水通道蛋白与脑

在哺乳动物内已发现有十种水通道蛋白(AQP0～AQP9)分布于水代谢活跃的器官.每种水通道蛋白都具有组织分布特异性,不同水通道蛋白之间的结构相似,均以四聚体形式存在,每一单体构成一个功能单位(水通道),具有转运水的功能.脑组织中的水通道蛋白主要为AQP4和AQP1,其分子结构与功能在基因水平存在动态调节.深入研究它们在脑内的分布与功能,尤其与脑水肿之间的关系,对指导脑水肿治疗和开发新药均具有重要意义.     

水通道蛋白4在大鼠脑垂体中的表达

目的：研究水通道蛋白4(AQP4)及其mRNA在脑垂体中的表达,探讨其在脑垂体激素分泌过程中的作用。方法：应用免疫组织化学和原位杂交技术,观察成年Wistar大鼠脑垂体中AQP4及其mRNA的正常分布。结果：AQP4及其mRNA在成年大鼠神经垂体的垂体细胞上表达呈阳性,分布在毛细血管窦周围的垂体细胞表达尤为强烈。腺垂体的所有细胞均有AQP4的表达,胞质中AQP4 mRNA表达呈阳性。中间叶所有细胞AQP4及其mRNA的表达呈弱阳性,其中滤泡星形细胞表达较内分泌细胞强烈。结论：AQP4广泛分布于脑垂体的各种组织细胞表面,可能在垂体激素的正常分泌过程中起重要的调节作用。     

肺组织水通道蛋白的研究进展

水通道蛋白 (AQP)是一组与水通透有关的细胞膜转运蛋白。分布于肺组织中的水通道蛋白有6种 (AQP1 ,3 ,4 ,5,8,9) ,每种AQP在组织中有其特异的细胞定位。AQP的主要功能是转运水 ,但某些AQP也能转运小分子溶质如尿素和甘油。AQP在膜中以四聚体形式存在 ,每一单体形成一个功能性的水孔道。肺组织中AQP在基因水平上存在动态调节。深入研究AQP在肺组织内的分布、调节及生理和病理状态下的作用 ,有促于对某些肺疾病发病机制的进一步认识。     

异丙酚通过抑制p38激活下调氨处理的大鼠脑星形胶质细胞AQP4的表达并减轻细胞水肿

目的:研究异丙酚对氨处理的大鼠脑皮质星形胶质细胞水通道蛋白-4(AQP4)表达的影响以及相关机制。方法:原代培养Sprague Dawley大鼠大脑皮质星形胶质细胞。用细胞免疫荧光标记星形胶质细胞特异性的神经胶质酸性蛋白,星形胶质细胞纯度达95%以上的细胞备用。实验分为正常对照组(N)、氯化铵(5mmol/L)处理24h组(NH4Cl-24h)、p38抑制剂SB203580(10μmol/L)预处理组(SB)、异丙酚(10μmol/L)预处理组(P)和溶剂二甲亚砜(DMSO)对照组(C)。p38抑制剂、异丙酚和溶剂均预处理星形胶质细胞30min后再加入氯化铵作用24h。用光学显微镜观察各组细胞水肿情况,同时用Western blotting检测AQP4、p38和磷酸化p38的表达情况。结果:光学显微镜观察发现氯化铵处理后星形胶质细胞较正常对照组细胞肿胀明显,异丙酚和p38抑制剂预处理能明显减轻星形胶质细胞水肿。Western blotting检测发现各组星形胶质细胞总p38蛋白表达无明显变化(P0.05)。异丙酚预处理组和SB预处理组磷酸化p38蛋白和AQP4蛋白表达均较氯化铵处理组和溶剂对照组明显降低(P0.05)。结论:AQP4蛋白表达受p38信号途径的调节,异丙酚可通过抑制p38信号途径的激活,降低氨引起的星形胶质细胞AQP4蛋白表达上调,减轻细胞水肿。     

肾脏AQP2表达及穿梭调节的分子机制

水通道蛋白是位于细胞膜或细胞内囊泡能够通透水的蛋白质分子 ,其中水通道蛋白 2 (AQP2 )主要分布于肾脏集合管主细胞的管腔侧膜及近管腔侧的胞浆囊泡内 ,是抗利尿激素敏感型水通道。抗利尿激素可调节肾脏集合管AQP2的表达及穿梭 ,在肾脏水平衡调节中起重要作用     

水通道蛋白-4在大鼠睾丸和附睾中的表达

目的 观察水通道蛋白-4（AQP4）在雄性大鼠睾丸和附睾的表达及分布特点,为探讨水通道蛋白在睾丸和附睾中的作用提供形态学基础。方法 采用免疫组织化学的方法,检测AQP4在睾丸和附睾的表达分布。结果 AQP4在睾丸中表达于各级生精细胞、支持细胞以及间质细胞中;在附睾管内表达于柱状上皮细胞,且腔面表达更明显。结论 AQP4可能参与了精子的生成及成熟过程,在激素的分泌调节中发挥作用。     

水通道蛋白4和人星形胶质细胞瘤在体内增殖和瘤周水肿的相关性研究

目的：研究水通道蛋白4（aquaporin-4, AQP4）和人星形胶质细胞瘤在体内增殖和瘤周水肿的相关性。方法建立人星形胶质细胞瘤裸鼠模型; EdU法检测细胞增殖;免疫组织化学法检测肿瘤组织内CD34阳性细胞表达,检测肿瘤组织微血管密度;免疫印迹法测AQP4蛋白表达;以人脑正常星形胶质细胞为阴性对照。结果细胞皮下注射一周后可见肿瘤长出, EdU法检测肿瘤增殖能力随肿瘤恶性程度增加而增大,免疫组织化学检测提示高度恶性组较低度恶性组具备更高的微血管密度, AQP4蛋白表达随恶性程度增加而增加。结论水通道蛋白4的表达水平和人星形细胞瘤增殖和瘤周水肿程度密切相关。     

水通道蛋白1、5在星形细胞瘤中的表达变化

目的:观察水通道蛋白1、5(AQP1、5)在人不同病理级别星形细胞瘤组织中的表达差异,探讨星形细胞瘤增殖、生长的分子机制.方法:收集人各个病理级别星形细胞瘤标本55例,以肿瘤周围相对正常脑组织作为对照,采用H-E染色诊断分级,石蜡切片免疫组织化学、免疫印迹分析及逆转录聚合酶链式反应观察AQP1、5及其mRNA的表达变化.结果:与正常脑组织相比,人星形胶质瘤组织中AQP1及其mRNA表达上调,随着星形细胞瘤病理级别的升高,AQP1及其mRNA表达增强,胶质母细胞瘤组织表达最强烈;而AQP5及其mRNA仅在高恶性星形细胞瘤组织中表达增强.结论:AQP1在人星形细胞瘤组织中表达与其病理级别相关,而AQP5仅在人高恶性星形细胞瘤组织中表达增强,提示不同病理级别胶质瘤组织中AQP的表达规律不尽相同.     

水通道蛋白-4 mRNA沉默可抑制离体缺氧星形胶质细胞水通道蛋白-4的表达

目的:研究水通道蛋白-4(AQP4) mRNA沉默对体外缺氧星形胶质细胞AQP4表达的影响.方法: 用氯化钴诱导体外星形胶质细胞缺氧,建立AQP4 mRNA沉默缺氧星形胶质细胞模型.随机分为正常组、对照组、缺氧组和干扰组,观察星形胶质细胞形态,免疫细胞化学、荧光定量PCR、免疫印迹法检测AQP4 mRNA 及蛋白表达....     

水通道蛋白5在大鼠大脑组织中的分布及表达

目的:观察水通道蛋白5(AQP5)在大鼠脑组织中的分布和表达,为研究脑组织中水分子的运输和平衡机制提供形态学基础。方法:运用免疫组织化学和免疫荧光技术,观察正常成年Wistar大鼠脑组织中AQP5的分布;运用免疫印迹观察AQP5的表达,并与AQP4在脑组织的分布和表达进行比较。结果:AQP5分布于大脑皮质的软脑膜、脉络丛、血管周围、海马锥体细胞层、齿状回颗粒细胞层、视上核、视交叉上核内和大脑纵裂两侧皮质深部,与AQP4分布范围相似;除此以外,AQP5还独自分布于大脑皮质下神经细胞。免疫印迹显示AQP5表达明显弱于AQP4。结论:AQP5在大鼠脑组织中分布广泛,可能协同AQP4在脑组织水运输平衡,脑脊液产生与回流及渗透压的调节过程中,发挥重要作用。     

Differential expressions of aquaporin subtypes in astroglia in the hippocampus of chronic epileptic rats

In order to elucidate the roles of aquaporins (AQPs) in astroglial responses, we investigated AQP expressions in the experimental epileptic hippocampus. In control animals, AQP1 protein expression was restricted to the ventricular-facing surface of the choroid plexus. AQP4 was expressed in astrocyte foot processes near blood vessels and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, and in astrocytes. Six to eight weeks after status epilepticus (SE), AQP1 expression was mainly, but not all, detected in vacuolized astrocytes, which were localized in the stratum radiatum of the CA1 region. AQP4 was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in non-vacuolized astrocytes was increased as compared to control level. AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes. Therefore, our findings suggest that AQP subunits may play differential roles in various astroglial responses (including astroglial swelling and astroglial loss) in the chronic epileptic hippocampus.     

Cell locations for AQP1, AQP4 and 9 in the non-human primate brain

The presence of three water channels (aquaporins, AQP), AQP1, AQP4 and AQP9 were observed in normal brain and several rodent models of brain pathologies. Little is known about AQP distribution in the primate brain and its knowledge will be useful for future testing of drugs aimed at preventing brain edema formation. We studied the expression and cellular distribution of AQP1, 4 and 9 in the non-human primate brain. The distribution of AQP4 in the non-human primate brain was observed in perivascular astrocytes, comparable to the observation made in the rodent brain. In contrast with rodent, primate AQP1 is expressed in the processes and perivascular endfeet of a subtype of astrocytes mainly located in the white matter and the glia limitans, possibly involved in water homeostasis. AQP1 was also observed in neurons innervating the pial blood vessels, suggesting a possible role in cerebral blood flow regulation. As described in rodent, AQP9 mRNA and protein were detected in astrocytes and in catecholaminergic neurons. However additional locations were observed for AQP9 in populations of neurons located in several cortical areas of primate brains. This report describes a detailed study of AQP1, 4 and 9 distributions in the non-human primate brain, which adds to the data already published in rodent brains. This relevant species differences have to be considered carefully to assess potential drugs acting on AQPs non-human primate models before entering human clinical trials.     

AQP9在组织中的分布和功能

AQP9是一种选择性水通透膜转运蛋白 ,属于主体内在蛋白家族的成员之一。AQP9不但对水具有通透性 ,而且对其它一些中性溶质也具有通透性。现已发现 ,AQP9分布在多种器官组织中。在脑组织 ,AQP9主要分布在星形胶质细胞 ,参与水的代谢和渗透压调节 ,还可能与脑的某些水代谢疾病有关。在肝脏 ,AQP9的分布有明显的性别差异 ,其功能可能与尿素清除有关。在睾丸 ,分布在生精小管和Leyding细胞 ,与液体的重吸收及雄性激素产生功能有关。研究AQP9的分子结构 ,生化特性 ,病理生理变化对于了解水代谢疾病的发生机制以及指导临床水代谢疾病的治疗具有十分重要的意义     

Aquaglyceroporin 9 in brain pathologies

Aquaglyceroporins belong to the aquaporin family and are permeable to water and also to small solutes such as glycerol and urea. In this review, we will compare the expression of aquaporin 9 (AQP9), an aquaglyceroporin, with that of AQP4, a pure water channel, in pathological conditions. In astrocytes, AQP4 is mainly involved in water and ionic homeostasis. Its expression is highly modified in several brain disorders and it plays a key role in cerebral edema formation. AQP9 is expressed in astrocytes and in catecholaminergic neurons. The level of expression of brain AQP9 is under the control of blood insulin concentrations, and its expression is increased in diabetes, suggesting that AQP9 could be involved in brain energy metabolism. The induction of AQP9 in astrocytes is observed over time after stroke onset, suggesting participation in the clearance of excess lactate in the extracellular space. In some models, AQP9 is also induced in non-catecholaminergic neurons after global ischemia and in the periphery of gliomas, however functional roles are still unclear. The review of literature underlies that each AQP has several distinctive roles which depend on the AQP and cell types.     

Distribution and possible roles of aquaporin 9 in the brain

Aquaporin 9 (AQP9) is a member of the aquaporin channel family involved in water flux through plasma membranes and exhibits the distinct feature of being also permeable to monocarboxylates, such as lactate, and various solutes, including glycerol, carbamides, purines, pyrimidines, and urea. AQP9 is constitutively expressed at high levels in the liver. In the brain under physiological conditions, AQP9 was first observed in tanycytes, and then in astrocytes. Only recently, its expression was also shown in neurons. Neurons expressing AQP9 are catecholaminergic and glucose sensitive. The expression of neuronal AQP9 can be negatively regulated by insulin and in diabetic animals an increase in AQP9 expression is observed in the catecholaminergic nuclei of the hindbrain, similar to the regulation of AQP9 by insulin in the liver. Furthermore, after transient brain ischemia, AQP9 expression is increased in astrocytes and its regulation may implicate the MAP-kinase pathways stimulated in such pathological conditions. Despite these new data, the exact role of AQP9 in the brain is still unclear. However, we may hypothesize that AQP9 is implicated in brain energy metabolism, as a neutral solute channel. AQP9 could facilitate the diffusion of lactate from the astrocyte to the neuron. In glucose sensitive neurons, diffusion of lactate and glycerol could stimulate these neurons in a similar manner to glucose and could regulate the energy balance. In pathological conditions, induction of AQP9 in astrocytes could participate in the clearance of excess lactate in the extracellular space. These hypotheses concerning the function of brain AQP9 are still speculative and open new areas of investigation.     

Biology of AQP4 and Anti‐AQP4 Antibody: Therapeutic Implications for NMO

The water channel aquaporin‐4 (AQP4) is the target of the immunoglobulin G autoantibody (AQP4‐IgG) in neuromyelitis optica (NMO). AQP4 is expressed in foot processes of astrocytes throughout the central nervous system, as well as in skeletal muscle and epithelial cells in kidney, lung and gastrointestinal organs. Phenotype analysis of AQP4 knockout mice indicates the involvement of AQP4 in water movement into and out of the brain, astrocyte migration, glial scar formation and neuroexcitatory phenomena. AQP4 monomers form tetramers in membranes, which further aggregate to form supramolecular assemblies called orthogonal arrays of particles. AQP4‐IgG is pathogenic in NMO by a mechanism involving complement‐ and cell‐mediated astrocyte cytotoxicity, which produces an inflammatory response with oligodendrocyte injury and demyelination. AQP4 orthogonal arrays are crucial in NMO pathogenesis, as they increase AQP4‐IgG binding to AQP4 and greatly enhance complement‐dependent cytotoxicity. Novel NMO therapeutics are under development that target AQP4‐IgG or AQP4, including aquaporumab monoclonal antibodies and small molecules that block AQP4‐IgG binding to AQP4, and enzymatic inactivation strategies to neutralize AQP4‐IgG pathogenicity.     

Lactic acid increases aquaporin 4 expression on the cell membrane of cultured rat astrocytes

The water channel protein aquaporin (AQP) may play roles in the homeostasis of water content in the brain and brain edema. One possible mechanism of brain edema is glial swelling due to lactic acidosis associated with ischemia. Here, we investigated the effect of lactic acid on the expression and cellular distribution of AQP 4 in cultured rat astrocytes. After 24h of incubation, the AQP4 expression level increased maximally with 35mM lactic acid. The AQP4 expression levels also increased with hydrochloric acid or acetic acid. In contrast, with sodium lactate, the AQP4 levels did not increase. The increase in AQP4 expression level occurred without a significant increase in AQP4 mRNA expression level by lactic acid. Under the conditions of de novo protein synthesis inhibition with cycloheximide, lactic acid increased the AQP4 expression level. Furthermore, lactic acid increased the AQP4 expression level on the cell surface of the astrocytes, as determined by a cell surface biotinylation assay and immunocytochemical examination. The increase in AQP4 expression level on the cell membrane of astrocytes induced by lactic acid may be a new regulation mechanism of AQP4 in the brain.     

Expression of the water channel protein aquaporin-9 in malignant brain tumors.

Recently, many studies seen concerning the expression and distribution of aquaporins and K channels in the central nervous system, and their physiological and pathophysiologic roles in water and ion homeostasis. Whereas most data were collected on aquaporin-4 (AQP4) in astrocytes, only little attention was paid to AQP9 which is a water channel transporting glycerol, mannitol, and urea as well. This is the first study describing AQP9 in human brain and human brain tumors. For comparison, we also investigated the immunohistochemical distribution of AQP9 in the rat glioma RG2. Whereas in the normal rat brain AQP9 is only weakly expressed by astrocytes, the anti-AQP9 immunoreactivity was found to be increased at the tumor border, but not within the tumor. In contrast, in human glioblastoma, most glioma cells throughout the tumor revealed a strong anti-AQP9 immunoreactivity across the whole surface of the cell. In the discussion, the increase of the anti-AQP9 immunoreactivity in glioma cells is suggested to reflect an upregulation and to counteract the glioma-associated lactic acidosis by clearance of glycerol and lactate from the extracellular space. In addition, the increased level of AQP9 immunoreactivity could be involved in the energy metabolism of the glioma and/or surrounding neuronal cells.     

Functional and molecular interactions between aquaporins and Na,K-ATPase

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Astrocytes play a key role in the clearance of both potassium (K⁺) and glutamate released during neuronal activity. Emerging evidence suggests that AQP4 facilitates K⁺ clearance by astrocytes and contributes to recovery of neuronal excitability. Here we report that AQP4 can assemble with its regulator metabotropic glutamate receptor 5 (mGluR5) and with Na,K-ATPase; the enzyme responsible for active K⁺ transport and for establishing the electrochemical gradient across the cell plasma membrane. We have, by use of pull down assays in rat brain tissue, identified the segment in the AQP4 NH₂-terminus containing the amino acid residues 23–32 as the site for interaction with Na,K-ATPase catalytic subunit and with mGluR5. Mutagenesis studies revealed that the AQP4 amino acids K27 and W30 are of key importance for interaction with both Na,K-ATPase and mGluR5. To confirm that interaction also occurs within intact cells, we have performed fluorescence resonance energy transfer (FRET) studies in primary astrocytes derived from rat striatum. The results indicate close proximity of wild type AQP4 and Na,K-ATPase in the plasma membrane of rat astrocytes. FRET efficiencies observed with the mutants AQP4 K27A and AQP4 W30A were significantly lower, highlighting the importance of these residues for the interaction between AQP4 and Na,K-ATPase. We conclude that AQP4/Na,K-ATPase/mGluR5 can form a macromolecular complex/transporting microdomain in astrocytes. This complex may be of functional importance for the regulation of water and K⁺ homeostasis in the brain, as well as for neuron-astrocyte metabolic crosstalk.