TNF-R1 and FADD mediate UVB-Induced activation of K+ channels in corneal epithelial cells.

The goal of this study was to elucidate the role of Fas, TNF-R1, FADD and cytochrome c in UVB-induced K+ channel activation, an early step in UVB-induced apoptosis, in human corneal limbal epithelial (HCLE) cells. HCLE cells were treated with Fas, TNF-R1 or FADD siRNA and exposed to 80 or 150 mJ/cm2 UVB. K+ channel activation and loss of intracellular K+ were measured using whole-cell patch-clamp recording and ion chromatography, respectively. Cytochrome c was measured with an ELISA kit. Cells in which Fas was knocked down exhibited identical UVB-induced K+ channel activation and loss of intracellular K+ to control cells. Cells in which TNF-R1 or FADD were knocked down demonstrated reduced K+ channel activation and decreased loss of intracellular K+ following UVB, relative to control cells. Application of TNF-α, the natural ligand of TNF-R1, to HCLE cells induced K+ channel activation and loss of intracellular K+. Cytochrome c was translocated to the cytosol by 2 h after exposure to 150 mJ/cm2 UVB. However, there was no release by 10 min post-UVB. The data suggest that UVB activates TNF-R1, which in turn may activate K+ channels via FADD. This conclusion is supported by the observation that TNF-α also causes loss of intracellular K+. This signaling pathway appears to be integral to UVB-induced K+ efflux, since knockdown of TNF-R1 or FADD inhibits the UVB-induced K+ efflux. The lack of rapid cytochrome c translocation indicates cytochrome c does not play a role in UVB-induced K+ channel activation.

Apoptosis of Corneal Epithelial Cells Caused by Ultraviolet B-induced Loss of K(+) is Inhibited by Ba(2.).

UVB exposure at ambient outdoor levels triggers rapid K(+) loss and apoptosis in human corneal limbal epithelial (HCLE) cells cultured in medium containing 5.5 mM K(+), but considerably less apoptosis occurs when the medium contains the high K(+) concentration that is present in tears (25 mM). Since Ba(2+) blocks several K(+) channels, we tested whether Ba(2+)-sensitive K(+) channels are responsible for some or all of the UVB-activated K(+) loss and subsequent activation of the caspase cascade and apoptosis. Corneal epithelial cells in culture were exposed to UVB at 80 or 150 mJ/cm(2). Patch-clamp recording was used to measure UVB-induced K(+) currents. Caspase-activity and TUNEL assays were performed on HCLE cells exposed to UVB followed by incubation in the presence or absence of Ba(2+). K(+) currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba(2+). When HCLE cells were incubated with 5 mM Ba(2+) after exposure to UVB, activation of caspases-9, -8, and -3 and DNA fragmentation were significantly decreased. The data confirm that UVB-induced K(+) current activation and loss of intracellular K(+) leads to activation of the caspase cascade and apoptosis. Extracellular Ba(2+) inhibits UVB-induced apoptosis by preventing loss of intracellular K(+) when K(+) channels are activated. Ba(2+) therefore has effects similar to elevated extracellular K(+) in protecting HCLE cells from UVB-induced apoptosis. This supports our overall hypothesis that elevated K(+) in tears contributes to protection of the corneal epithelium from adverse effects of ambient outdoor UVB.

Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts.

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal-limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenylarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3-4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself.

Gene expression in human accessory lacrimal glands of Wolfring.

PURPOSE: The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. METHODS: Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. RESULTS: Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. CONCLUSIONS: The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs.

Stratified corneal limbal epithelial cells are protected from UVB-induced apoptosis by elevated extracellular K⁺.

The goal of this study was to determine whether elevated [K(+)] protects stratified corneal epithelial cells from entering apoptosis following exposure to ambient levels of UVB radiation. Human corneal limbal epithelial (HCLE) cells were stratified to form multilayered constructs in culture. The cells were exposed to UVB doses of 100-250 mJ/cm(2) followed by incubation in medium with 5.5-100 mM K(+). The protective effect of K(+) was determined by measuring the caspase-3 and -8 activity and TUNEL staining of the stratified HCLE constructs. In response to UVB exposure, activation of apoptotic pathways peaked at 24 h. Caspase-8 in stratified cells was activated by exposure to UVB at 100-250 mJ/cm(2), and activity was significantly reduced in response to 50 or 100 mM K(+). Caspase-3 was activated in the stratified cells in response to 100-250 mJ/cm(2) UVB and showed a significant reduction in activity in response to 25, 50 or 100 mM K(+). DNA fragmentation, as indicated by TUNEL staining, was elevated after exposure to 200 mJ/cm(2) UVB, and decreased following incubation with 25-100 mM K(+). These results show that in a culture system that models the intact corneal epithelium, elevated extracellular K(+) can reduce UVB-induced apoptosis which is believed to be initiated by loss of K(+) from cells. This is the basis of damage to the corneal epithelium caused by UVB exposure. Based on these observations it is suggested that the relatively high K(+) concentration in tears (20-25 mM) may play a role in protecting the corneal epithelium from ambient UVB radiation.

Effects of peroxide-based contact lens-disinfecting systems on human corneal epithelial cells in vitro.

OBJECTIVE: To evaluate the effects of residual hydrogen peroxide (H2O2) in neutralized H2O2-based contact lens-disinfecting solutions on morphology, viability, and barrier function of monolayer and stratified human corneal-limbal epithelial (HCLE) cells. METHODS: Cells were exposed to contact lens formulations containing 0.01% H2O2 for 10, 20, or 60 minutes. The morphology of monolayer or stratified cells was observed by microscopy. Monolayer or stratified cell viability was determined using a live/dead assay, and monolayer cell viability was quantified using flow cytometry. Effects of formulations on barrier function of stratified HCLE cells were evaluated by measuring fluorescein permeability and transepithelial resistance of cultures grown on membrane inserts. To determine the sensitivity of the tests to peroxide damage, stratified cells were also exposed to 0.01% to 0.3% H2O2 in culture medium. RESULTS: All formulations caused swelling of monolayer cells. Formulations with or without H2O2 at pH 7.9 caused mild decreases in monolayer cell viability but did not affect viability or barrier function of stratified HCLE cells. H2O2 (≥0.1%) in culture medium caused damage without recovery to stratified HCLE cells. CONCLUSIONS: Although tests on stratified cells are capable of detecting damage caused by H2O2 in culture medium, residual H2O2 in neutralized ophthalmic formulations had no effect on stratified cells in vitro. These data suggest that H2O2, used appropriately, is a safe disinfectant. Data comparing monolayer and stratified cultures suggest that monolayers are more sensitive to peroxide damage and that the effects of neutralized formulations on stratified cells may better predict the intact corneal epithelial response.

Inhibition of UV-B induced apoptosis in corneal epithelial cells by potassium channel modulators.

The goal of this study was to determine whether prevention of K(+) loss can protect human corneal-limbal epithelial (HCLE) cells from UV-B induced apoptosis. Immunostaining for activated caspase-3 of HCLE cells exposed to 150-200 mJ/cm(2) UV-B demonstrated induction of apoptosis 6 h after exposure. The number of apoptotic cells was decreased by incubation in medium with 25 or 100 mM K(+). If this protection is due to a reduction of UV-induced K(+) loss then K(+) channel blockers should also protect HCLE cells from UV-B. Caspase-8 activity induced by exposure to UV-B at 150 mJ/cm(2) was significantly reduced when the cells were incubated in 0.3 microM BDS-I or 0.05-1 mM quinidine. Caspase-3 was also activated by UV-B and a reduction in activity was observed after incubation in 0.1-0.3 microM BDS-I and 0.1-1 mM quinidine. Induction of DNA fragmentation, as measured by the TUNEL assay, was decreased by treatment with 0.3 microM BDS-I and 0.01-0.05 mM quinidine. Patch-clamp recording showed activation of K(+) channels after exposure to UV-B and a decrease in outward K(+) current was observed following application of BDS-I. Quinidine did not block K(+) currents in HCLE cells, suggesting that the protective effect of quinidine occurs by a mechanism other than via K(+) channels. The effect of the K(+) channel blocker BDS-1 on HCLE cells exposed to UV-B confirms that preventing K(+) efflux protects corneal epithelial cells from apoptosis. This suggests the elevated [K(+)] in tears may protect the corneal epithelium from effects of ambient UV-B.

Elevated extracellular K+ inhibits apoptosis of corneal epithelial cells exposed to UV-B radiation.

The goal of this study was to determine if the high [K(+)] in tears, 20-25 mM, serves to protect corneal epithelial cells from going into apoptosis after exposure to ambient UV-B radiation. Human corneal-limbal epithelial (HCLE) cells in culture were exposed to UV-B at doses of 50-200 mJ/cm(2) followed by measurement of K(+) channel activation and activity of apoptotic pathways. Patch-clamp recording showed activation of K(+) channels after UV-B exposure at 80 mJ/cm(2) or 150 mJ/cm(2) and a decrease in UV-induced K(+) efflux with increasing [K(+)](o). The UV-activated current was partially blocked by the specific K(+) channel blocker, BDS-1. DNA fragmentation, as measured by the TUNEL assay, was induced after exposure to UV-B at 100-200 mJ/cm(2). DNA fragmentation was significantly decreased when cells were incubated in 25, 50 or 100mM K(o)(+) after exposure to UV-B. The effector caspase, caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), but there was a significant decrease in activation when the cells were incubated in 25, 50 or 100mM K(o)(+) following exposure to UV-B. A decrease in mitochondrial potential, a possible activator of caspase-3, occurred after exposure to UV-B at 100-200 mJ/cm(2). This decrease in mitochondrial potential was prevented by 100mM K(o)(+); however, 25 or 50mM K(o)(+) provided minimal protection. Caspase-9, which is in the pathway from mitochondrial potential change to caspase-3 activation, showed little activation by UV-B radiation. Caspase-8, an initiator caspase that activates caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), and this UV-activation was significantly reduced by 25-100mM K(o)(+). The data show that the physiologically relevant [K(+)](o) of 25 mM can inhibit UV-B induced activation of apoptotic pathways. This suggests that the relatively high [K(+)] in tears reduces loss of K(+) from corneal epithelial cells in response to UV exposure, thereby contributing to the protection of the ocular surface from ambient UV radiation.

Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells.

PURPOSE: To compare gene expression profiles of lacrimal gland duct and acinar cells after laser capture microdissection (LCM) and identify molecular networks related to K+ secretion, testing the hypothesis that duct cells are responsible for high K+ levels in tears. METHODS: Frozen sections of lacrimal glands from five rats were subjected to LCM to isolate pure samples of duct and acinar cells. RNA was extracted, amplified, reverse transcribed, and hybridized to rat cDNA microarrays. Paired arrays from ducts and acini of the five animals were scanned and analyzed with in-house software. Gene expression was confirmed with fluorescent antibodies and confocal microscopy. RESULTS: A list of 10,294 genes expressed in ducts and acini was searched using gene ontologies related to ion transport. From a list of 55 genes that were expressed in ducts, a panel of genes hypothesized to be involved in basolateral-to-apical transport of K+ and Cl- was chosen for validation by immunofluorescence and confocal microscopy. This analysis confirmed translation of the genes of interest and showed that NKCC1, Na+,K+-ATPase and the M3 cholinergic receptor are expressed on the basolateral membrane of duct cells, whereas KCC1, IK(Ca)1, CFTR, and ClC3 are apically localized. CONCLUSIONS: Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying lacrimal gland duct cells about which relatively little is known at the molecular level. As demonstrated in a proposed model, the polarized expression of transporters and channels on lacrimal gland duct membranes is consistent with the hypothesis that duct cells secrete the relatively high K+ in lacrimal fluid.

Pre-clinical investigation of the efficacy of an artificial tear solution containing hydroxypropyl-guar as a gelling agent.

PURPOSE: Pre-clinical studies of a new artificial tear product (Systane Lubricating Eye Drops Alcon Laboratories, Inc., Fort Worth, TX) containing the novel gelling agent hydroxypropyl-guar (HP-guar) and two demulcents, polyethylene glycol 400 (PEG) and propylene glycol (PG) were conducted to determine the ability of the product to protect ocular surface epithelial cells from desiccation in vivo and in vitro, and to promote recovery of the damaged corneal epithelial barrier in vivo. Other leading artificial tear products were also evaluated as comparators to determine the relative effectiveness of different polymer systems. METHODS: Damage due to desiccation was assessed by measuring corneal uptake of methylene blue compared to untreated corneas. Corneas of anesthetized rabbits were treated with the new artificial tear product and subjected to desiccation by holding the eyelids open for 2 hours with a speculum. Control eyes were subjected to desiccation without application of a tear formulation. To measure recovery of the corneal epithelium from damage, corneas of anethesthetized rabbits were exposed to 0.01% benzalkonium chloride (BAC) for 5 minutes to increase epithelial permeability. The corneas were exposed to the new artificial tear for 1.5 hours followed by measurement of uptake of 5,6 carboxyfluorescein (CF). In the desiccation and CF uptake experiments, the new tear product was also compared to a tear product formulation without HP-guar and to a commercially available artificial tear containing carboxymethyl cellulose (CMC) and Purite. In a third set of experiments, immortalized human corneal epithelial cells and Chang conjunctival cells in culture were exposed to the PEG/PG/HP-guar tear product, the control formulation without HP-guar, a tear formulation preserved with BAC, or the artificial tear containing CMC/Purite for 15min. The tear formulation was removed and the cells were exposed to desiccating conditions in a laboratory safety hood for 10 or 30min. Cell viability was determined using the MTT assay. RESULTS: The in vivo desiccation model, showed that the new tear product, Systane, offered complete protection of the cornea from desiccation (methylene blue uptake not different than naïve control). Following exposure to 0.01% BAC, the new artificial tear product also provided an environment in which the corneal epithelium recovered completely from damage (CF uptake not different than normal, untreated cornea). This level of protection was not observed when corneas were treated with other formulations. Results from the in vitro desiccation procedure indicated that viability of corneal epithelial and Chang cells treated with the PEG/PG/HP-guar product was significantly greater than viability of cells treated with the tear product without HP-guar or the tear products containing BAC or CMC/Purite. CONCLUSIONS: The tear product containing HP-guar, PEG 400 and propylene glycol satisfies several pre-clinical criteria for an appropriate artificial tear formulation. It gives long-term desiccation protection of the intact cornea and also epithelial cells in culture and has no apparent deleterious affects on cells. It also provides conditions in which a damaged corneal epithelium can recover normal barrier function. The combination of ingredients in the formulation appears to provide an effective mucomimetic artificial tear product. These pre-clinical data suggest that the product will be effective in providing superior relief for the dry eye sufferer.