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[Purpose] To investigate the effectiveness of screening and subsequent intervention for age-related macular degeneration (AMD) in Japan. [Study design] Best-case-scenario analysis using a Markov model. [Methods] The clinical effectiveness and cost-effectiveness of screening for AMD were assessed by calculating the reduction proportion of blindness and the incremental cost-effectiveness ratio (ICER). The Markov model simulation began at screening at the age of 40 years and ended at screening at the age of 90 years. The first-eye and second-eye combined model assumed annual state-transition probabilities in the development and treatment of AMD. Data on prevalence, morbidity, transition probability, utility value, and treatment costs were obtained from previously published reports. Sensitivity analysis was performed to assess the influence of the parameters. [Results] In the base-case analysis, screening for AMD every 5 years, beginning at age 40 years and ending at age 74 years (reflecting the screening ages of the current Japanese legal “Specific Health Checkups”) showed a decrease of 40.7% in the total number of blind patients. The screening program reduced the number of blind people more than did the additional AREDS/AREDS2 formula supplement intake. However, the ICER of screening versus no screening was ¥9, 846, 411/QALY, which was beyond what people were willing to pay (WTP) in Japan. Sensitivity analysis revealed that neither OCT nor AI improved the ICER, but the scenario in which the prevalence of smoking decreased by 30% improved the ICER (¥4, 655, 601/QALY) to the level under the WTP. [Conclusions] Ophthalmologic screening for AMD is highly effective in reducing blindness but is not cost-effective, as demonstrated by a Markov model based on real-world evidence from Japan.

To evaluate the changes in ocular surface mucins with 2%rebamipide ophthalmic suspension treatment in soft contact lens (SCL) wearers. Rebamipide suspension is a mucin secretagogue approved for the treatment of dry eye syndrome in Japan. In this study, the fluorescence intensity of wheat germ agglutinin conjugate of fluorescein (F-WGA) was used as a marker of membrane-associated mucins, and sialic acid concentration in tear fluids as a marker of secreted mucins.Thirty-two eyes of 16 SCL wearers with discomfort were treated with rebamipide suspension at a dose of one drop in each eye four times daily for two weeks. The parameters of clinical efficacy were tear break-up time, fluorescein staining scores for the cornea and conjunctiva, and Schirmer test values. Fluorescence intensities in the central cornea were measured by fluorophotometry after the application of 5% F-WGA solution. Tears collected by Schirmer test strips were analyzed by high-performance liquid chromatography, and the concentrations of sialic acid, total protein, and the four major tear proteins, namely secretory IgA, lactoferrin, lipocalin-1, and lysozyme were measured.Significant increases in F-WGA fluorescence intensities (p 0.005) were seen in the corneal surfaces. Sialic acid concentrations increased over time; however, the differences were not statistically significant. Except for a slight increase in kerato-conjunctival staining scores (p 0.05) and secretory IgA (p 0.05), no other significant differences were seen among clinical parameters or tear proteins.Topical application of rebamipide suspension significantly increased F-WGA intensity, a marker of membrane-associated mucins in SCL wearers.

To establish a method for the measurement of 5-fluorouracil (5-FU), and tegafur (FT) in tear samples from patients treated with oral fluoropyrimidine anticancer agent S-1. Cross-sectional study. High performance liquid chromatographic (HPLC) method reported for plasma samples was modified for tear samples. Simulated-tear solutions containing lactoferrin, lysozyme and standard solution containing 5-FU or FT were prepared for preliminary measurements. Tear samples from seven patients treated with S-1 were included. The tears were collected following S-1 administration using Schirmer’s strips. 5-FU and FT concentrations of pure standard solution without extraction process were detected as original concentration. However, on extraction, FT samples in simulated-tear solution showed a peak for 5-FU but not for FT. FT was converted to 5-FU in the extraction process. Decomposition from FT to 5-FU was suppressed when 50 mg/mL bovine serum albumin was added during extraction. The mean concentrations of 5-FU and FT in tears during S-1 treatment were 0.17 ± 0.11 and 1.94 ± 0.71 μg/mL, respectively. A simple HPLC method to determine 5-FU and FT in tear samples was established.

The optical properties of 2,5-diamino-3,6-dicyanopyrazine derivatives bearing singly methyl- or halogen-substituted benzyl groups were studied in both solution and crystalline phases. In solution, all derivatives showed similar absorption and fluorescence properties, since the electronic state of their fluorophore was not altered by substituents attached to the benzyl groups. In contrast, crystalline compounds exhibited a variety of fluorescence colours (yellow, orange, and red), since their solid-state absorption and fluorescence properties were correlated with a change in the electron-donating ability of amino groups due to crystallisation. The absolute fluorescence quantum yields of crystalline dyes (0.14–0.91) showed broad variability, depending on the nature and positions of terminal substituents.

The crystal structures of the ortho-X-benzyl derivatives, where X=F, Cl, Br, I, and Me, of 2,5-bis(N,N-dibenzylamino)-3,6-dicyanopyrazine dyes (C34H24N6X4) were analysed to evaluate the effect of a systematic series of structures on the occurrence of polymorphism. Detailed crystal structure analysis indicated that the thermally stable forms of the polymorphic derivatives (Cl and Br derivatives) were close-packed, whereas those of the non-polymorphic derivatives (F and I derivatives) were stabilised by an intermolecular interaction involving the ortho-substituents. In the thermally metastable forms of the polymorphic derivative, halogen-halogen and halogen-nitrogen interactions contributed to the stabilisation of these crystals in the same way as the thermally stable form of the non-polymorphic derivatives. This indicated that the ease of polymorph occurrence would require an appropriate balance between the crystal energy of the close-packed structure and that of the crystal structure generated mainly by the electrostatic interactions involving the halogens in these halogenated pyrazine derivatives. In addition, the similar tendency of the occurrence of polymorphs in these halogenated pyrazine derivatives was found in 19 sets of halogenated compounds having known crystal structures of F, Cl, Br and I derivatives including at least one polymorphic derivative in the crystal structure database.

Polymorphism-dependent fluorescence, a phenomenon in which a compound shows various fluorescence properties among its polymorphs, contributes to understanding the relationship between the crystal structure and solid-state fluorescence properties. Here we report a variety of solid-state fluorescence efficiencies brought about by benzyl groups in polymorphs of an organic pyrazine dye, where the polymorphs showed quite different fluorescence efficiencies (fluorescence quantum yield: 14% vs. 64%) despite their similar molecular conformations and packing motifs in the crystal structures. Crystal structure analysis and optical measurements revealed that local loose packing caused by the benzyl groups, which are not a part of the pyrazine fluorophore, greatly influenced the fluorescence efficiency in the polymorphs. This result showed that a benzyl substituent can be used as an effective molecular modification for tuning solid-state fluorescence efficiency.

Tear fluids are a mixture of secretions derived from lacrimal glands, accessory lacrimal glands, conjunctiva, and meibomian glands. Compositional changes to tears occur in the normal state and during ocular surface disease, such as dry eye conditions. We have investigated compositional changes to tears after topical application of ophthalmic solutions, with regard to tear-specific proteins (secretory immunoglobulin A, lactoferrin, lipocalin-1, and lysozyme) and ocular surface mucin in normal and dry eye conditions using high-performance liquid chromatography. After application of saline solution (0.9% sodium chloride) in normal subjects, transient but significant decreases in all tear components were observed. The recovery of protein concentrations took up to 30 minutes and lasted longer when the saline solution was applied more frequently. When applying ophthalmic solutions, a balance between washout and dilutional effects should be considered in addition to the therapeutic effect. Investigation of the effect of diquafosol solution (3%) in normal subjects revealed a significant increase in sialic acid concentration, a marker of ocular mucin, at 5 minutes after application, whereas a significant decrease was observed with saline. This result indicates the accelerated secretion of mucin from ocular tissues induced by diquafosol. A clinical study to determine the efficacy of diquafosol in patients with dry eye revealed improvements in tear breakup time, keratoconjunctival staining scores, and Schirmer test score, accompanied by an increase in sialic acid concentration in tears. Investigating normal and dry eye conditions through tear analysis may clarify the pathophysiology of dry eye conditions and support the efficacy of treatments.

Crystal structures of three bisazomethine dyes with dipropyl, dibutyl, and dihexyl substituents on their terminal amino groups are reported. To systematically interpret the effects of the terminal dialkyl substituents on the low-dimensional arrangements of the π–π stacked molecules, the structural features of the molecular geometries and the low-dimensional arrangements were compared with those in the reported crystal structure of two bisazomethine dyes, i.e. with terminal dimethylamino and diethylamino groups. Lattice energy calculations were also carried out to interpret the substitution effects from an energetic perspective. In the crystal structures of all five dyes, one-dimensional arrangements of the π–π stacked molecules were found. The slip angles between the π–π stacked molecules constituting the characteristic one-dimensional arrangements of the five bisazomethine dyes were distributed in the range of 24.66(4)–79.34(7)°. The lengths of the alkyl chains and projections of the terminal dialkyl substituents from the molecular planes in the five bisazomethine dyes were found to play significant roles in determining the slip angles between the π–π stacked molecules and the distances between the molecules aligned along the long molecular axes.

A metal-free pyrazine dye exhibited vapoluminescence when exposed to benzene vapour. Single-crystal X-ray analysis revealed that its flexible bulky groups and weak interactions involving halogens play an essential role in the occurrence of the observed vapoluminescence.

Conformational polymorphism of a series of 2,5-bis(N,N-dibenzylamino)-3,6-dicyanopyrazine dyes, with high molecular flexibility in a non-hydrogen-bonding system, is described. Three derivatives having a substituent at the 4-position of the benzyl groups exhibited several crystal forms with different colours (red, orange, and yellow), whereas a single form was found in the unsubstituted benzyl derivative and the derivative containing 2-naphthylmethyl groups. The effects of conformational flexibility and the substituents on conformational polymorphism were evaluated through structural, energetic, and statistical analyses based on all of the observed crystal structures. They were divided into four conformational groups according to their conformational similarities, and the major difference among the groups was attributed to the amino geometry. Statistical research using the Cambridge Structure Database suggested that a dibenzylamino group connected to an aromatic ring had a suitable potential surface for the occurrence of conformational polymorphs, and had at least two potential wells with respect to the amino geometry. This result revealed that the flexibility of the dibenzylamino group in the pyrazine derivatives enabled the formation of various molecular shapes. However, the preference of occurrence of polymorphs was different in these derivatives. Crystal structure analysis indicated that the similarity in the molecular shapes was significant for the closed packing in the thermally stable forms, whereas the terminal substituents on the dibenzyl groups played an important role in the crystal structures of the thermally metastable forms via the formation of weak interactions such as halogen interactions.

Unstable tear film characterized by shorting of tear break-up time (BUT) is associated with discomfort and dryness in contact lens wearers. The glycocalyx is thought to be crucial in maintaining the wettability and lubrication of the ocular surface. We evaluated the ocular surface glycocalyx in soft contact lens (SCL) wearers using a fluorescein-labeled wheat germ agglutinin (F-WGA) as a marker to demonstrate the ocular surface glycoconjugates in vivo.Twenty experienced SCL wearers and 20 healthy volunteers with no history of CL wearing (controls) were enrolled in the study. After applying a 5% F-WGA solution to the eyes of study individuals, fluorescent intensities in their respective central corneas were measured by fluorophotometry. The relationship between F-WGA intensity in the corneal surface and clinical parameters associated with contact lens wear were analyzed.F-WGA fluorescence intensity in the SCL group was 418.5 ± 103.3, which was significantly lower than that of the controls (825.0 ± 179.8; p 0.0001, Mann-Whitney test). F-WGA fluorescence intensity was not correlated with Schirmer's test values or age, whereas a statistically significant correlation between F-WGA fluorescence intensity and tear film BUT was observed (r = 0.77, p 0.0001). The decrease in F-WGA fluorescence intensity could be reversed by discontinuation of SCL use.Reduction and/or compositional alteration of ocular surface glycocalyx may be one of the causative factors of SCL-induced eye dryness.

To evaluate the cost-effectiveness for a screening interval longer than 1 year detecting diabetic retinopathy (DR) through the estimation of incremental costs per quality-adjusted life year (QALY) based on the best available clinical data in Japan.A Markov model with a probabilistic cohort analysis was framed to calculate incremental costs per QALY gained by implementing a screening program detecting DR in Japan. A 1-year cycle length and population size of 50,000 with a 50-year time horizon (age 40-90 years) was used. Best available clinical data from publications and national surveillance data was used, and a model was designed including current diagnosis and management of DR with corresponding visual outcomes. One-way and probabilistic sensitivity analyses were performed considering uncertainties in the parameters.In the base-case analysis, the strategy with a screening program resulted in an incremental cost of 5,147 Japanese yen (¥; US$64.6) and incremental effectiveness of 0.0054 QALYs per person screened. The incremental cost-effectiveness ratio was ¥944,981 (US$11,857) per QALY. The simulation suggested that screening would result in a significant reduction in blindness in people aged 40 years or over (-16%). Sensitivity analyses suggested that in order to achieve both reductions in blindness and cost-effectiveness in Japan, the screening program should screen those aged 53-84 years, at intervals of 3 years or less.An eye screening program in Japan would be cost-effective in detecting DR and preventing blindness from DR, even allowing for the uncertainties in estimates of costs, utility, and current management of DR.

To evaluate the efficacy of topical application of 3% diquafosol tetrasodium solution for the treatment of soft contact lens (SCL) wearers with dryness. In addition to clinical tests and subjective symptoms, we assessed the fluorescence intensity of wheat germ agglutinin conjugate of fluorescein (F-WGA) used as a marker of membrane-associated mucins and sialic acid concentration in tear fluids as a marker of secreted mucins.Twelve SCL wearers with dryness symptoms were treated with diquafosol for 4 weeks. Clinical tests included the tear film break-up time (BUT), corneal and conjunctival fluorescein staining scores, and Schirmer values. Subjective symptoms were evaluated by the Dry Eye-Related Quality-of-Life Score (DEQS). Fluorescence intensities in the central cornea were measured by fluorophotometry at 5 minutes after a 5% F-WGA solution was applied to the eye. The tears collected by the Schirmer test strips were analyzed by high-performance liquid chromatography (HPLC), and the concentrations of sialic acid; total protein; and the four major tear proteins secretory IgA, lactoferrin, lipocalin-1, and lysozyme proteins were measured.Comparing the results before and after diquafosol treatment, BUT (p0.01), kerato-conjunctival staining score (p0.05), corneal staining score (p0.05), and DEQS score (p0.01) showed statistically significant improvements. The F-WGA fluorescence intensities (p0.0001) significantly increased after treatment, whereas the concentrations of sialic acid and tear proteins remained unchanged.Topical application of diquafosol solution to the SCL wearers with dryness improved biomarker of membrane-associated mucins, BUT, staining of cornea and conjunctiva, and subjective symptoms.

Purpose: This study was performed to determine the levels of lipids deposited on in vivo worn silicone hydrogel lenses. Methods: Three silicone hydrogel materials, galyfilcon A, senofilcon A, and asmofilcon A, were worn for 2 weeks by 35 normal subjects. Total lipid deposition was determined by the sulfo-phospho-vanillin reaction. Cholesterol was estimated by a colorimetric probe through enzymatic oxidation. Phospholipid level was estimated by determining phosphorus with ammonium molybdate through enzymatic digestion. Results: The total lipid content recovered from galyfilcon A, senofilcon A, and asmofilcon A was 32.9 ± 33.8, 42.1 ± 14.0, and 36.6 ± 31.9 μg/lens, respectively. The cholesterol content recovered from galyfilcon A, senofilcon A, and asmofilcon A was 26.2 ± 26.9, 28.6 ± 19.4, and 31.1 ± 21.1 μg/lens, respectively. There were no statistically significant differences in total lipids and cholesterol among the contact lens types. However, the quantity of phospholipid recovered from the asmofilcon A (7.0 ± 5.5 μg/lens) lenses was significantly higher than from galyfilcon A (1.1 ± 0.8 μg/lens) and senofilcon A (2.4 ± 0.8 mg/lens) lenses (p < 0.05, Mann-Whitney test). Conclusions: The quantity of total lipid and cholesterol deposited on the 3 silicone hydrogel lenses tested did not differ. However, there were significant differences in the amounts of phospholipid deposited among the 3 silicone hydrogel lenses, of which clinical significance should be explored in the future study.

To evaluate the clinical efficacy of 3 % diquafosol sodium ophthalmic solution for dry eye, and to analyze the concentration of tear proteins and mucin-like substances after the treatment. Fifty eyes of 25 patients with dry eye syndrome were prospectively enrolled. The patients were treated with diquafosol solution at a dose of 1 drop in each eye 6 times daily for 4 weeks. The parameters of clinical efficacy were tear osmolarity, tear breakup time (BUT), fluorescein staining scores for the cornea and conjunctiva, Schirmer test values, and subjective symptoms evaluated using the ocular surface disease index (OSDI). Tears collected with Schirmer test strips were analyzed by high-performance liquid chromatography, and the concentrations of the total protein and the 4 major tear proteins, namely, secretory IgA, lactoferrin, lipocalin-1, lysozyme, and N-acetyl-neuraminic acid (Neu5Ac), were measured. Neu5Ac is a major sialic acid, a marker of secretory mucins. The BUT, keratoconjunctival staining scores, and Schirmer test values were improved with statistical significance after the treatment with diquafosol solution, while changes in the other parameters, including tear osmolarity, corneal staining scores, and OSDI scores were not significant. The Neu5Ac concentration was significantly increased, which was not accompanied by changes in tear proteins. Topical application of diquafosol significantly improved the clinical parameters of the BUT, keratoconjunctival staining scores, and Schirmer test values and was accompanied by increased sialic acid content in the tears of patients with dry eye.

This study was undertaken to determine the effect of topical application of diquafosol tetrasodium on proteins and mucin-like substances from tears of clinically healthy subjects.Tears were collected from both the eyes of 10 healthy volunteers. Diquafosol tetrasodium solution (3%) was applied once to the right eye and 0.9% sodium chloride solution (saline) once to the left eye. Tear samples were collected by Schirmer test strips before application and 5, 15, 30 and 60 min after application. Sialic acid, a marker of mucin-like substances, and major tear proteins including secretory IgA, lactoferrin, lipocalin-1, and lysozyme were measured by high performance liquid chromatography.Levels of total protein, sIgA and lysozyme were transiently decreased in both groups but returned to baseline levels within 15 min after application. The concentration of lactoferrin and lipocalin-1 did not change significantly in both groups. Sialic acid in tears was significantly decreased 5 min after saline application, but significantly increased 5 min after diquafosol application. No significant difference in sialic acid was seen after 15 min in both groups.Topical application of saline and diquafosol resulted in transient decrease of tear proteins possibly due to wash out or dilution effects. In contrast, diquafosol application significantly increased sialic acid, although the effect was transient. This suggests diquafosol stimulates the secretion of mucins from ocular tissues of healthy human subjects.

To investigate vision-related quality of life (VRQoL) and associated factors in patients with cataracts and the outcomes of cataract surgery using the newly developed VRQoL instrument: the visual function questionnaire, 11-item Japanese version (the VFQ-J11). A total of 457 patients scheduled for cataract surgery at 12 clinical sites from November 2008 through February 2010 were included in the study. The patients completed the VFQ-J11 before and 3 months after surgery. The VFQ-J11 was used to investigate factors associated with VRQoL of the cataract patients, the outcome of cataract surgery, and the predictors of improved VRQoL due to cataract surgery. In a multiple regression model, the VFQ-J11 score was significantly associated with corrected distance visual acuity in the better-seeing eye (better eye VA), and improvement in the VFQ-J11 score after cataract surgery was associated not only with improvement in the better eye VA, but also with improvement in the worse eye VA. Compared to one-eye cataract surgery, both-eyes surgery had a greater impact on VFQ-J11 score improvement. The VFQ-J11 is a good measure of VRQoL in cataract patients. The present study indicates that by including the domains measured in the VFQ, the VFQ-J11 can provide valid data on VRQoL and be less of a burden for patients.

To determine and compare the effects of single and frequent topical applications of saline solution on tear protein concentration in clinically normal subjects.Tears were collected from both eyes of 11 normal volunteers using Schirmer's strips. Saline solution (0.9% sodium chloride) was applied once in the right eye and five times with an interval of 1 min each in the left eye. Tears were collected before and 5, 15, 30 and 60 min after application of the solution. Total tear protein concentration in the samples was measured by the Bradford method and major tear protein concentration (secretory immunoglobulin A, lactoferrin, lipocalin-1, lysozyme and sialic acid) was measured using a high-performance liquid chromatography assay.A significant decrease was observed in the concentration of total tear protein, major tear proteins and sialic acid after topical application of saline solution. This decrease was attributed to the washout and dilution effect. A low protein concentration persisted longer with more frequent application of the solution. The concentration of secretory immunoglobulin A and sialic acid concentration recovered slowly compared with that of other proteins.Even a single application of saline solution resulted in significant changes in major tear protein and sialic acid concentration in the tears of normal subjects. Differences in the recovery of tear protein concentration may be related to the process of protein production and secretion. A balance between normal tear function and the therapeutic effects of ophthalmic solutions should be considered when deciding the frequency of application, particularly in patients with dry eye.

OBJECTIVE: To evaluate with the best available clinical data in Japan the cost-effectiveness of cataract surgery through the estimation of the incremental costs per quality-adjusted life years (QALYs) gained. METHODS: A Markov model with a probabilistic cohort analysis was constructed to calculate the incremental costs per QALY gained by cataract surgery in Japan. A 1-year cycle length and a 20-year horizon were applied. Best available evidence in Japan supplied the model with data on the course of cataract surgery. Uncertainty was explored using univariate and probabilistic sensitivity analysis. RESULTS: In base case analysis, cataract surgery was associated with incremental costs of Japanese yen (¥) 551,513 (US$ 6,920) and incremental effectiveness of 3.38 QALYs per one cataract patient. The incremental cost effectiveness ratio (ICER) was ¥ 163,331 (US$ 2,049) per QALY. In Monte Carlo simulation, the average patient with cataract surgery accrued 4.65 [95 % confidence interval (CI): 2.75-5.69] more QALYs than patients without surgery, giving an ICER of ¥ 118,460 (95 % CI: 73,516-207,926) (US$ 1,486) per QALY. CONCLUSIONS: Cataract surgery in Japan is highly cost-effective even when allowing for the uncertainty of the known variability that exists in estimates of the costs, utilities, and postoperative complication rate.

Because human corneal endothelial cells do not proliferate once the endothelial monolayer is formed, corneal wound healing is thought to be mediated by cell enlargement or migration rather than proliferation. However, the cellular mechanisms involved in corneal wound healing have not been fully determined. Because transforming growth factor-β(2) (TGF-β(2)) isoform is present in high concentrations in normal human aqueous humor, it may play a role in human corneal endothelial cell wound healing. The purpose of this study was to determine the effect of TGF-β(2) on the proliferation and migration of cultured human corneal endothelial cells (HCECs). To achieve this, we first examined the effect of TGF-β(2) on the wound closure rate in an in vitro HCEC wound healing model. However, unexpectedly TGF-β(2) had no effect on the wound closure rate in this model. Therefore, a real-time cell electronic sensing (RT-CES) system and the BrdU incorporation assay were used to determine the effect of TGF-β(2) (0.1-10 ng/ml) on cultured HCEC proliferation during in vitro wound healing. The specificity of this effect was confirmed by adding the TGF-β receptor I kinase inhibitor. TGF-β(2) inhibited the proliferation of HCECs in a dose dependent way and was blocked by TGF-β receptor I kinase inhibitor. Next, the Boyden chamber assay was used to determine how TGF-β(2) (10 ng/ml) affect HCEC migration. Exposure to TGF-β(2) increased cell migration, and a synergistic effect was observed when FGF-2 was added. To determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the migration of HCECs, western blot analysis and Bio-Plex™ suspension array were used to detect phosphorylation of Erk1/2, p38, and JNK in HCECs stimulated by TGF-β(2) and/or FGF-2. The effect of the p38 MAPK inhibitor, SB239063 (10 μM), on TGF-β(2) and/or FGF-2-induced cellular migration was determined by the Boyden chamber assay. Both TGF-β(2) and FGF-2-induced p38 phosphorylation, and a synergistic effect was observed with exposure to both growth factors. SB 239063 inhibited TGF-β(2) and FGF-2-induced migration of HCECs. These results indicate that TGF-β(2) reduces proliferation but stimulates migration of cultured HCECs. In addition, TGF-β(2) and FGF-2 may have synergistic effects on the migration of HCECs mediated by p38 MAPK phosphorylation.

Purpose The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Methods Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Results Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. Conclusions These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.