Sodium ion uptake into isolated plasma membrane vesicles: indirect effects of other ions.
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Vesicles derived from plasma membrane of corneal endothelium were agitated to their minimum size distribution. When isotonic salt solutions surrounding the vesicles were changed there were alterations to the vesicle size distribution: the modal point of the logarithmic distribution did not change but the log variance did, indicating that substantial fission and fusion of vesicles occurred depending upon the nature of the surrounding solute. Orientation and total membrane area was conserved in the transformed population of vesicles. Although the ions added to the external isotonic salt solutions in the present series of experiments have no direct effect upon sodium membrane transporters in these membranes, kinetics of sodium accumulation into the vesicles were affected in a way that correlated with changes to the vesicle size distribution. Early-saturating (<1 min) intravesicular concentrations of sodium corresponded with apparently stable populations. Late-saturating (>1 min) intravesicular concentrations of sodium corresponded with significant vesicle distribution shifts and included a few seconds of delay. During the linear accumulation phase, both populations showed similar magnitudes of sodium transport. The significance of these data is discussed. (+info)
Estimation of corneal endothelial pump function in long-term contact lens wearers.
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PURPOSE: To study the effects of long-term contact lens wear on morphologic and physiologic properties of corneal endothelial cells. METHODS: The endothelial permeability to fluorescein and the rate of corneal deswelling from hypoxia-induced edema were measured in 20 long-term (mean, 17+/-9 years; range, 5-33 years) contact lens wearers and 20 age-matched control subjects. From these data, the relative endothelial pump rate in each subject was estimated, based on the pump-leak hypothesis of corneal hydration control. Corneal autofluorescence and the aqueous humor flow rate were determined by fluorescein fluorophotometry. Images of corneal endothelial cells were recorded by using specular microscopy, and morphologic indices (cell density, coefficient of variation of cell area, percentage of hexagonal cells, and skewness) were determined. RESULTS: No statistically significant differences were found between the contact lens and control groups in endothelial permeability, corneal deswelling, relative endothelial pump rate ([mean +/- SD] 1.07+/-0.33 relative pump units versus 1.01+/-0.25 relative pump units; contact lens versus control; P = 0.57), and endothelial cell density. Contact lens wearers had a significantly higher aqueous humor flow rate (3.57+/-1.03 microl/min versus 2.77+/-0.51 microl/min; P = 0.005), coefficient of variation of cell area (0.35+/-0.09 versus 0.28+/-0.04; P = 0.006), and corneal autofluorescence (3.1+/-0.6 ng/ml versus 2.3+/-0.3 ng/ml fluorescein equivalents; P < 0.001) than did non-contact lens wearers. CONCLUSIONS: Despite the known effects of long-term contact lens wear on corneal endothelial morphometry, no effect on endothelial function was found. (+info)
Central corneal endothelial guttae and age-related macular degeneration: is there an association?
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The similarities between the corneal endothelium and retinal pigment epithelium in terms of their embryology, barrier function and predilection to age-related degeneration prompted this investigation into a possible association between central corneal guttae (CCG) and age-related macular degeneration (ARMD). 50 patients with clinically significant CCG were prospectively evaluated for the presence of ARMD. 51 age-matched patients attending for unrelated ailments who did not have CCG were also evaluated for the presence of drusen and other signs of ARMD. Of the 50 patients with CCG, 23 had bilateral ARMD and 4 had unilateral ARMD. In the control group, 9 patients had bilateral and 4 had unilateral ARMD. There was significant difference in the prevalence of ARMD between patients with CCG and those with no CCG (p = 0.017 and p < 0.001 for right and left eyes respectively). We found an association between CCG and ARMD. The presence of CCG in a patient may imply increased risk for the presence of ARMD. In a patient with CCG requiring cataract or corneal surgery, the successful outcome may be compromised by the presence of ARMD. (+info)
Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells.
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PURPOSE: The loss of corneal endothelial cells associated with aging and possibly other causes has been speculated to be related to exposure to reactive oxygen species (ROS). The current study was conducted to investigate, by use of photosensitizers, the underlying mechanisms involved in the death of bovine corneal endothelial cells (BCENs) caused by ROS. METHODS: BCEN cells in primary culture were treated with a photosensitizer (riboflavin or rose bengal) with light exposure. The patterns of cell damage and death were assessed using an acridine orange-ethidium bromide differential staining method, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. Antioxidants, including catalase, L-histidine, salicylic acid, and superoxide dismutase, were used to determine the types of ROS involved. Activation of nuclear factor (NF)-kappaB was examined by fluorescent immunocytochemistry with anti-p65 antibody. RESULTS: Light-irradiated riboflavin or rose bengal resulted in a significant decrease in viability of BCEN cells. Chromosomal condensation and fragmentation were observed in apoptotic cells, and membrane lysis and damage of cell ultrastructures were observed in necrotic cells. Riboflavin induced apoptosis at 30 minutes and thereafter and induced necrosis after 2 hours. Rose bengal was shown to cause similar effects within half the time required for the effects of riboflavin. Catalase and salicylic acid were found to provide protection for BCENs from cytotoxic effects of riboflavin, and L-histidine was found to protect BCENs from cytotoxicity induced by rose bengal. Kinetic studies using immunocytochemistry showed that NF-kappaB was translocated into the nucleus within 15 minutes and 30 minutes after treatment with rose bengal and riboflavin, respectively. CONCLUSIONS: The cytotoxic effects of photo-irradiated riboflavin and rose bengal are shown to be mediated by two distinct but parallel pathways, one leading to apoptosis and the other to necrosis. Possible involvement of NF-kappaB in cell death is suggested. These findings provide potential leads for future investigation into the molecular mechanisms of loss of corneal endothelial cells related to aging, oxidative stress, and possibly other similar causes. (+info)
Results of small incision extracapsular cataract surgery using the anterior chamber maintainer without viscoelastic.
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AIMS: To assess the efficacy of extracapsular cataract surgery using the anterior chamber maintainer (ACM) without the use of viscoelastic. To compare the effects of this surgical technique on non-diabetic and diabetic patients. METHODS: A prospective single armed clinical trial of 46 eyes in 46 patients undergoing cataract surgery using the ACM without viscoelastic. Patients were assessed preoperatively and at 3 weeks, 3 months, and 12 months postoperatively. The main outcome variables included visual acuity, surgically induced astigmatic change (SIAC), changes in endothelial cell density (ECD), and morphology affecting the central and superior regions of the cornea. RESULTS: Postoperatively, 56% and 70% of patients had unaided visual acuities of 6/12 or better at 3 weeks and 3 months respectively. Even after excluding those patients with pre-existing maculopathy (including diabetic maculopathy), there remains a significant difference between the non-diabetic and diabetic groups in terms of the proportion of patients attaining an unaided visual acuity of 6/12 or better at both 3 weeks (p = 0.003) and 3 months (p = 0.001). Three months postoperatively, the SIAC based upon the keratometric and refractive data was 1.1 dioptres (D) and 1.3 D respectively. There was no statistically significant difference in the SIAC when the non-diabetic and diabetic groups were compared. The mean central and superior endothelial cell losses at 3 months postoperatively were 16% and 22% respectively and at 12 months postoperatively were 20% and 25% respectively. The diabetic group demonstrated greater endothelial cell losses and a more marked and protracted deviation of endothelial cell morphology from normality when compared with the non-diabetic group; however, the differences did not reach statistical significance. CONCLUSIONS: The efficacy of small incision cataract surgery using the ACM in terms of visual outcome and induced astigmatism is comparable with the results obtained using other techniques that utilise a similar size of incision. However, in view of the magnitude and range of the endothelial cell losses associated with this technique the concurrent use of viscoelastic is suggested. There does not appear to be a statistically or clinically significant difference between non-diabetic and diabetic patients in terms of the magnitude of the endothelial cell losses or in the wound healing response in the 12 months after cataract surgery using the ACM. (+info)
Molecular identification and immunolocalization of the water channel protein aquaporin 1 in CBCECs.
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PURPOSE: Water channel proteins are important pathways for water movements across cell membranes, including those in the corneal endothelium that contribute to the fluid transport mechanism essential in maintaining corneal transparency. This study was conducted to identify and locate the water channel protein(s) in cultured bovine corneal endothelial cells (CBCECs). METHODS: Poly(A)+ RNA was isolated from CBCECs, and MMLV reverse transcriptase and random hexamer primers were used to generate a cDNA pool by reverse transcription-polymerase chain reaction (RT-PCR). Two specific degenerate primers were synthesized based on consensus sequences from the major intrinsic lens protein superfamily; a "touchdown" PCR protocol accommodated the degeneracy. Immunolocalization was performed by incubating sections of CBCECs with an antibody against human aquaporin 1 (AQP1). Cryosections (0.85 microm) of CBCECs were used for light microscopy, and 800-A ultrathin cryosections were used for electron microscopy (EM). RESULTS: A 372-bp fragment was isolated. Its encoded amino acid sequence was 100% identical with that of bovine AQP1 (AQP2_bovin). CBCECs reacted strongly with the anti-AQP1 antibody, and the labeling was selectively localized to the plasma membrane by light microscopy. Subcellular localization by EM revealed immunoreactivity with the inner leaflets of the plasma membrane. CONCLUSIONS: The identity of the aquaporin, its abundance, and its membrane location suggest that it is a major pathway for fluid flow across endothelial cell membranes. This is consistent with transcellular endothelial fluid transport. (+info)
Clinical estimation of corneal endothelial pump function.
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PURPOSE: To develop a technique to estimate the corneal endothelial pump rate in human subjects. METHODS: Corneal hydration control is thought to be maintained by a pump-leak mechanism whereby the leak of solutes and fluid across the endothelial barrier into the stroma is, in the steady state, exactly balanced by the pumping of solutes and passive fluid transfer across the endothelium to the aqueous humor. Overall corneal hydration control can be measured from the rate at which the swollen cornea thins (deswells), and a measure of the leak can be obtained simultaneously from the endothelial permeability to fluorescein. From the pump-leak hypothesis, the deswelling rate is directly proportional to the pump rate and inversely proportional to the leak rate. The relative endothelial pump rate can be estimated as the product of the normalized deswelling rate and the normalized endothelial permeability. This procedure was used to obtain the relative endothelial pump rate in 41 patients with diabetes mellitus, 12 patients with long-term corneal transplants, 20 long-term wearers of contact lenses, and 19 normal volunteer subjects after the short-term administration of topical dorzolamide. RESULTS: The relative endothelial pump rate did not differ significantly from that of control subjects in diabetics, in contact lens wearers, and after dorzolamide administration, but was markedly decreased in the patients with corneal transplants, despite a reduction in permeability (reduced leak). CONCLUSIONS: This method allows the estimation of both the barrier and pump arms of corneal endothelial function and should be useful in the investigation of causes and mechanisms of functional endothelial insufficiency. (+info)
Hsp47-dependent and -independent intracellular trafficking of type I collagen in corneal endothelial cells.
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PURPOSE: Type I collagen is post-translationally regulated in corneal endothelial cells (CEC): CEC synthesize procollagen I and degrade it intracellularly. We investigated whether there is a Hsp47-independent pathway during intracellular trafficking of procollagen I. METHODS: Specific inhibitors were used to block intracellular transport of procollagen I and Hsp47. Immunocytochemical analysis was performed to determine the intracellular localization of the proteins of interest. RESULTS: When cells were treated with alpha,alpha'-dipyridyl, this specific inhibitor for collagen promoted retention in the endoplasmic reticulum (ER) of some of the underhydroxylated procollagen I, which was colocalized with Hsp47 in CEC. At the same time, another fraction of the alpha,alpha'-dipyridyl-induced underhydroxylated procollagen I was not located in the ER. When CEC were treated with brefeldin A, procollagen I and Hsp47 demonstrated a high degree of colocalization at the ER, whereas the inhibitor had less of an effect on the compartmentalization of procollagen I and prolyl 4-hydroxylase. When CEC were treated with either monensin or bafilomycin A1, procollagen I and Hsp47 were not colocalized: procollagen I was mostly localized at the Golgi area, while Hsp47 predominantly showed ER distribution. When colocalization of procollagen I and prolyl 4-hydroxylase was examined, a major population of procollagen I was not colocalized with prolyl 4-hydroxylase in the ER. CONCLUSIONS: These results indicate that some procollagen I and Hsp47 travel together from the ER to the cis-Golgi compartment and that a major population of procollagen I that may not be properly hydroxylated may be destroyed intracellularly via the Hsp47-independent pathway in CEC. (+info)