酸敏感离子通道与脑梗死

急性脑梗死约占全部脑卒中的70%,病死率和致残率高,且极易复发。但目前针对急性脑梗死在时间窗内溶栓、抗凝等治疗手段不能从根本上切实有效地修复受损脑组织,且伴有出血等风险。寻找脑梗死形成发展的原因并予以治疗迫在眉睫。酸中毒是引起缺血性脑损伤的重要机制。大量实验研究表明,酸中毒能加重神经元的缺血性损伤,且其梗死面积与酸中毒的程度直接相关。但缺血产生的酸中毒如何引起神经元损伤的确切机制尚不明确。最近研究发现酸中毒能激活一种在中枢及周围神经中广泛存在的膜通道,即酸敏感离子通道,它对Ca^2＋通透,能引起细胞内Ca^2＋超载,同时能激活胞内酶引起细胞内蛋白质、脂类及核酸的降解,加重缺血后脑损伤。本文就酸敏感离子通道1a与脑梗死做一综述。     

酸敏感离子通道研究进展

组织酸化是生理和病理下常见的现象.神经元可以通过酸敏感的离子通道（ASICs）来感受细胞周围的pH值的降低.ASICs属于NaC/DEG家族的一个成员.目前,已发现了6个ASICs亚基,它们在外周和中枢神经系统中广泛表达,其同聚体和异聚体通道有着各种不同的电生理学特性.ASICs在机体感觉尤其是痛觉中起着至关重要的作用.     

酸敏感离子通道的功能及其相关调控

酸敏感离子通道（ASICs）是一类由胞外酸化所激活的阳离子通道.目前,已发现了6个ASICs亚基,它们在外周和中枢神经系统中广泛表达.利用基因敲除等技术,已证明它们在触觉、痛觉、酸味觉以及学习记忆中具有重要作用.同时,它们也参与某些病理反应.ASICs可以被神经肽、温度、金属离子和缺血相关物质等调控,从而整合细胞周围的多种信号以行使其功能.     

酸敏感离子通道外源性配体研究进展

酸敏感离子通道(ASICs)属于上皮 Na+ 通道/退化蛋白超家族,对细胞外 H+ 浓度变化敏感,其受多种外源性配体调控,产生 不同生理和病理学效应。越来越多研究发现,ASICs 参与脑缺血、炎症、肿瘤等具有酸化改变的病理过程。简介 ASICs 的结构及其配体 作用位点以及各亚基的组织分布和电生理特性,主要对各类 ASICs 外源性配体的研究进展作一综述。     

大鼠脊髓背角神经元中酸敏感离子通道的特性和功能研究

酸敏感离子通道（ASICs）是一类能被细胞外酸所激活的配体门控离子通道。本文综合报道大鼠脊髓背角神经元中ASICs的亚基组成及其功能性调节：（1）脊髓背角主要表达ASIC1a、ASIC2a和ASIC2b,但不表达ASIC1b和ASIC3;（2）在脊髓背角神经元中酸诱导电流可能由ASIC1a同聚体通道所介导;（3）胞外痛觉信号如实验性缺血和神经肽FMRF可以通过不同的机制增强脊髓背角神经元酸诱导电流;（4）炎症痛可以上调脊髓背角ASICs在转录和蛋白水平的表达。上述各点提示,在生理或病理情况下脊髓背角ASICs对脊髓水平的感觉信息传递特别是痛觉的传导可能发挥着重要作用。     

机械敏感离子通道Piezo1在心血管系统中的作用

机械敏感离子通道(mechanosensitive ion channels, MSC)是一类受机械压力影响而产生兴奋电信号的离子通道,广泛分布于生物各组织器官中,参与生物体内的多种生理过程。最近在哺乳动物体内发现了一种新型的MSC蛋白Piezo1,它与其他MSC蛋白不具有同源性,在细胞感应机械应力的过程中发挥着重要作用。大量研究结果表明,Piezo1在动脉血压的控制、红细胞体积的改变、心脏相关因子的分泌等生理过程中扮演了重要角色,与心血管系统关系密切。在哺乳动物心血管系统中,心脏、动脉血管、毛细微血管和红细胞等都可感受来自细胞外环境机械应力刺激,而Piezo1将机械应力转化为生物电信号,进而影响后续的生理过程。本文介绍了Piezo1在心血管系统中的作用,并总结Piezo1蛋白的具体作用机制及其差异,以期为进一步的研究提供有益参考。     

Aβ1-42对大鼠小胶质细胞酸敏感离子通道的表达和电生理特性的影响

Aβ_1-42与小胶质细胞之间相互作用诱发的神经炎症反应已被视为阿尔茨海默病(AD)的重要病理指征,酸敏感离子通道(ASIC)参与了小胶质细胞的炎症应答和神经炎症免疫调节。为了探究Aβ_1-42对小胶质细胞ASIC的表达和电生理特性的影响,本研究利用Aβ_1-42孵育原代培养的SD大鼠大脑皮层小胶质细胞,免疫印迹技术和免疫荧光技术检测小胶质细胞ASIC1、ASIC2a、ASIC3的蛋白表达和分布情况,全细胞膜片钳记录Aβ_1-42对ASIC电流特性的影响,钙离子成像技术分析ASIC钙离子通透性的变化。结果显示：Aβ_1-42处理小胶质细胞后,能够诱导ASIC1的蛋白表达量增加,且其在细胞浆和细胞核中均有明显增加;胞外pH值快速降低诱发的ASIC电流,小胶质细胞胞内钙离子浓度明显增强,这种增强效应可被ASIC非特异性抑制剂阿米洛利和ASIC1特异性抑制剂PcTx1抑制。本研究结果表明,Aβ_1-42诱导小胶质细胞ASIC1蛋白功能性表达增加,使得ASIC1易于被胞外快速...     

细胞外调控分子对酸敏感离子通道的功能调控及其机制

酸敏感离子通道(acid-Sensing ion channels,ASlCs)是一类由细胞外质子(H )激活的配体门控阳离子通道.迄今为止,人们在哺乳动物体内已经发现了6种ASICs亚基蛋白,它们分布在多种组织器官中.越来越多的研究表明:ASICs参与了机体的生理、病理过程,如:学习、记忆、痛觉、脑中风和肿瘤.在过去的10年中,人们发现多种内源性或外源性分子可以调控ASICs通道活性.由于这些细胞外调控分子与多种生理和病理功能有关,因此研究细胞外调控分子对ASICs的调控及其分子机制,可以帮助我们更多地了解ASICs功能以及结构信息,也为人们设计ASICs靶点特异性药物提供了理论依据.文章将系统地介绍细胞外调控分子对ASICs的功能调控及其作用机制,特别是该研究领域的最新进展.     

酸感受性离子通道生物学特性及其调控

酸感受离子通道(ASICs)为H -门控的阳离子通道,是一类新的配体门控性离子通道,属于钠通道超家族的新成员.作为近来研究的热点,ASICs具有许多重要的生物学功能,并很有可能成为抗癫痫、镇痛、提高学习记忆能力和保护神经元缺血损伤作用药理学新靶点.近来,ASICs各个亚基已被克隆,它们在生物体内分布、表达、功能和相关调节因素的研究正受到广泛重视.     

细胞外基质刚度调控压电型机械敏感离子通道组件1对神经干细胞分化的影响

目的 本研究旨在探讨细胞外基质刚度变化对神经干细胞（neural stem cells,NSCs）分化的影响及其作用机制。方法 本研究基于成功构建脊髓损伤大鼠模型,并制备不同刚度（0.7 kPa、40 kPa）的聚丙烯酰胺凝胶基底,将大鼠原代NSCs于不同刚度基底上培养。压电型机械敏感离子通道组件1（piezo type mechanosensitive ion channel component 1,Piezo1）shRNA质粒转染NSCs细胞。免疫荧光染色检测神经元标志物双皮质醇（doublecortion,DCX）和星形胶质细胞标志物胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）阳性细胞百分比。免疫组织化学及蛋白质免疫印迹（Western blot）法检测损伤组织及NSCs细胞中Piezo1蛋白的表达水平。结果 与0.7 kPa基质刚度组相比,40 kPa基质刚度组中DCX阳性细胞数增加,而GFAP阳性细胞数减少,Piezo1蛋白表达量上升。脊髓损伤大鼠损伤组织Piezo1蛋白表达显著高于空白对照（sham）组。40 kPa基质刚度条件下沉默Piezo1后,DCX阳性细胞数减少,而GFAP阳性细胞数增加,差异具有统计学意义（P<0.05）。机制研究发现,沉默Piezo1导致IV型胶原及纤连蛋白表达下降。重组纤连蛋白逆转了Piezo1 shRNA对NSCs分化的影响,即DCX阳性细胞数增加,而GFAP阳性细胞数减少。结论 综上可见,硬基底刚度通过促进Piezo1蛋白表达,上调IV型胶原及纤连蛋白表达,从而调控NSCs细胞分化。本研究为基于生物材料治疗脊髓损伤提供了新的视角。     

Ca2+-Permeable Acid-sensing Ion Channels and Ischemic Brain Injury

Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain unclear. Recent studies have demonstrated that activation of Ca²⁺-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent, neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca²⁺, and an activation of these channels induces increases in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i). Activation of ASICs with resultant [Ca²⁺]_i loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca²⁺ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing [Ca²⁺]_o. In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca²⁺-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients.     

A Method for Activation of Endogenous Acid-sensing Ion Channel 1a (ASIC1a) in the Nervous System with High Spatial and Temporal Precision

Protons activate acid-sensing ion channel 1a (ASIC1a) in the central nervous system (CNS) although the impact of such activation on brain outputs remains elusive. Progress elucidating the functional roles of ASIC1a in the CNS has been hindered by technical difficulties of achieving acidification with spatial and temporal precision. We have implemented a method to control optically the opening of ASIC1a in brain slices and also in awake animals. The light-driven H⁺ pump ArchT was expressed in astrocytes of mouse cortex by injection of adenoviral vectors containing a strong and astrocyte-specific promoter. Illumination with amber light acidified the surrounding interstitium and led to activation of endogenous ASIC1a channels and firing of action potentials in neurons localized in close proximity to ArchT-expressing astrocytes. We conclude that this optogenetic method offers a minimally invasive approach that enables examining the biological consequences of ASIC1a currents in any structure of the CNS and in the modulation of animal behaviors.     

S-nitrosylation of PTEN Invovled in Ischemic Brain Injury in Rat Hippocampal CA1 Region

The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is not only a protein, but also a lipid phosphatase that can negatively regulate the serine/threonine kinase Akt. It has been reported that PTEN can be regulated by means of phosphorylation. However, whether PTEN can be regulated by another post-translational protein modification (S-nitrosylation) was not fully elucidated. In this study, we investigated the S-nitrosylation of PTEN during transient cerebral ischemia/reperfusion in rat hippocampus. Transient brain ischemia was induced by the four-vessel occlusion in Sprague–Dawley rats. Our data show that S-nitrosylation of PTEN was increased significantly after 12 h of reperfusion compared with sham control. Pretreatment with the inhibitor of nNOS (7-NI) and the inhibitor of iNOS could inhibit PTEN’s activity and decrease S-nitrosylation of PTEN. Taken together, these results indicate that nitric oxide could regulate PTEN’s activity via S-nitrosylation during transient global ischemia in rat hippocampus. The authors D.-S. Pei, Y.-F. Sun contribute equally to this work.     

Kynurenine Pathway Enzymes in Brain: Responses to Ischemic Brain Injury Versus Systemic Immune Activation

Accumulation of l -kynurenine and quinolinic acid (QUIN) in the brain occurs after either ischemic brain injury or after systemic administration of pokeweed mitogen. Although conversion of l -[¹³C₆]tryptophan to [¹³C₆]-QUIN has not been demonstrated in brain either from normal gerbils or from gerbils given pokeweed mitogen, direct conversion in brain tissue does occur 4 days after transient cerebral ischemia. Increased activities of enzymes distal to indoleamine-2,3-dioxygenase may determine whether l -kynurenine is converted to QUIN. One day after 10 min of cerebral ischemia, the activities of kynureninase and 3-hydroxy-3,4-dioxygenase were increased in the hippocampus, but local QUIN levels and the activities of the indoleamine-2,3-dioxygenase and kynurenine-3-hydroxylase were unchanged. By days 2 and 4 after ischemia, however, the activities of all of these enzymes in the hippocampus as well as QUIN levels were significantly increased. Kynurenine aminotransferase activity in the hippocampus was unchanged on days 1 and 2 after ischemia but was decreased on day 4, at a time when local kynurenic acid levels were unchanged. A putative precursor of QUIN, [¹³C₆]anthranilic acid, was not converted to [¹³C₆]-QUIN in the hippocampus of either normal or 4-day postischemic gerbils. Gerbil macrophages stimulated by endo-toxin in vitro converted l -[¹³C₆]tryptophan to [¹³C_e]QUIN. Kinetic analysis of kynurenine-3-hydroxylase activity in the cerebral cortex of postischemic gerbils showed that V_max increased, without changes in K_m. Systemic administration of pokeweed mitogen increased indoleamine-2,3-dioxygenase and kynureninase activities in the brain without significant changes in kynurenine-3-hydroxylase or 3-hydroxyanthranilate-3,4-dioxygenase activities. Increases in kynurenine-3-hydroxylase activity, in conjunction with induction of indoleamine-2,3-dioxygenase, kynureninase, and 3-hydroxyanthranilate-3,4-dioxygenase in macro-phage infiltrates at the site of brain injury, may explain the ability of postischemic hippocampus to convert l -[¹³C₆]tryptophan to [¹³C₆]QUIN.     

Nicotinamide Attenuates Focal Ischemic Brain Injury in Rats: With Special Reference to Changes in Nicotinamide and NAD+ Levels in Ischemic Core and Penumbra

We investigated the neuroprotective action of nicotinamide in focal ischemia. Male spontaneously hypertensive rats (5–7 months old) were subjected to photothrombotic occlusion of the right distal middle cerebral artery (MCA). Either nicotinamide (125 or 250 mg/kg) or vehicle was injected IV before MCA occlusion. Changes in the cerebral blood flow (CBF) were monitored using laser-Doppler flowmetry, and infarct volumes were determined with TTC staining 3 days after MCA occlusion. In another set of experiments, the brain nicotinamide and nicotinamide adenine dinucleotide (NAD⁺) levels were analyzed by HPLC using the frozen samples dissected from the regions corresponding to the ischemic core and penumbra. In the 250-mg/kg nicotinamide group, the ischemic CBF was significantly increased compared to that the untreated group, and the infarct volumes were substantially attenuated (–36%). On the other hand, the ischemic CBF in the 125 mg/kg nicotinamide group was not significantly different from the untreated CBF, however, the infarct volumes were substantially attenuated (–38%). Cerebral ischemia per se did not affect the concentrations of nicotinamide and NAD⁺ both in the penumbra and ischemic core. In the nicotinamide groups, the brain nicotinamide levels increased significantly in all areas examined, and brain NAD⁺ levels increased in the penumbra but not in the ischemic core. Increased brain levels of nicotinamide are considered to be primarily important for neuroprotection against ischemia, and the protective action may be partly mediated through the increased NAD⁺ in the penumbra.     

Neuroprotective Effects of Ischemic Preconditioning in Brain Mitochondria Following Cerebral Ischemia

Numerous studies support the hypothesis that reperfusion following cerebral ischemia contributes substantially to ischemic injury and that mitochondrial dysfunction plays a central role. Defining the mechanisms by which mitochondrial dysfunction occurs may be important for the development of new therapies against delayed neuronal cell death. Ischemic preconditioning (IP) increases an organ's resistance to ischemic injury. There are two windows for IPC, one that requires several hours to develop and another one with a rapid setting (rapid window). However, the rapid window only provides neuroprotection for few days. We have recently determined that this lack of chronic protection by the rapid window was due to lack of protection against mitochondrial dysfunction.     

Amiloride Docking to Acid-sensing Ion Channel-1

Amiloride is a small molecule diuretic, which has been used to dissect sodium transport pathways in many different systems. This drug is known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as sodium/hydrogen antiporters and sodium/calcium exchangers. The exact structural basis for these interactions has not been elucidated as crystal structures of these proteins have been challenging to obtain, though some involved residues and domains have been mapped. This work examines the interaction of amiloride with acid-sensing ion channel-1, a protein whose structure is available using computational and experimental techniques. Using molecular docking software, amiloride and related molecules were docked to model structures of homomeric human ASIC-1 to generate potential interaction sites and predict which analogs would be more or less potent than amiloride. The predictions made were experimentally tested using whole-cell patch clamp. Drugs previously classified as NCX or NHE inhibitors are shown to also inhibit hASIC-1. Potential docking sites were re-examined against experimental data to remove spurious interaction sites. The voltage sensitivity of inhibitors was also examined. Using the aggregated data from these computational and experimental experiments, putative interaction sites for amiloride and hASIC-1 have been defined. Future work will experimentally verify these interaction sites, but at present this should allow for virtual screening of drug libraries at these putative interaction sites.