Acute energy reduction induces caspase-dependent apoptosis and activates p53 in retinal ganglion cells (RGC-5)

The energy reduction-induced death of retinal ganglion cells is associated with many ophthalmic diseases. The present study was designed to investigate the apoptosis pathway of retinal ganglion cells (RGC-5) following acute ATP reduction by using glucose deprivation (GD). RGC-5 cells were cultured in glucose-free or normal DMEM for 3 days. The changes in intracellular ATP and cell viability were monitored by ATP assay and MTT assay. APOPercentage™ and in situ TUNEL assays were used to determine the cell death pattern. The involvement of oxidative stress was assessed by measuring intracellular ROS generation, the HO-1 expression, the effect of antioxidants, and the ratio of GSSG to total GSH. The activation of p53 and apoptosis markers was evaluated by Western blotting. We found that glucose deprivation caused an acute decline of intracellular ATP level, concomitantly decreasing cell viability. The cell death exhibited typical features indicative of apoptosis, including cell shrinkage, phosphatidylserine externalization and DNA fragmentation. Oxidative stress was involved in the cell death process; an antioxidant significantly protected the cells against glucose deprivation. p53 and apoptosis markers, caspase-3 and PARP-1 were activated after RGC-5 cells were cultured in glucose-free media for 32 h. Z-VAD-fmk, a pan-caspase inhibitor, was sufficient to prevent apoptosis. These results suggest that acute energy reduction induced by glucose deprivation triggers caspase-dependent apoptosis and activates p53. Blocking the critical steps in this cell death pathway may have therapeutic effects, rescuing the retinal ganglion cells from damages associated with acute energy reduction.     

Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells

The purpose of this study was to determine the viability of cell-based delivery of brain-derived neurotrophic factor (BDNF) from genetically modified mesenchymal stem cells (MSCs) for neuroprotection of RGC-5 cells. RGC-5 cells were differentiated with the protein kinase inhibitor staurosporine (SS) and exposed to the cellular stressors glutamate or H₂O₂. As a neuroprotective strategy, these cells were then co-cultured across a membrane insert with mesenchymal stem cells (MSCs) engineered with a lentiviral vector for production of BDNF (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to stressed RGC-5 cells. After SS-differentiation RGC-5s developed neuronal-like morphologies, and a significant increase in the proportion of RGC-5s immunoreactive for TuJ-1 and Brn3a was observed. Differentiated RGC-5s also had prominent TrkB staining, demonstrating expression of the high-affinity BDNF receptor. Treatment of SS-differentiated RGC-5s with glutamate or H₂O₂, produced significant cell death (56.0 ± 7.02 and 48.90 ± 4.58% of control cells, respectively) compared to carrier-solution treated cells. BDNF-delivery from MSCs preserved more RGC-5 cells after treatment with glutamate (80.0 ± 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 ± 1.89%, p < 0.01). BDNF-MSCs also protected more RGC-5s after treatment with H₂O₂ (65.6 ± 3.47%) than GFP-MSCs (46.0 ± 4.20%, p < 0.01). We have shown survival of differentiated RGC-5s is reduced by the cellular stressors glutamate and H₂O₂. Additionally, our results demonstrate that genetically modified BDNF-producing MSCs can enhance survival of stressed RGC-5 cells and therefore, may be effective vehicles to deliver BDNF to retinal ganglion cells affected by disease.     

Depletion of optineurin in RGC-5 cells derived from retinal neurons causes apoptosis and reduces the secretion of neurotrophins

Optineurin is a Golgi complex-associated ubiquitous protein with high expression levels in retinal ganglion cells (RGCs). Mutations in optineurin have been observed in rare hereditary cases of primary open-angle glaucoma and in amyotrophic lateral sclerosis. We explored the possibility that optineurin deficiency will compromise neuronal exocytosis leading to a diminished secretion of neurotrophic factors that are critically required for neuronal survival. To this end, we used RNA interference to induce depletion of optineurin in RGC-5 cells derived from retinal neurons. SiRNA specific for optineurin was transiently transfected. Moreover, a stable cell line with constitutive optineurin deficiency (RGC-5 pSilencer OPTN) was generated. In addition, we investigated the subcellular localization of optineurin in primary RGCs in retinal cell cultures isolated from eyes of mature mice. In RGC-5 cells, optineurin localized to the periphery of the Golgi complex and was observed in vesicular structures throughout the cytoplasm and close to the plasma membrane. A comparable Golgi-associated localization of optineurin was observed in cultured primary RGCs that were identified by TUJ1 labeling. Optineurin deficiency caused a marked increase in the number of RGC-5 cells with fragmented Golgi complex. RGC-5 pSilencer OPTN with stable optineurin deficiency showed a pronounced increase in the number of cells undergoing apoptotic cell death. Furthermore, the amounts of secreted neurotrophin-3 (NT-3) and ciliary neurotrophic factor were significantly lower in culture medium of RGC-5 pSilencer OPTN cells when compared to controls. Adding exogenous NT-3 to the culture medium to achieve amounts seen in control cultures completely prevented the increase in apoptotic cell death. We propose that lack of neurotrophic support due to impaired secretion of neurotrophic proteins is a critical factor that causes or contributes to RGC or motor neuron death in patients with mutated optineurin.     

Occludin independently regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells

PURPOSE: The aim of this study was to determine the function of the tight junction protein occludin in the control of permeability, under diffusive and hydrostatic pressures, and its contribution to the control of cell division in retinal pigment epithelium. METHODS: Occludin expression was inhibited in the human retinal pigment epithelial cell line ARPE-19 by siRNA. Depletion of occludin was confirmed by Western blot, confocal microscopy, and RT-PCR. Paracellular permeability of cell monolayers to fluorescently labeled 70 kDa dextran, 10 kDa dextran, and 467 Da tetramethylrhodamine (TAMRA) was examined under diffusive conditions or after the application of 10 cm H2O transmural pressure. Cell division rates were determined by tritiated thymidine incorporation and Ki67 immunoreactivity. Cell cycle inhibitors were used to determine whether changes in cell division affected permeability. RESULTS: Occludin depletion increased diffusive paracellular permeability to 467 Da TAMRA by 15%, and permeability under hydrostatic pressure was increased 50% compared with control. Conversely, depletion of occludin protein with siRNA did not alter diffusive permeability to 70 kDa and 10 kDa RITC-dextran, and permeability to 70 kDa dextran was twofold lower in occludin-depleted cells under hydrostatic pressure conditions. Occludin depletion also increased thymidine incorporation by 90% and Ki67-positive cells by 50%. Finally, cell cycle inhibitors did not alter the effect of occludin siRNA on paracellular permeability. CONCLUSIONS: The data suggest that occludin regulates tight junction permeability in response to changes in hydrostatic pressure. Furthermore, these data suggest that occludin also contributes to the control of cell division, demonstrating a novel function for this tight junction protein.     

siRNA靶向沉默载脂蛋白2(Lcn2)对缺氧诱导视网膜神经节细胞-5细胞凋亡的作用及机制

目的 研究载脂蛋白2(lipocalin 2,Lcn 2)对缺氧诱导视网膜神经节细胞-5(retinal ganglion cells,RGC-5)损伤的抑制作用及可能机制.方法 将RGC-5细胞置于缺氧环境下处理(0h、4h、8h、12 h、24 h、48 h),采用实时定量PCR和Western blot 法检测Lcn 2的表达水平.将细胞分为4组:对照组、缺氧组、siNC+缺氧组(转染siRNA阴性对照后进行缺氧处理24 h)和siL-cn2+缺氧组(细胞转染Lcn 2 siRNA后进行缺氧处理24 h).ELISA检测细胞凋亡率和Caspase-3活性,DCFH-DA试剂盒检测活性氧(reactive oxygen speciesin,ROS)产生情况,线粒体膜电位检测试剂盒评价线粒体膜电势,Western blot检测半胱氨酸天冬氨酸蛋白酶3(cleaved-Caspase-3,c-Caspase-3)、聚腺苷二磷酸核糖聚合酶(cleaved-PARP,c-PARP)、Bax、Bcl-2和细胞色素C(cytochrome C,Cyto C)蛋白表达.结果 与0h组相比,缺氧组(4h、8h、12h、24 h和48 h)Lcn 2 mRNA表达水平均升高(均为P＜0.05),同时缺氧组(12 h、24h和48 h)的Lcn 2蛋白表达水平均较0h组升高(均为P＜0.05).与对照组细胞凋亡率(99.66％±2.86％)比较,缺氧组(138.33％±13.76％)显著升高(P＜0.05);siLcn 2+缺氧组细胞凋亡率(105.02％±8.60％)较siNC+缺氧组(142.33％±6.54％)显著下降(P＜0.05).此外,缺氧组较对照组Caspase-3相对活性增强、c-Caspase-3和c-PARP表达均上调(均为P＜0.05),与siNC+缺氧组比较,siLcn 2+缺氧组Caspase-3相对活性水平、c-Caspase-3和c-PARP相对表达水平均降低(均为P＜0.05).与对照组ROS相对荧光强度(1.03±0.11)比较,缺氧组(4.26±0.63)增强(P＜0.05),siLcn 2+缺氧组ROS相对荧光强度(3.10±0.24)较siNC+缺氧组(4.73±0.26)显著减弱(P＜0.05).且siLen 2+缺氧组较siNC+缺氧组线粒体膜电势增加、Cyto C蛋白表达水平及Bax和Bcl-2相对比率减少(均为P＜0.05).结论 沉默Lcn 2抑制缺氧诱导的细胞凋亡及ROS产生,可能是通过抑制线粒体凋亡信号通路实现的.     

sigma-1 receptors protect RGC-5 cells from apoptosis by regulating intracellular calcium, Bax levels, and caspase-3 activation

PURPOSE: sigma-1 Receptor ligands prevent neuronal death associated with glutamate excitotoxicity both in vitro and in vivo. However, the molecular mechanism of the neuroprotective effect remains to be elucidated. The present study was undertaken to determine whether sigma-1 receptor agonists provide neuroprotection by decreasing glutamate-induced calcium mobilization and preventing apoptotic gene expression. METHODS: Cell death was measured by using a calcein-AM/propidium iodide cell-survival assay. Western blot analysis determined the expression levels of Bax in normal RGC-5 cells. Caspase-3 activation after glutamate treatment was determined with a carboxyfluorescein caspase-3 detection kit. Glutamate-induced intracellular calcium mobilization was measured by using ratiometric calcium imaging. RESULTS: sigma-1 Receptor-overexpressing RGC-5 (RGC-5-S1R) cells had lower glutamate-induced intracellular calcium mobilization than did normal RGC-5 cells, and the sigma-1 receptor ligand (+)-SKF10047 reduced the glutamate calcium response in normal and RGC-5-S1R cells. (+)-SKF10047 protected RGC-5 cells from glutamate-induced cell death, and the RGC-5-S1R cells showed a significant resistance to glutamate-induced apoptosis compared with the control RGC-5 cells. BD1047, a sigma-1 receptor antagonist, blocked the protective effect of (+)-SKF10047. Western blot analysis showed that (+)-SKF10047 inhibited the increase in Bax after glutamate treatments. Glutamate-mediated cell death involved activation of caspase-3, and sigma-1 receptor activation prevented an increase in caspase-3 expression. CONCLUSIONS: The results suggest that sigma-1 receptors regulate intracellular calcium levels and prevent activation of proapoptotic genes, thus promoting retinal ganglion cell survival. The sigma-1 ligands appear to be neuroprotective and are a potential target for neuroprotective therapeutics.     

Elevated intraocular pressure and transgenic applications in the mouse

Exploitation of the mouse as a model for glaucoma has been advanced by the development of methods to measure mouse intraocular pressure (IOP), identification of mutant mouse strains in which IOP spontaneously increases, and the development of treatments to induce elevated IOP. These developments enable investigations that directly test the influence of specific gene product alterations on the progression of glaucoma. Moreover, new transgenic mouse models have been produced with genetic mutations that parallel human gene mutations that have been linked to the onset of glaucoma. These new mouse models and technologies have potential for uncovering the biological basis of glaucoma as well as for evaluating new treatments.     

Elevated intraocular pressure and optic nerve injury models in the rat

Models of experimentally elevated intraocular pressure in rats provide valuable opportunities to discover and study mechanisms of pressure-induced optic nerve damage. The structure and vasculature of the rat optic nerve head have several anatomic similarities and differences from the primate that allow useful comparisons and insights into human glaucoma. Specifically, the ultrastructural relationship between astrocytes, retinal ganglion cell axons and the connective tissues of the optic nerve head appear quite similar to the primate, and have a high potential for revealing cellular mechanisms of axonal injury. Three widely used models of creating elevated IOP in rats exist. However, they are not all equivalent and appear to differ in the relationship they exhibit between the level of pressure and extent of optic nerve damage. This indicates that these models may differ in the mechanisms by which they produce elevated eye pressure. All of these models are amenable to a variety of methods for evaluating damage. These include objective and subjective histologic assessment of the optic nerve, counting cells in the retinal ganglion cell layer of the retina and the use of retinal whole mounts to count retinal ganglion cells that have been back-labeled with dye applied to the superior colliculus. In the decade since their introduction, these versatile models have provided important insights into mechanisms of pressure-induced optic nerve damage using sensitive molecular biology techniques. They have also allowed the evaluation of several potential strategies for neuroprotection in glaucoma, ranging from currently available drugs to gene transfer studies.     

The effect of elevated hydrostatic pressure on expression level of type III collagen mRNA by lamina cribrosa cells in vitro

OBJECTIVE: To investigate the effect of elevated hydrostatic pressure on the expression level of type III collagen mRNA by lamina cribrosa cells (LCCs) and approach the action of changes of extracellular stroma on glaucomatous optic nerve damage. METHODS: LCCs were cultured and submitted to elevated hydrostatic pressure (4.67 - 5.33 kPa). We observed collagen type III mRNA level by dot-blot hybridization and computer imaging analysis. RESULTS: Under the pressure for 3 days, the amounts of collagen III mRNA were 0.5828 +/- 0.0517 in optical density (OD), compared with the same period controls (0.2832 +/- 0.0613), the difference was statistically significant (P < 0.05). When exposed to pressure up to 6 days, the collagen III mRNA level remained somewhat higher than that of the control, but there was no significant difference (P > 0.05). After 9 to 12 days of exposure to pressure, the mRNA amounts were decreased respectively to 0.3246 +/- 0.0957 and 0.2164 +/- 0.0171 in OD that were lower than that of the control in the respective same periods, and the differences between the experimental and the control had no significance (P > 0.05). CONCLUSION: Elevated hydrostatic pressure may increase expression level of collagen III mRNA by LCCs in a certain period.     

Oxidative damage to human lens epithelial cells in culture: estrogen protection of mitochondrial potential,ATP, and cell viability

PURPOSE: Epidemiologic studies demonstrate a higher incidence of cataracts in estrogen-deprived postmenopausal women, but the mechanism for the increased risk of cataracts is unclear. An elevated level of H(2)O(2) in aqueous humor and whole lenses has been associated with cataractogenesis. In the present study, for the first time, the protective effect of estrogens against oxidative stress were tested in cultured human lens epithelial cells (HLECs). METHODS: To investigate the involvement of 17beta-estradiol (17beta-E(2)) in protection against oxidative stress, HLECs were exposed to insult with H(2)O(2) at a physiological level (100 microM) over a time course of several hours, with and without pretreatment with 17beta-E(2). Cell viability was measured by calcein AM assay, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to determine intracellular reactive oxygen species (ROS). Intracellular adenosine triphosphate (ATP) level was quantified with a luciferin- and luciferase-based assay and mitochondrial potential (deltapsi(m)) was monitored by a fluorescence resonance energy-transfer technique. RESULTS: H(2)O(2) caused a dose-dependent decrease in mitochondrial membrane potential, intracellular ATP levels, and cell viability. Dose-dependent increases in cell viability and intracellular ATP level were observed with pretreatment of 17beta-E(2) for 2 hours before oxidative insult. At 1 nM, 17beta-E(2) increased cell viability from 39% +/- 4% to 75% +/- 3%, and at 100 nM or higher, it increased survival to greater than 95%. The level of intracellular ATP approached normal with 17beta-E(2) at 100 nM or higher. Pretreatment with 17beta-E(2) did not diminish intracellular ROS accumulation after exposure to H(2)O(2). Moreover, two nonfeminizing estrogens, 17alpha-E(2) and ent-E(2), both of which do not bind to either estrogen receptor alpha or beta, were as effective as 17beta-E(2) in the recovery of cell viability. The estrogen receptor antagonist, ICI 182,780, did not block protection by 17beta-E(2). Both 17beta- and 17alpha-E(2) moderated the collapse of deltapsi(m) in response to either H(2)O(2) or excessive Ca(2+) loading. CONCLUSIONS: The present study indicates that both 17alpha- and 17beta-E(2) can preserve mitochondrial function, cell viability, and ATP levels in human lens cells during oxidative stress. Although the precise mechanism responsible for protection by the estradiols against oxidative stress remains to be determined, the ability of nonfeminizing estrogens, which do not bind to estrogen receptors, to protect against H(2)O(2) toxicity indicates that this conservation is not likely to be mediated through classic estrogen receptors.     

Cornea fluid dynamics. I: measurement of hydrostatic and colloid osmotic pressure in rabbits

Interstitial fluid pressure (Pi) has been measured in vivo in ocular tissues of rats and rabbits, and in vitro in corneas of cows and humans with micropipettes connected to a servocontrolled counterpressure system. The in vivo measurements were performed after anaesthesia and careful immobilization of the head. In all species Pi became gradually more negative from the limbus to the cornea centre; in rabbits the change was from -17.8 mmHg 0.5 mm centrally to the limbus to -42.7 mmHg in central cornea. Cornea and sclera interstitial fluid was isolated by centrifugation, and was used for analysis of colloid osmotic pressure (both tissues) and Na(+)-concentration and electrophoresis (cornea only). During low speed centrifugation (3000 rpm), a wet weight fraction of 2-4% was isolated from cornea, having a colloid osmotic pressure not significantly different from that of plasma (18.9 mmHg). Na+ analysis suggested that the isolated fluid did not derive from corneal cells. Electrophoresis showed a protein pattern in cornea fluid similar to that of plasma except for a band with MW corresponding to 39,000 found in cornea only. The present study demonstrates a pressure gradient from sclera to cornea, and along cornea from periphery to centre, and suggests that proteins in cornea fluid contribute significantly to swelling pressure.     

Effects of interleukin-1beta and dexamethasone on the expression of matrix metalloprotease mRNA by trabecular cells exposed to elevated hydrostatic pressure

PURPOSE: We investigated the effects of interleukin-1beta (Il-1beta) and dexamethasone (Dex) on the expression of matrix metalloprotease-1, -2, -3 and -14 (membrane type-1 MMP-MT1-MMP) as well as tissue inhibitors of matrix metalloproteases (TIMP-1 and -2) mRNA by trabecular cells exposed not only to normal, but also to elevated levels of hydrostatic pressure. METHODS: Confluent primary cultures of porcine trabecular cells were incubated in a serum-free medium (SFM) as controls, or in SFM containing either 10 ng/ml Il-1beta or 10 nm Dex and exposed to pressures of 15 mmHg or 50 mmHg (corresponding to normal and high intraocular pressure, respectively) in specially designed pressure chambers. After 72 hours, total RNA was extracted from the harvested cells, reverse transcribed and amplified using primers specific to MMP-1, -2, -3 and -14, and TIMP-1 and -2. RESULTS: The most significant changes were detected in the levels of MMP-3 mRNA in control cells (2.4-fold increase), of TIMP-1 and -2 mRNA in cells treated with Il-1beta (2.6-fold increase) and of MMP-3 mRNA in cells treated with Dex (3.5-fold increase) exposed to 50 mmHg pressure. CONCLUSION: Because MMP-3 (stromelysin) mRNA showed the highest upregulation, our findings suggest that trabecular cells preferentially degrade and turn over the proteoglycan components of the extracellular matrix in response to short-term exposure to increased hydrostatic pressure with and without Dex as a homeostatic mechanism.     

In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells

PURPOSE: To determine whether primate embryonic stem (ES) cell-derived pigment epithelial cells (ESPEs) have the properties and functions of retinal pigment epithelial (RPE) cells in vitro and in vivo. METHODS: Cynomolgus monkey ES cells were induced to differentiate into pigment epithelial cells by coculturing them with PA6 stromal cells in a differentiating medium. The expanded, single-layer ESPEs were examined by light and electron microscopy. The expression of standard RPE markers by the ESPEs was determined by RT-PCR, Western blot, and immunocytochemical analyses. The ESPEs were transplanted into the subretinal space of 4-week-old Royal College of Surgeons (RCS) rats, and the eyes were analyzed immunohistochemically at 8 weeks after grafting. The effect of the ESPE graft on the visual function of RCS rats was estimated by optokinetic reflex. RESULTS: The expanded ESPEs were hexagonal and contained significant amounts of pigment. The ESPEs expressed typical RPE markers: ZO-1, RPE65, CRALBP, and Mertk. They had extensive microvilli and were able to phagocytose latex beads. When transplanted into the subretinal space of RCS rats, the grafted ESPEs enhanced the survival of the host photoreceptors. The effects of the transplanted ESPEs were confirmed by histologic analyses and behavioral tests. CONCLUSIONS: The ESPEs had morphologic and physiological properties of normal RPE cells, and these findings suggest that these cells may provide an unlimited source of primate cells to be used for the study of pathogenesis, drug development, and cell-replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction.     

Transplantation of cells from eye-like structures differentiated from embryonic stem cells in vitro and in vivo regeneration of retinal ganglion-like cells

Background An embryonic stem (ES) cell-derived eye-like structure, made up of neural retinal lineage cells, retinal pigment epithelial (RPE) cells, and lens cells was constructed in our laboratory. We have shown that cells from these eye-like structures can be integrated into the developing optic vesicle of chicks. The purpose of this study was to determine whether the cells from these eye-like structures can differentiate into retinal ganglion cells (RGCs) when transplanted into the vitreous of an injured adult mouse retina. Methods ES cells were induced to differentiate into eye-like structures in vitro for 6 or 11 days. Recipient mouse eyes were injected with NMDA to injure the RGCs prior to the transplantation. Sham-treated eyes received the same amount of carrier vehicle. Cells were extracted from the eye-like structures and transplanted into the vitreous of damaged and control eyes. The host eyes were analyzed both qualitatively and quantitatively by immunohistochemistry 10 days or 8 weeks after transplantation. Results Cells from the ES cell-derived eye-like structures were integrated into the RGC layer, and differentiated into neurons when transplanted into control (non-NMDA-treated) adult eyes. However, they rarely expressed RGC markers. When they were transplanted into NMDA-treated eyes, the cells spread on the surface of the retina and covered a relatively large area of the host RGC layer that had been injured by the NMDA. The cells from the ES cell-derived eye cells frequently differentiated into cells expressing RGC-specific markers, and formed a new RGC layer. In addition, a small number of these ES cell-derived cells were observed to extend axon-like processes toward the optic disc of the host. However, visually evoked responses could not be recorded from the visual cortex. Discussion These findings suggest that ES cell-derived eye-like structures contain cells that can differentiate into RG-like cells and regenerate a new RGC layer. These cells also appeared to be integrated into the retina and extend axon-like processes toward the optic nerve head. This study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.We had full control of all primary data and we all agree to allow Graefe’s Archive for Clinical and Experimental Ophthalmology to review our data upon request.     

Elevated intraocular pressure, pigment dispersion and dark hypopyon in endogenous endophthalmitis from Listeria monocytogenes.

Listeria monocytogenes endophthalmitis occurred in an immunologically competent patient with no identifiable extraocular septic focus. The patient presented with a dark hypopyon and markedly elevated intraocular pressure, and the diagnosis was established by culture and histopathologic examination of ocular fluid. Four of the fourteen reported cases of Listeria monocytogenes endophthalmitis also presented with a dark hypopyon, and all cases had markedly elevated intraocular pressure. The presence of a dark hypopyon and elevated intraocular pressure may indicate endogenous intraocular infection with Listeria monocytogenes, even in an apparently healthy host.     

细胞外ATP诱导晶体上皮细胞的钙内流和钙动员

目的观察细胞外ＡＴＰ对离体晶体上皮细胞内钙离子的影响。方法钙指示剂ｆｌｕｏ－３着染离体晶体上皮细胞，用激光扫描共聚焦显微镜观察不同条件下细胞内荧光强度的变化。结果正常离体晶体上皮细胞对ＡＴＰ的刺激反应表现为双波型。对不同浓度的ＡＴＰ反应不同。乙二醇双（２－二氨基乙基）乙醚－四乙酸［ｅｔｈｙｌｅｎｅｇｌｙｃｏｌｂｉｓ（２－ａｍｉｎｏｅｔｈｙｌ）ｅｔｈｅｒｔｅｔｒａｃｅｔｉｃａｃｉｄ，ＥＧＴＡ］螯合细胞外钙后，ＡＴＰ刺激晶体上皮细胞出现单波型。结论ＡＴＰ能刺激离体晶体上皮细胞，引起细胞外钙内流和细胞内钙动员，晶体上皮细胞对ＡＴＰ的刺激反应有剂量依赖性，晶体上皮细胞膜中可能存在ＡＴＰ的特殊受体（Ｐ２－嘌呤受体）。