水通道蛋白与眼病

水通道蛋白（aquaporin,AQPs)是存在于动植物及微生物细胞膜上转运水的特异孔道，目前已鉴定出8种水通道蛋白，不同水通道蛋白之间具有类似的特征，众多的研究证实这类蛋白质对维持机体的正常状态至关重要，且与水平衡紊乱所致的临床症状密切相关，本综述了近年来水通道蛋白的研究进展，并重点讨论水通道蛋白在眼的分布及其与眼病的可能关系。     

水通道蛋白-1在人眼睫状突无色素上皮细胞及眼组织的表达

水通道蛋白(aquaporin，AQP)即存在于动植物及微生物细胞膜上转运水的特异孔道，该孔道是由一系列具有同源性的内在膜蛋白家族成员所形成，介导着不同类型细胞膜的跨膜水转运，目前已从哺乳动物的组织中鉴定8种水通道蛋白，广泛分布于各个组织和器官，在维持机体的水平衡中起着重要的作用，且与水平衡紊乱所造成的一些疾病密切相     

水通道蛋白-1在培养的牛角膜内皮细胞中的表达研究

目的研究水通道蛋白-1在培养的牛角膜内皮细胞中的表达情况及意义。方法用免疫组织化学方法,检测水通道蛋白-1在培养的牛角膜内皮细胞中的表达。结果培养的牛角膜内皮细胞中有水通道蛋白-1的表达,且主要位于细胞膜上。结论牛角膜内皮细胞中的水通道蛋白-1在角膜内皮液体转运中起着重要作用,其表达改变可能导致角膜水肿和功能改变。     

水通道蛋白与眼病

水通道蛋白 (aquaporin ,AQPs)是存在于动植物及微生物细胞膜上转运水的特异孔道。目前已鉴定出 8种水通道蛋白 ,不同水通道蛋白之间具有类似的特征。众多的研究证实这类蛋白质对维持机体的正常状态至关重要 ,且与水平衡紊乱所致的临床症状密切相关。本文综述了近年来水通道蛋白的研究进展 ,并重点讨论水通道蛋白在眼的分布及其与眼病的可能关系。     

水通道蛋白-1在牛角膜内皮细胞液体转运中作用研究

目的:研究水通道蛋白-1在牛角膜内皮细胞中的表达及在角膜内皮液体转运中的作用.方法:用免疫组织化学方法,检测水通道蛋白-1在培养的牛角膜内皮细胞中的表达,并用水通道蛋白阻滞剂对氯汞苯磺酸酯(pCMBS)处理培养的牛角膜内皮细胞,观察处理前后角膜内皮细胞的渗透性水通透率(Pf)改变.结果:培养的牛角膜内皮细胞中有水通道蛋白-1的表达,主要位于细胞膜上.汞剂处理前后的角膜内皮细胞Pf分别为(0.044±0.005)cm/s(n=15)和(0.017±0.003)cm/s(n=15),其差异有统计学意义(P＜0.01).结论:水通道蛋白-1在牛角膜内皮细胞液体转运中起着重要作用,其表达的改变会导致角膜水肿和功能改变.     

水通道蛋白-1在角膜内皮细胞液体转运中的作用研究

目的 研究水通道蛋白-1在牛角膜内皮细胞中的表达及在角膜内皮液体转运中的作用.方法 用免疫组织化学方法,检测水通道蛋白-1在培养的牛角膜内皮细胞中的表达,并用水通道蛋白阻滞剂对氯汞苯磺酸酯（pCMBS）处理培养的牛角膜内皮细胞,观察处理前后角膜内皮细胞的渗透性水通透率（Pf）改变.结果 培养的牛角膜内皮细胞中有水通道蛋白-1的表达,主要位于细胞膜上.汞剂处理前后的角膜内皮细胞Pf分别为（0.044±0.005）cm/s（n=15）和（0.017±0.003）cm/s（n=15）,其差异有统计学意义（P＜0.01）.结论 水通道蛋白-1在牛角膜内皮细胞液体转运中起着重要作用,其表达的改变能够导致角膜水肿和功能改变.     

水通道蛋白1在人角膜内皮细胞的表达

目的 研究水通道蛋白1(water channel protein1,AQP1)抗体在人角膜中的表达及水转运机制。方法 运用免疫组织化学法测定人角膜中AQP1的表达。结果 在角膜内皮细胞和基质细胞中有AQP1的表达，而角膜上皮细胞不表达AQP1。结论 本实验证实了AQP1在角膜内皮的表达，为角膜的水转运及角膜水肿机制的研究提供了分子生物学基础。     

水通道蛋白与白内障研究进展

水通道蛋白(aquaporins,AQPs)主要介导自由水沿渗透压梯度的被动跨生物膜转运,对水有高度选择性。晶状体只有两种水通道蛋白,晶状体上皮细胞表达的AQP1和晶状体纤维细胞表达的AQP0,它们共同调节晶状体水代谢,维持晶状体生理功能及透明性,其异常表达可导致白内障的发生。术文综述了近年来对水通道蛋白0,1的研究,并讨论水通道蛋白与白内障发生的关系。     

水通道蛋白与眼科疾病

水通道蛋白(aquaporin,AQP)是一类广泛存在于哺乳动物上皮和内皮细胞膜上的特异性转运水蛋白,在眼部组织中大量存在,并参与包括维持眼压、促进上皮修复等在内的一系列病理生理过程.近年研究发现,其与眼科疾病的发生发展也密不可分,AQP0广泛大量存在于晶状体中,其基因突变导致先天性白内障发生,其后天表达改变会导致年龄相关性白内障的加速发展;眼内介导水分运输的AQP表达失调,会导致糖尿病视网膜病变的黄斑水肿、青光眼的高眼压及葡萄膜炎、视神经炎等一系列与水分子运输失衡相关的眼病.如果在眼内可以使用安全有效的技术手段从数量及空间分布上改变AQP的表达,这很可能成为治疗眼科疾病的一个新靶点.     

P-糖蛋白在眼晶状体中的表达与功能

P-糖蛋白是一种与肿瘤多药耐药有关的膜蛋白，并且也存在于眼晶状体上，与Cl^-通道转运的关系密切，对维持晶状体透明性及内环境的稳定有重要意义。本文对P-糖蛋白的生物学特点，在晶状体的表达，及其与Cl^-通道、糖性白内障发生的关系作一综述。     

Molecular identification of functional water channel protein in cultured human nonpigmented ciliary epithelial cells

PURPOSE: Water channel proteins are important pathways for water movements across cell membranes, including those in the ciliary epithelium, which is the major site of aqueous humor secretion. In this study, we aimed to demonstrate the expression of functionally active aquaporin-1 (AQP1) water channels in cultured human ciliary epithelial cells. METHODS: Poly A(+) RNA was isolated from cell cultures of Simian Virus 40 (SV-40) transformed human nonpigmented ciliary epithelium (NPE) subjected to RT-PCR reaction using primers specific to AQP1. Northern analysis was used to define the expression of AQP1 in NPE cells. Western immunoblotting with polyclonal antibody raised against AQP1 was used to evaluate the AQP1 protein expression in the plasma membranes of human NPE cells. Light scattering method was used to determine the osmotic water permeability in the suspension of NPE cells. RESULTS: RT-PCR using specific primers for AQP1, Northern analysis and Western immunoblot using AQP1 specific antibody demonstrated the expression of AQP1 in the plasma membranes of NPE cells. Osmotic water permeability (P( f)) measurements confirmed that functional AQP1 water channels are expressed in human NPE cells and the P(f) for these cells was 9.8 x 10( -3) cm/s at 10 degrees C. CONCLUSIONS: The presence of AQP1 in human NPE cells suggests that it may have a role in the fluid flow across epithelial membranes. In addition, the existence of AQP1 in the human NPE cells provide an excellent in vivo model to study the regulation of aquaporins and their possible role in the aqueous humor secretion.     

Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume.

PURPOSE. Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS. An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS. AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001). CONCLUSIONS. In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo.     

Functions of aquaporins in the eye

The aquaporins (AQPs) are integral membrane proteins whose main function is to transport water across cell membranes in response to osmotic gradients. At the ocular surface, AQP1 is expressed in corneal endothelium, AQP3 and AQP5 in corneal epithelium, and AQP3 in conjunctival epithelium. AQPs are also expressed in lens fiber cells (AQP0), lens epithelium (AQP1), ciliary epithelium (AQP1, AQP4) and retinal Müller cells (AQP4). Mutations in AQP0 produce congenital cataracts in humans. Analysis of knockout mice lacking individual AQPs suggests their involvement in maintenance of corneal and lens transparency, corneal epithelial repair, intraocular pressure (IOP) regulation, retinal signal transduction and retinal swelling following injury. The mouse phenotype findings implicate AQPs as potential drug targets for therapy of elevated IOP and ocular disorders involving the cornea, lens and retina. However, much research remains in defining cell-level mechanisms for the ocular AQP functions, in establishing the relevance to human eye disease of conclusions from knockout mice, and in developing AQP-modulating drugs.     

Aquaporin-1 channels in human retinal pigment epithelium: role in transepithelial water movement

PURPOSE: Aquaporin (AQP) is a hexahelical integral membrane protein that functions as a constitutive channel for water and regulated channel for cations in fluid transporting tissues, including many in the eye. Although AQP1 has been cloned from a cDNA library prepared from cultures of retinal pigment epithelial (RPE) cells isolated from human fetal tissue, three separate studies failed with various immunochemical techniques to detect AQP1 protein in adult human or rat RPE preparations. The purpose of this study was to examine specifically the expression and distribution of AQP1 in adult human RPE in situ by using alternative methodologies and model systems and to determine the contribution of AQP1 to water movement across cultured RPE cells isolated from human cadaveric and fetal eyes. METHODS: AQP1 in human RPE in situ was determined after biotinylation of proteins on cell surfaces and streptavidin chromatography, followed by immunoblot analyses. AQP1 distribution in a polarized in vitro RPE model was determined with indirect immunofluorescence confocal microscopy. The role of channel-mediated transport of water across RPE cell monolayers on filters was assessed by osmotic challenge assay. Expression levels of AQP1 were controlled with an adenovirus expression system and monitored by immunoblot analyses. RESULTS: AQP1 protein was detected in human RPE in situ and in cultures of human adult and fetal RPE cells. In functional assays, AQP1 facilitated water movement across RPE monolayers in an expression-dependent manner in two complementary model systems. CONCLUSION: The expression of AQP1 by RPE in vivo probably contributes to the efficient transepithelial water transport across RPE, maintains retinal attachment, and prevents subretinal edema.     

A developmental switch in the expression of aquaporin-4 and Kir4.1 from horizontal to Müller cells in mouse retina

PURPOSE: In adult retina, aquaporin-4 (AQP4) and inwardly rectifying K(+) (Kir4.1) channels localize to astrocyte and Müller cell membranes facing vascular and vitreous compartments, optimizing clearance of extracellular K(+) and water from the synaptic layers. However, it is unknown whether these channels are expressed at early developmental stages, before gliogenesis or angiogenesis take place in the neural retina. This study was conducted to determine the presence of AQP4 and Kir4.1 proteins in the developing mouse retina. METHODS: Simultaneous AQP4 and Kir4.1 immunodetection was performed in postnatal mice 1, 9, 15, and 30 days of age. Confocal microscopy was used to identify the cellular distribution of AQP4 and Kir4.1 proteins, as well as their coexpression with the cell-selective immunomarkers Prox-1, calbindin, and neurofilament. RESULTS: AQP4 and Kir4.1 proteins were coexpressed in calbindin- and Prox1-expressing retinal neurons at birth. These neurons were identified as horizontal cells based on their position and morphology. By P15, when vision starts, AQP4 and Kir4.1 localization coordinately switched from horizontal cells to Müller glial cells. CONCLUSIONS: The findings showed that AQP4 and Kir4.1 protein expression is confined to differentiating horizontal cells before its expression in Müller cells. The finding of AQP4 in neurons is novel, since AQP4 expression within the central nervous system is restricted to glia. Also, the results demonstrated that AQP4 is a horizontal cell-specific immunomarker in neonatal retina. The transitory coexpression of AQP4 and Kir4.1 proteins by differentiating horizontal interneurons suggests that these cells mediate K(+) and water transcellular uptake until the initiation of phototransduction, when glial cells assume these functions.     

Tear secretion by lacrimal glands in transgenic mice lacking water channels AQP1, AQP3, AQP4 and AQP5

The expression of three aquaporin (AQP)-type water channels has been reported in the lacrimal gland: AQP5 in the apical membranes of acinar and duct cells, AQP4 in the basolateral membranes of acinar cells, and AQP1 in microvascular endothelia. Recent experiments indicate that water movement through AQP5 in the salivary gland is important in saliva secretion. To investigate the role of aquaporins in lacrimal gland function, basal and pilocarpine-stimulated tear secretion was compared in wildtype mice and knockout mice lacking AQP1, AQP4 and AQP5, as well as AQP3, which was found here to be expressed in the basolateral membrane of acinar cells. Tear fluid was collected in anesthetized mice using microcapillary tubes before and at 4 min intervals after pilocarpine administration. Tear fluid volumes were (in microliter per 4 min, S.E.): 0.69 +/- 0.06 (wildtype mice), 0.70 +/- 0.07 (AQP1 -/-), 0.81 +/- 0.13 (AQP3 -/-), 0.62 +/- 0.14 (AQP4 -/-), and 0.78 +/- 0.09 (AQP5 -/-) (differences not significant). Chloride concentrations (average 155 +/- 13 mM) measured by a fluorescence assay were also not different in tear fluid collected from wildtype vs aquaporin null mice. These findings provide direct evidence against an essential role for aquaporins in lacrimal gland fluid secretion. The requirement for aquaporins in salivary but not lacrimal gland secretion, may involve the substantially slower fluid secretion rate across lacrimal gland acinar cells.     

Water permeability of C-terminally truncated aquaporin 0 (AQP0 1-243) observed in the aging human lens

PURPOSE: To first assess the distribution of posttranslationally truncated products of aquaporin 0 (AQP0) in dissected sections of a normal human lens and to determine the effect of backbone cleavage on the water permeability of AQP0. METHODS: A 27-year-old lens was concentrically dissected into six sections. Membrane protein was isolated from each section and cleaved with cyanogen bromide, and the peptides were separated and analyzed by reverse-phase (RP)-HPLC-mass spectrometry (MS). The sites of posttranslational AQP0 C-terminal truncation were determined by mass spectrometry. Truncated forms of AQP0 were expressed in a Xenopus laevis oocyte system, and the effect of truncation on AQP0 water permeability was assessed in an oocyte osmotic swelling assay. RESULTS: The extent of truncation at many sites within the C terminus increased with fiber cell age, and the effects of truncations after residues 234, 238, and 243 on AQP0 water permeability were examined. Truncation after residue 243 resulted in an approximate 15% decrease in permeability compared with the full-length protein, AQP0 1-263. However, rather than a direct effect on water transport, analysis of surface protein expression indicated that the decrease in permeability was a result of less efficient protein trafficking to the oocyte surface and that the permeabilities of full-length and 1-243 AQP0 were indistinguishable. Further, C-terminal truncation of AQP0 to 1-234 and 1-238, completely impaired trafficking into the plasma membrane, precluding the measurement of permeability. CONCLUSIONS: These data provide evidence that loss of 20 amino acids from the C terminus may not directly affect the ability of AQP0 to transport water.     

Aquaporin-1-facilitated keratocyte migration in cell culture and in vivo corneal wound healing models

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24 h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.     

Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: Effects on lens transparency

Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0^−/−) mouse? To investigate, we generated a transgenic wild-type mouse line expressing AQP1 in the fiber cells using αA-crystallin promoter. These transgenic mice (TgAQP1^+/+) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1^+/+/AQP0^−/−) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal model to demonstrate that AQP0 may have an additional function, possibly cell-to-cell adhesion.