Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells

PURPOSE: Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells. METHODS: Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa). RESULTS: RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632. CONCLUSIONS: Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.     

Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells

Increased contractility of the actin cytoskeleton by phosphorylation of the regulatory myosin light chain (MLC) results in a loss of barrier integrity in corneal endothelial cells. This study has investigated the effect of extracellular ATP, which may influence both Ca2+ and cAMP signalling, on MLC phosphorylation and barrier integrity in cultured bovine corneal endothelial cells (BCEC) known to express A2B and P2Y purinergic receptors, and ecto-nucleotidases. Extracellular ATP (100 microM) promoted MLC dephosphorylation (pMLC=61.8% at 18 min; n=9). Pre-exposure to ARL-67156, an ecto-nucleotidase inhibitor, prevented ATP-induced dephosphorylation. Other P2Y agonists, UTP and ATPgammaS, also induced MLC dephosphorylation but to a lesser degree compared to ATP. Thrombin (2 U/ml), which activate Rho kinase through PAR-1 receptors in the endothelium, induced MLC phosphorylation (pMLC=129.2%; n=14). This phosphorylation was completely abolished by concomitant exposure to ATP. When cells were pretreated with adenosine (100 microM; A2B agonist) or forskolin (10 microM), thrombin-induced phosphorylation was suppressed. ATP also led to a significant increase in cAMP (> 3-fold compared to 10 microM adenosine). Thrombin-induced increase in trans-endothelial flux of horseradish peroxidase (44 kDa) and disruption of the cortical actin were suppressed by ATP. These findings indicate that in BCEC (1) ATP induces elevated cAMP through its metabolite adenosine leading to MLC dephosphorylation, (2) Stimulation of P2Y2 receptors also leads to activation of MLCP since UTP- and ATPgammaS caused MLC dephosphorylation, and (3) ATP is antagonistic to thrombin since the latter inhibits MLCP through increased activity of Rho kinase. These findings further emphasize the role of contractility of the actin cytoskeleton in regulating the barrier integrity of corneal endothelium.     

Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells

PURPOSE: Enhanced contractility of the actin cytoskeleton in trabecular meshwork (TM) cells is implicated in increased resistance to aqueous humor outflow. In this study, we have investigated effects of forskolin, which is known to elevate cAMP and also enhance aqueous humor outflow, on myosin light chain (MLC) phosphorylation, a biochemical marker of actin contractility. METHODS: Experiments were performed using cultured bovine TM cells. Phosphorylated MLC (pMLC), expressed as the % of untreated cells, was assessed by urea-glycerol gel electrophoresis and Western blotting. RhoA activity was determined by affinity precipitation of RhoA-GTP to RhoA binding domain of an effector of RhoA. Intracellular cAMP levels were measured by ELISA. RESULTS: Exposure to LPA (lysophosphatidic acid) led to increased MLC phosphorylation (LPA: pMLC=133%) and activation of RhoA. These responses of LPA were suppressed by co-treatment with forskolin (LPA+forskolin: pMLC=88%). Similarly, ET-1 and nocodazole-induced MLC phosphorylation (ET-1: pMLC=145%; nocodazole: pMLC=145%) as well as RhoA activation were suppressed by co-treatment with forskolin (ET-1+forskolin: pMLC=99%; nocodazole+forskolin: pMLC=107%). Exposure to forskolin alone led to MLC dephosphorylation (pMLC=68%). Forskolin alone led to a 4-fold increase in cAMP levels. This increase was not affected when co-treated with LPA or ET-1. CONCLUSIONS: Forskolin prevents MLC phosphorylation induced by LPA, ET-1, and nocodazole through inhibition of RhoA-Rho kinase axis. MLC dephosphorylation and consequent relaxation of actin cytoskeleton in TM cells presumably underlies the increased outflow facility reported in response to forskolin.     

Benzalkonium chloride induces dephosphorylation of Myosin light chain in cultured corneal epithelial cells

PURPOSE: Phosphorylation of myosin light chain (MLC) is essential for the contractility of the actin cytoskeleton, which regulates barrier integrity, adhesion, and migration. This study was conducted to investigate the effect of benzalkonium chloride (BAK), a preservative in topical ophthalmic formulations, on MLC phosphorylation in primary cultures of bovine corneal epithelial cells (BCECs). METHODS: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by Western blot analysis. Activation of RhoA, which inhibits MLC phosphatase through Rho kinase, was examined by immunoprecipitation. The release of adenosine triphosphate (ATP) was measured by the luciferase-luciferin bioluminescence technique. RESULTS: Positive expression of MLC kinase (MLCK) was found at the mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Exposure to BAK for 10 to 20 minutes at concentrations of 0.0005%, 0.001%, and 0.003% reduced MLC phosphorylation by more than 30%. In addition, BAK led to thinning of the cortical actin and a decrease in cell adhesion. However, RhoA activity was found to increase with BAK treatment. Similar to BAK, ATP-depletion (induced by both antimycin-A and hypoxia) led to MLC dephosphorylation. BAK exposure also showed acute ATP release. CONCLUSIONS: BAK induces acute ATP release and concomitant MLC dephosphorylation in bovine corneal epithelial cells. The dephosphorylation, presumably due to ATP loss, is indicative of a loss of contractility of the actin cytoskeleton that could affect cellular functions contributing to the maintenance of epithelial barrier integrity.     

Characterization of adenosine receptors in bovine corneal endothelium

Previous studies indicated that adenosine can increase [cAMP](i) and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP](i), [Ca(2+)](i) and effects on Cl(-) permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agents on adenosine stimulated Cl(-) transport, [cAMP](i) and [Ca(2+)](i). RT-PCR indicated the presence of A(1) and A(2b) adenosine receptors, while A(2a) and A(3) were negative. Western blot (WB) confirmed the presence of A(2b) ( approximately 50 kDa) and A(1) ( approximately 40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP](i) by 2.7-fold over control and this was inhibited 66% by 10 microm alloxazine, a specific A(2b) blocker. A(1) activation with 1 micromN(6)-CPA (a specific A(1) agonist) or 100 nm adenosine decreased [cAMP](i) by 23 and 6%, respectively. Adenosine had no effect on [Ca(2+)](i) mobilization. Indirect immunofluorescence localized A(2b) receptors to the lateral membrane and A(1) to the apical surface in cultured BCE. Adenosine significantly increased apical Cl(-) permeability by 2.2 times and this effect was nearly abolished by DMPX (10 microm), a general A(2) blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A(1) and A(2b) adenosine receptors. A(1), preferentially activated at <1 microm adenosine, acts to decrease [cAMP](i) and A(2b), activated at >1 microm adenosine, increase [cAMP](i).     

压力对体外培养的牛角膜内皮细胞的影响

目的观察压力对体外培养的牛角膜内皮细胞形态结构及细胞活力的影响。方法对体外培养的牛眼角膜内皮细胞施以2.67kPa、5.33kPa及8.00kPa的压力各6、12、24、36、48h,以不加压组作为对照组。用倒置相差显微镜及电镜观察细胞形态结构,以台盼蓝染色观察比较48h时各组细胞活力的差别,用单因素方差分析比较不同压力下阳性细胞率。结果当作用于细胞的压力为2.67kPa(1kPa=7.5mmHg),作用时间为6、12、24、36、48h时,与对照组比较,细胞形态结构均无明显变化;48h时台盼蓝计数与对照组比较差异无统计学意义(P=0.776)。当压力为5.33kPa,作用时间为24h时,细胞形态结构出现轻度的损伤,在36、48h时加重;48h时台盼蓝计数与对照组比较,差异有统计学意义(P=0.00)。当压力为8.00kPa时,6h时即出现明显细胞形态结构损害,随着压力和作用时间的进一步增加,细胞的损伤程度也进一步加重;48h时台盼蓝计数与对照组比较差异有统计学意义(P=0.000)。结论牛角膜内皮细胞在加压48h时只能承受2.67kPa的压力,当压力为5.33kPa及8.00kPa,施压48h时,将造成细胞...     

Effects of prostaglandin F2alpha, latanoprost and carbachol on phosphoinositide turnover, MAP kinases, myosin light chain phosphorylation and contraction and functional existence and expression of FP receptors in bovine iris sphincter

A potential role for myosin light chain kinase (MLCK) in regulating intraocular pressure and outflow function has recently been reported in living monkey eye and rabbit eye. There is little information about the effects of the ocular hypotensive agents, prostaglandin F2alpha (PGF2alpha) and latanoprost on this signaling pathway in ocular tissues. The aim of this study was to determine the agonist activity of PGF2alpha, latanoprost and carbachol (CCh) on the MLCK pathway in isolated bovine iris sphincter and furthermore to investigate the existence of the FP receptor in this tissue. In the present studies on the MLCK pathway four signal transduction mechanism assays were employed, phosphoinositide (PI) turnover, p42/p44 MAP kinase phosphorylation and activation, MLC phosphorylation and contraction. In the studies on the existence of the FP receptor in the bovine iris sphincter, the pharmacology and expression of the FP receptor protein, using a polyclonal anti-FP-receptor antibody and Western blot analysis, were determined. The data obtained on the MLCK pathway showed that the three agonists stimulated the biochemical and pharmacological responses in a concentration and time-dependent manner and that the order of potency and efficacy is PGF2alpha>latanoprost>CCh. The EC50 values in the PI turnover, MAP kinase phosphorylation, MLC phosphorylation and contraction assays were for PGF2alpha: 9, 42, 200 and 140 nM, respectively, for latanoprost: 13, 59, 250 and 828 nM, respectively, and for CCh: 22, 200, 630 and 910 nM, respectively. Wortmannin, a selective inhibitor of MLCK, dose-dependently inhibited MLC phosphorylation and contraction induced by PGF2alpha, demonstrating a close relationship between activation of the MLCK pathway and contraction. The pharmacological studies showed that in the concentration range of 1 nM to 10 microM, the FP-receptor agonists caused concentration-response curves with the following order of potencies: 17-phenyl trinor PGF2alpha (bimatoprost acid)>PGF2alpha>cloprostenol>latanoprost>latanoprost acid>bimatoprost amide>fluprostenol. Immunoblot analysis of the FP receptor demonstrated expression of the prostaglandin FP receptor protein in this smooth muscle. These results clearly indicate that the MLCK signaling pathway is involved in the FP-receptor function of the bovine iris sphincter and furthermore demonstrate that functional FP receptors exist and are expressed in this tissue.     

Gap junctional intercellular communication in bovine corneal endothelial cells

Gap junctions and/or paracrine mediators, such as ATP, mediate intercellular communication (IC) in non-excitable cells. This study investigates the contribution of gap junctions toward IC during propagation of Ca(2+) waves in cultured bovine corneal endothelial cells (BCEC) elicited by applying a point mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were visualized using the fluorescent dye Fluo-4. The area reached by the Ca(2+) wave, called the active area (AA), was determined as a measure of efficacy of IC. RT-PCR and Western blotting showed expression of Cx43, a major form of connexin, in BCEC. In scrape-loading (using lucifer yellow) and fluorescence recovery after photobleaching (FRAP; using carboxyfluorescein) protocols, significant dye transfer of the hydrophilic dyes was evident indicating functional gap junctional IC (GJIC) in BCEC. Gap27 (300 microM), a connexin mimetic peptide that blocks gap junctions formed by Cx43, reduced the fluorescence recovery in FRAP experiments by 19%. Gap27 also reduced the active area of the Ca(2+) wave induced by point mechanical stimulation from 73,689 microm(2) to 26,936 microm(2), implying that GJIC contribution to the spread of the wave is at least approximately 63%. Inhibitors of ATP-mediated paracrine IC (PIC), such as a combination of apyrase VI and apyrase VII (5U/ml each; exogenous ATPases), suramin (200 microM; P2Y antagonist), or Gap26 (300 microM; blocker of Cx43 hemichannels) reduced the active area by 91%, 67%, and 55%, respectively. Therefore, estimating the contribution of GJIC from the residual active area after PIC inhibition appears to suggest that GJIC contributes no more than approximately 9% towards the active area of the Ca(2+) wave. Gap27 did not affect the enhancement in active area induced by ARL-67156 (200 microM, ectonucleotidase inhibitor), ATP release induced by point mechanical stimulation, and zero [Ca(2+)](o)-induced lucifer yellow uptake, indicating that the peptide has no influence on PIC. Exposure to Gap27 in the presence of PIC inhibitors led to a significant further inhibition of the Ca(2+) wave. The finding that the residual active area after inhibition of PIC by apyrases was much smaller than the reduction of the active area by Gap27, provides evidence for interaction between GJIC and PIC. These findings together suggest that functional gap junctions are present in BCEC, that both GJIC and PIC contribute significantly to IC, and that the two pathways interact.     

Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells

PURPOSE: To investigate histamine-induced changes in the phosphorylation of myosin light chain (MLC) and its influence on the barrier integrity of corneal endothelial cells through altered contractility of the actin cytoskeleton. METHODS: Experiments were performed in cultured bovine corneal endothelial cells (BCECs). Phosphorylation of MLC, which increases contractility of the actin cytoskeleton through actomyosin interaction, was assessed by urea-glycerol gel electrophoresis and Western blot analysis. Immunocytochemistry was used to locate phosphorylated MLC in relation to tight junctions. Phosphorylation of the 17-kDa PKC-potentiated inhibitory protein of type 1 protein phosphatase (CPI-17), which inhibits MLC phosphatase, was studied using Western blot analysis. The cortical actin cytoskeleton was visualized by staining with Texas-red phalloidin. Barrier integrity was determined by quantifying horseradish peroxidase (HRP; 44 kDa) flux across cells grown on porous filters. RESULTS: RT-PCR and Western blot analysis confirmed the expression of Galphaq/11-coupled H1 receptors in BCECs. Exposure to histamine (100 microM; 10 minutes) led to phosphorylation of MLC (134% relative to untreated cells) and of CPI-17. Histamine also increased the flux of HRP by sevenfold and disrupted the assembly of the dense cortical actin found in resting cells. PKC activation by phorbol 12-myristate 13-acetate (PMA; 100 nM; 30 minutes) caused phosphorylation of both MLC and CPI-17. The histamine-induced MLC phosphorylation was reduced by pre-exposure to either ML-7 (50 microM), an MLCK (MLC kinase) inhibitor, or chelerythrine (10 microM), an inhibitor of PKC. Cotreatment with agents that elevate cAMP in BCECs prevented the histamine-induced MLC phosphorylation and the disruption of the actin cytoskeleton, and increased HRP flux. Phosphorylated MLC in response to histamine or PMA was found in a punctate form in close proximity to ZO-1, a marker of the tight junctional complex. CONCLUSIONS: Histamine induces MLC phosphorylation by activating MLCK and partly inhibiting MLC phosphatase. The latter is facilitated by the phosphorylation of CPI-17. Localization of phosphorylated MLC in proximity to ZO-1 suggests increased contractility of the cortical actin at the tight junctional complex. This contractility oppose the tethering forces and lead to a breakdown of the barrier integrity. Last, elevated cAMP prevents histamine-induced loss of the barrier integrity, not only by blocking inactivation of MLC phosphatase but also by inactivating MLCK.     

体外培养中牛角膜内皮细胞和虹膜色素上皮细胞的FasL表达

目的：明确FasL在体外培养的角膜内皮细胞和虹膜色素上皮细胞（IPE）中FasL的表达情况及其意义,材料和方法：采用酶辅助的显微分离法分离新生牛眼IPE细胞,酶消化法分离角膜内皮细胞,进行体外细胞培养,抗FasL染色呈阳性;IPE细胞及对照组均为阴性。结论：在前房免疫赦免的形成中,角膜内皮细胞主要通过表达和分泌FasL发挥作用,而PE细胞则主要通过表达和分泌有免疫抑制作用的细胞因子以及通过一种非FasL－FasL诱导细胞凋亡的通路来阻止T细胞活化、增殖。     

人脐静脉血管内皮细胞在体移植替代猴角膜内皮细胞的形态学分析

目的　使用去除后弹力层的恒河猴自体角膜为细胞载体,将培养的人脐静脉血管内皮细胞（ｈｕｍａｎｕｍｂｉｌｉｃａｌｖｅｉｎｅｎ-ｄｏｔｈｅｌｉａｌｃｅｌｌｓ,ＨＵＶＥＣ）种植到角膜内表面,观察ＨＵＶＥＣ替代恒河猴角膜内皮细胞的功能情况以及ＨＵＶＥＣ在恒河猴眼内生长的情况。方法　取恒河猴６只随机分为３组:实验组（３只）、实验对照组（２只）、空白对照组（１只）。实验组:用离心沉淀法将培养的ＨＵＶＥＣ移植到去除后弹力层的恒河猴自体角膜内表面,之后将自体角膜缝回植床;实验对照组:将撕除部分后弹力层的术眼角膜植片原位缝回植床;空白对照组:取下术眼角膜植片不做任何处理原位缝回植床。术后观察各组角膜植片透明情况;实验组及实验对照组于术后３０ｄ及６０ｄ、空白对照组于术后６０ｄ行术眼摘除,标本做病理切片、ＣＤ３４免疫组化及扫描电镜,观察房角结构及ＨＵＶＥＣ在角膜植片内表面形态分布。结果　实验组角膜植片维持了一定的厚度和透明性,而实验对照组角膜植片发生严重大泡性改变。病理切片示实验组角膜内表面可见一层细胞生长,ＣＤ３４染色阳性,提示为血管内皮细胞;实验对照组角膜内表面未见任何细胞生长;空白对照组角膜植片保留完整后弹力层及内皮细胞层。扫描电镜示实验组有ＨＵＶＥＣ单层在角膜内表面生长但有大量白细胞聚集及少量细胞碎片嵌顿于小梁网;实验对照组角膜内表面残留胶原纤维样物质,无细胞生长;空白对照组见完整六边形角膜内皮细胞层。结论　ＨＵＶＥＣ能够在撕除后弹力层的恒河猴角膜内表面生长并发挥一定的屏障作用,维持角膜的厚度和透明性,但会产生较重的排斥反应。     

超声乳化白内障手术对年龄相关性白内障患者角膜内皮细胞的影响

目的:探讨年龄相关性白内障患者超声乳化白内障吸除术后角膜内皮细胞的变化及角膜内皮形态结构的变化。方法:选择在我院行超声乳化白内障吸除折叠人工晶状体植入术的年龄相关性白内障患者79例91眼,按晶状体核密度分为两组,其中Ⅲ级以下核(包括Ⅲ级核)43眼,Ⅲ级以上核48眼。在术前及术后3d时,应用非接触式角膜内皮显微镜观察其中央角膜的内皮细胞密度(CD值)及角膜内皮形态结构的变化。结果:CD值较术前均有不同程度的下降,Ⅲ级核以下(包括III级核)CD值下降6%,Ⅲ级核以上CD值下降13%。CD值随着晶状体核硬度上升,呈现下降趋势。术后细胞形态也发生改变,角膜内皮细胞变异系数(CV)值则增高,Ⅲ级核以下(包括Ⅲ级核)内皮细胞面积平均值及CV值变化均小于III级核以上。结论:老年患者角膜内皮对白内障超声乳化手术产生的损伤敏感,术前应将角膜内皮细胞镜作为常规检查,术中术者可根据患者核硬度随时调整能量和负压的参数组合,尽可能降低超声能量,缩短超声时间,最大限度地保护角膜内皮的正常功能,以提高手术成功率。     

Microtubule disassembly breaks down the barrier integrity of corneal endothelium

Increased contractility of the peri-junctional actomyosin ring (PAMR) breaks down the barrier integrity of corneal endothelium. This study has examined the effects of microtubule disassembly on Myosin Light Chain (MLC) phosphorylation, a biochemical marker of actomyosin contraction, and barrier integrity in monolayers of cultured bovine corneal endothelial cells (BCEC). Exposure to nocodazole, which readily induced microtubule disassembly, led to disruption of the characteristically dense assembly of cortical actin cytoskeleton at the apical junctional complex (i.e., PAMR) and dispersion of ZO-1 from its normal locus. Nocodazole also led to an increase in phosphorylation of MLC. Concomitant with these changes, nocodazole caused an increase in permeability to HRP and FITC dextran (10 kDa) and a decrease in trans-endothelial electrical resistance (TER). Y-27632 (a Rho kinase inhibitor) and forskolin (known to inhibit activation of RhoA through direct elevation of cAMP) opposed the nocodazole-induced MLC phosphorylation, decrease in TER, and dispersion of ZO-1. Thrombin, which breaks down the barrier integrity of BCEC monolayers, also induced microtubule disassembly and MLC phosphorylation. Pre-treatment with paclitaxel to stabilize microtubules opposed the thrombin effects. These results suggest that microtubule disassembly breaks down the barrier integrity of BCEC through activation of RhoA and subsequent disruption of the PAMR. The thrombin effect also highlights that signaling downstream of GPCRs can also influence the organization of microtubules.     

Research progress on the negative factors of corneal endothelial cells proliferation

The human corneal endothelium forms a boundary layer between anterior chamber and corneal stoma. The corneal endothelial cells are responsible for maintaining cornea transparency, which is very vital for our visual acuity, via its pump and barrier functions. The adult corneal endothelial cells in vivo lack proliferation in response to the cell loss caused by outer damages and diseases. As a result, in order to compensate for cell loss, corneal endothelial cells migrate and enlarge while not via dividing to increase the endothelial cell density. Therefore, it is not capable for corneal endothelium to restore the corneal clarity. Some researches have proved that in vitro the corneal endothelial maintained proliferation ability. This review describes the current research progress regarding the negative factors that inhibit proliferation of the corneal endothelial cells. This review will mainly present several genes and proteins that inhibit the proliferation of the corneal endothelial cells, of course including some other factors like enzymes and position.     

Intra-cellular potential of rabbit corneal endothelial cells.

A stable potential difference across corneal endothelial cell membranes (?45±2 mV, 301 impalements) was measured by minimizing mechanical disturbances and penetrating the cells with microelectrodes of 50–150 MΩ resistance. A microelectrode tip could be kept in a cell for as long as 40 min. The highest stable potential difference obtained was ?75 mV. In addition, the input resistance of the cells was 29 MΩ in the average (20 impalements; range 3·5 to 93 MΩ). Treatment with 10^?4m ouabain and high [K⁺] in the external medium decreased the intra-cellular potential difference.     

白内障超声乳化对糖尿病患者角膜内皮的影响

目的:探讨白内障超声乳化对糖尿病患者角膜内皮细胞的影响。方法:糖尿病组:白内障合并糖尿病患者50例(58眼);对照组:白内障非糖尿病患者64例(70眼)。应用角膜内皮显微镜于术前及术后1d;1wk;1,3mo测量角膜内皮细胞密度、六角形细胞比率,分析结果。结果:与术前相比,糖尿病组术后角膜内皮细胞密度、六角形细胞比率均显著降低(P<0.05)。糖尿病组和对照组内皮细胞密度、六角形细胞比率术前差异无统计学意义(P>0.05),但在术后1d;1wk;1,3mo,糖尿病组患者角膜内皮细胞丢失均明显高于对照组(P<0.05),糖尿病组患者角膜内皮细胞中六角形细胞比率均明显低于对照组(P<0.05)。结论:白内障手术对角膜内皮细胞具有一定的损伤性,糖尿病患者较非糖尿病者角膜内皮易损伤。     

小梁切除术中透明质酸钠对角膜内皮细胞的影响

目的探讨小梁切除术中应用透明质酸钠对角膜内皮细胞数量和形态的影响。方法接受小梁切除术的原发性急性闭角型青光眼75例（75眼）,随机分为两组：治疗组37例（37眼）,前房内及巩膜瓣下注入1%透明质酸钠0.1ml并保留;对照组38例（38眼）,则注入平衡盐溶液（BSS）0.1ml。采用非接触型角膜内皮显微镜分别于术前及术后第1周、第3个月进行角膜内皮细胞检查并分析其数量和形态参数的变化,同时观察术后眼压、前房形成情况、滤过泡及并发症。结果治疗组术前、术后第1周及第3个月角膜内皮细胞密度分别为（2183±325）个/mm2、（2096±332）个/mm2、（2112±261）个/mm2;平均细胞面积分别为（462.7±42.8）μm2、（422.9±53.1）μm2、（430.8±55.2）μm2;细胞面积变异系数分别为（47.3±15.7）%、（48.1±12.5）%、（42.6±10.9）%;六角型细胞百分率分别为（52.3±14.8）%、（51.6±13.3）%、（55.7±11.2）%;术后第1周及第3个月各参数与术前比较,差异均无统计学意义（P〉0.05）。对照组术前、术后第1周及第3个月角膜内皮细胞密度分别为（2098±314）个/mm2、（1631±388）个/mm2、（1855±402）个/mm2;平均细胞面积分别为（462.7±42.8）μm2、（562.7±73.8）μm2、（536.6±65.1）μm2;细胞面积变异系数分别为（49.5±14.3）%、（59.2±12.6）%、（58.7±12.3）%;六角型细胞百分率分别为（53.2±16.1）%、（40.4±13.2）%、（42.6±11.8）%,术后第1周及第3个月的各参数与术前比较,差异均有统计学意义（P〈0.05）。术后第1周及第3个月,治疗组的角膜内皮细胞损失率分别为4.0%及3.3%,均低于对照组的17.5%及11.6%（P〈0.05）。治疗组术后浅前房发生的眼数、程度均低于对照组（P〈0.05）。术后第1周及第3个月,不同前房深度组的角膜内皮损失率之间均有统计学差异（P〈0.01）。术后前房深度越浅,角膜内皮细胞的损失率越大,两者有     

Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells

We have previously demonstrated the presence of a Na(+)-K(+)-2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive (86)Rubidium ((86)Rb) uptake in (86)Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k(1/2)) of 21.1 and 1.33 mM, respectively. Extracellular Cl- is also required for cotransport activation, but shows higher order kinetics; the k(1/2) for Cl- is 28.1 mM and the Hill coefficient 2.1. HCO(3)(-) exerts a modulating effect on cotransporter activity; at 0 HCO(3)(-) the bumetanide-sensitive K(+) uptake is reduced by 30% compared to that at 26 mm HCO(3)(-). Manipulations of the intracellular [Cl-] by preincubation in Cl- -free solution or inhibition of Cl- efflux resulted in increased uptake at low [Cl-](i) and decreased uptake at high [Cl-](i). To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k(1/2)=21 nM) and okadaic acid (k(1/2)=915 nM) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Ca(i)(2+) by preincubation in Ca2+ -free medium as well as by exposure to W-7, an inhibitor of the binding of Ca(2+) to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+ -calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+ -calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin-myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Cl- are requirements for the function of the CBCEC Na(+)-K(+)-2Cl(-) cotransporter, while intracellular Cl- and extracellular HCO(3)(-) modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.     

Effect of hydrocortisone on corneal endothelial cells in vitro

The effect of hydrocortisone on the growth potential of human and baboon corneal endothelial cells in tissue culture was evaluated. Hydrocortisone-21-sodium succinate was incorporated into growth medium at 10(-3) to 10(-7) M concentrations in a standard tissue culture medium. Inclusion of hydrocortisone into growth medium at higher concentrations (e.g. 10(-4) and 10(-3) M) resulted in a significantly reduced cell growth and a decreased level of DNA synthesis. Higher concentrations of hydrocortisone induced morphological alterations in the corneal endothelial cells in vitro. The incorporation of 5% dextran into the growth medium failed to alter the negative hydrocortisone effects. These experiments suggest the potential adverse effects of hydrocortisone for the corneal endothelium in vitro when used at higher concentrations.     

角膜内皮细胞体外培养的研究进展

角膜内皮是角膜的重要组成部分,由单层的角膜内皮细胞(corneal endothelial cell,CEC)组成,在维持角膜正常的透明度方面发挥着重要的作用.CEC在出生后即丧失增殖能力,一旦受损或丢失会导致不可逆性角膜疾病.但是,大量的研究表明CEC可以在体外增殖,这为体外培养人CEC用于角膜疾病的治疗提供了可行性,但至今仍没有建立一套标准的CEC体外培养方法.本文将归纳总结近年来CEC体外培养方法.以利于今后优化CEC体外培养体系.