~(2+) dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter. J Neurochem. 2008; 106(4):1563-76.
10. Djali S and and Dawson LA. Characterization of endogenous Amino acid efflux from hippocampal slices during chemically-induced ischemia. Neurochemical Research. 200126(2):135–143.
11. Fujimoto S, Katsuki H, Kume T, Kaneko S, Akaike A. Mechanisms of oxygen glucose deprivation-induced glutamate release from cerebrocortical slice cultures. Neurosci Res. 2004; 50(2):179-87.
12. Hagberg H, Gilland E, Bona E, et al. Enhanced expression ofinterleukin (IL)-I and IL-6 messenger RNA and bioactive protein after hypoxia-ischemia in neonatal rats. Pediatr Res. 1996; 40:603-609.
13. Haile M, Limson F, Gingrich K, Li YS, Quartermain D, Blanck T, Bekker A. Nimodipine prevents transient cognitive dysfunction after moderate hypoxia in adult mice. J Neurosurg Anesthesiol. 2009; 21(2):140-4.
14. Henrich M, Buckler KJ. Effects of anoxia and aglycemia on cytosolic calcium regulation in rat sensory neurons. J Neurophysiol. 2008; 100(1):456-73.
15. Hossain MA. Molecular mediators of hypoxic–ischemic injury and implications for epilepsy in the developing brain. Epilepsy & Behavior 2005; 7: 204–213.
16. Ichiba H, Tamai H, Negishi H, et al, for the Kansai Magnesium Study Group. Randomized controlled trial of magnesium sulfate infusion for severe birth asphyxia. Pediatr Int. 2002;44:505-509.
17. Grow J, Barks JD. Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: Current concepts. Clin Perinatol. 2002; 29:585-602.
18. Koinig H, Vornik V, Rueda C, Zornow MH. Lubeluzole inhibits accumulation of extracellular glutamate in the hippocampus during transient global cerebral ischemia. Brain Res. 2001; 898(2):297-302.
19. Kulik A, Trapp S, Ballanyi K. Ischemia but not anoxia evokes vesicular and Ca2+-independent glutamate release in the dorsal vagal complex in vitro. J Neurophysiol. 2000; 83(5):2905-15.
20. Lipton P. Ischemic cell death in brain neurons. Physiol Rev.1999; 79:1431-1568.
21. Lipton SA. Pathologically-activated therapeutics for neuroprotection: mechanism of NMDA receptor block by memantine and s-nitrosylation. Curr. Drug Targets, 2007, 8: 621-632.
22. Manning SM, Talos DM, Zhou C, Selip DB, Park HK, Park CJ, Volpe JJ, Jensen FE. NMDA receptor blockade with memantine attenuates white matter injury in a rat model of periventricular leukomalacia. J Neurosci. 2008;28(26):6670-8.
23. Mark LP, Prost RW, Ulmer JL, et al. Pictorial review of glutamate excitotoxicity: fundamental concepts for neuroimaging. Am J Neuroradiol 2001; 22:1813–1824.
24. McManus T, Sadgrove M, Pringle AK, Chad JE, Sundstrom LE. Intraischaemic hypothermia reduces free radical production and protects against ischaemic insults in cultured hippocampal slices. J Neurochem. 2004 91(2):327-36.
25. Mueller RN, Deyo DJ, Brantley DR, Disterhoft JF, Zornow MH. Lubeluzole and conditioned learning after cerebral ischemia. Exp Brain Res. 2003;152(3):329-34.
26. Muir KW, Lees KR. Excitatory amino acid antagonists for acute stroke. Cochrane Database Syst Rev. 2003;(3):CD001244. Review
27. Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies. Curr.Drug Targets, 2001, 2:241-271.
28. Palmer C. Hypoxic-ischemic encephalopathy. Therapeutic approaches against microvascular injury, and role of neutrophils, PAF, and free radicals. Clin Perinatol. 1995;22: 481-517.
29. Papazisis G, Kallaras K, Kaiki-Astara A, Pourzitaki C, Tzachanis D, Dagklis T, Kouvelas D. Neuroprotection by lamotrigine in a rat model of neonatal ypoxic-ischaemic encephalopathy.Int J Neuropsychopharmacol. 2008;11(3):321-9.
30. Perlman JM, MB, ChB. Intervention strategies for neonatal hypoxic-ischemic cerebral injury. Clin Ther. 2006; 28: 1353-1365.
31. Sahuquillo J and Vilalta A. Cooling the injured brain: how does moderate hypothermia influence the pathophysiology of traumatic brain injury. Curr Pharm Des., 2007; 13: 2310-2322.
32. Vannucci SJ, Hagberg H. Hypoxia–ischemia in the immature brain. J. Exp Biol. 2004; 207: 3149-3154.
33. Vidwans AS, Hewett SJ. Enhanced release of synaptic glutamate underlies the potentiation of oxygen-glucose deprivation-induced neuronal injury after induction of NOS-2. Exp Neurol. 2004; 190(1):91-101.
34. Volbracht C, van Beek J, Zhu C, Blomgren K, Leist M. Neuroprotective properties of memantine in different in vitro and in vivo models of excitotoxicity. Eur J Neurosci. 2006;23(10):2611-22.
35. Wardas J. Neuroprotective role of adenosine in the CNS. J. Pharmacol. 2002; 54: 313–326.
36. Waters KA, Machaalani R. NMDA receptors in the developing brain and effects of noxious insults. Neurosignals. 2004;13:162–174.
37. Yamaguchi S, Endo K, Kitajima T, Ogata H, Hori Y. Involvement of the glutamate transporter and the sodium-calcium exchanger in the hypoxia-induced increase in intracellular Ca2+ in rat hippocampal slices. Brain Res. 1998; 813(2):351-8.