Mikako Oka

Effects of Dorzolamide Hydrochloride on Ocular Tissues

We studied the intraocular pharmacokinetics of dorzolamide hydrochloride eye drops and the effect of dorzolamide on carbonic anhydrase activity and localization in ocular tissues. Carbonic anhydrase activity was detected in normal ocular tissues. The activity was inhibited in corneal endothelial cells, the ciliary body, lens epithelial cells, or the retina 1 to 8 hours after instillation of dorzolamide eye drops. In lens epithelial cells and the retina, the enzyme activity had not recovered even 10 hours after instillation of the drug. Immunostaining did not reveal any differences between the group administered dorzolamide eye drops and the control group administered a physiologically balanced solution. Time-related changes in dorzolamide concentrations in ocular tissues were measured by high-performance liquid chromatography (HPLC). In the cornea, anterior aqueous, iris, ciliary body and retina, drug concentrations increased 15 minutes after the instillation and peaked within 1 hour. These results suggest that dorzolamide immediately suppresses carbonic anhydrase activity in ocular tissues, and is rapidly distributed among the tissues of the eye when administered as eye drops.

Quantitative analysis of ascorbic acid permeability of aquaporin 0 in the lens

Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 μM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0. AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner. The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.

Ascorbate free radical reductase activity in vertebrate lenses of certain species

PURPOSE To clarify the function of ascorbate free radical (AFR) reductase in the antioxidation system of different vertebrate lenses. METHODS The soluble and insoluble fractions were prepared from bullfrog, guinea pig, rat, rabbit, swine, and bovine lenses, and membrane-bound enzymes in the insoluble fraction were extracted by 0.3% Triton X-100. Ascorbate free radical reductase and diaphorase activities in each fraction were determined. RESULTS Ascorbate free radical reductase activity in the lens soluble fraction was the highest in the bullfrog. That in the guinea pig and rabbit was at the next level. There was only a little activity in rat and swine lenses, and none was detected in the bovine lenses. However, a large species difference in AFR reductase activity was not observed in the 0.3% Triton X-100 extracts. Diaphorase activity was three to nine higher than AFR reductase activity in the soluble fractions of bullfrog, guinea pig, and rabbit. In the 0.3% Triton X-100 extracts of all animal species used, it was very high, 108 to 311 times the AFR reductase activity. CONCLUSION These results indicate that the lens soluble and membrane-bound AFR reductase in the different animals may be individual enzyme molecules and have different antioxidative functions. Because the lenses of bullfrog, guinea pig, and rabbit are known to contain a near-ultraviolet (UV) light-absorbing compound, reduced pyridine nucleotide, at a high concentration, the soluble AFR reductase activity is expected to be high in the vertebrate lenses with a near-UV light filter, to enhance the antiphoto-oxidation capacity of ascorbate.

λ-crystallin related to dehydroascorbate reductase in the rabbit lens

PURPOSE To evaluate the relationship of lambda-crystallin to reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydroascorbate (DHA) reductase found specifically in the rabbit lens. METHODS DHA reductase Fractions I-IV were separated from the lambda/betaL1-crystallin fraction of rabbit lens soluble protein by diethylaminoethyl (DEAE)-cellulose ion-exchange column chromatography, and then the enzyme was partially purified from Fraction II by rechromatography on the same ion-exchange column. The isolated DHA reductase fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, native isoelectric focusing and two-dimensional gel electrophoresis. RESULTS Using Western blot and a probe of antiserum to recombinant lambda-crystallin, the main 33-kDa protein band was strongly stained in all the rabbit lens DHA reductase fractions, and most of the additional protein bands of approximately 25-30 kDa were also detectable. In the partially purified enzyme, the 33-kDa subunit alone was identified as a distinct protein band by SDS-PAGE, and a main basic protein was found at pI 7.6 by native isoelectric focusing. In addition, many bands of more acidic proteins were separated from other enzyme fractions, and protein spots corresponding to the 33 and/or approximately 25-30-kDa subunits were detected in each of the more acidic proteins by two-dimensional gel electrophoresis. CONCLUSION These results suggest that lambda-crystallin is closely related to the DHA reductase in the rabbit lens. The above heterogeneity of the enzyme-crystallin may arise from posttranslational modifications.

The Extracellular C-loop Domain Plays an Important Role in the Cell Adhesion Function of Aquaporin 0.

ABSTRACT Purpose: Although aquaporin 0 (AQP0) is a member of the AQP family, it has limited water permeability compared with other members. AQP0 may also have cell adhesion-related functions, but the evidence is still limited. Here, we studied the relationship of AQP0 to cell adhesion and determined the region required for cell adhesion.Methods: L-cell fibroblasts stably expressing AQP0 or AQP1 (L-AQP0 or L-AQP1) were established. One group of cells was stained with CellTracker Red and cultured into a confluent monolayer, whereas the other group was loaded with CellTracker Blue and seeded over the monolayer. To study cell adhesion, the percentages of lower and upper layer cells were measured using flow cytometry. To determine the region of AQP0 required for adhesion, activity was done by pull-down assay using glutathione S-transferase fusion proteins. To study the water permeability, Xenopus laevis oocyte expressing AQP0 wild-type or AQP0 mutated in C-loop was transferred to a hypotonic solution and photographed, and the diameter was measured to calculate the volume.Results: More cells adhered to the lower cells in the L-AQP0 homotypic pair than other pairs such as L-AQP1 homotypic or L-AQP0/L-AQP1 heterotypic pairs. Pull-down assays revealed that AQP0 could bind to itself via the C-loop extracellular domain. Furthermore, we determined that 109Pro and 110Pro in the C-loop were important for cell adhesion. However, mutation of the C-loop in AQP0 did not affect its water permeability.Conclusions: AQP0 is known to bind lipids in the opposing membrane. Our data suggest that this cell-to-cell adhesion occurs not only in the AQP0/liquids but also via AQP0/AQP0 interaction through the C-loop domain. Mutations in the C-loop amino acids did not affect the water permeability of AQP0 but did affect its cell adhesion function. These independent dual functions of AQP0 are important for lens transparency.

Effect of hesperetin on chaperone activity in selenite-induced cataract

Abstract Background. Chaperone activity of α-crystallin in the lens works to prevent protein aggregation and is important to maintain the lens transparency. This study evaluated the effect of hesperetin on lens chaperone activity in selenite-induced cataracts. Methodology. Thirteen-day-old rats were divided into four groups. Animals were given hesperetin (groups G2 and G4) or vehicle (G1 and G3) on Days 0, 1, and 2. Rats in G3 and G4 were administered selenite subcutaneously 4 hours after the first hesperetin injection. On Days 2, 4, and 6, cataract grades were evaluated using slit-lamp biomicroscopy. The amount of a-crystallin and chaperone activity in water-soluble fraction were measured after animals sacrificed. Results. G3 on day 4 had developed significant cataract, as an average cataract grading of 4.6 ± 0.2. In contrast, G4 had less severe central opacities and lower stage cataracts than G3, as an average cataract grading of 2.4 ± 0.4. The a-crystallin levels in G3 lenses were lower than in G1, but the same as G4. Additionally, chaperone activity was weaker in G3 lenses than G1, but the same as in G4. Conclusions. Our results suggest that hesperetin can prevent the decreasing lens chaperone activity and a-crystallin water solubility by administered of selenite.

Co-administration of Magnesium Ion Prevents Indomethacin-Induced Intestinal Ulcerogenic Lesions in Adjuvant-Induced Arthritis Rats

In a study to find ways to prevent the side effects of indomethacin (IMC), we previously reported that magnesium ion (Mg2+) can prevent the onset of IMC-induced gastric mucosa in adjuvant-induced arthritis (AA) rats, a model for rheumatoid arthritis (RA). In this study we investigated whether the co-administration of IMC and Mg2+ prevents the formation and aggravation of intestinal ulcerogenic lesions in AA rats. The single oral administration of an excessive dose of IMC (40 mg/kg) induces hemorrhagic lesions and nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) in the jejunal and ileal mucosa of AA rats, and the extent of the lesions, as well as iNOS and NO levels in AA rats are higher than in normal rats. On the other hand, the co-administration of 200 mg/kg Mg2+ attenuates intestinal ulceration and the elevation in the iNOS and NO levels in AA rats. Further, hemorrhagic lesioning and enhanced iNOS and NO levels in AA rats also result from the repetitive oral administration of 3 mg/kg IMC (therapeutic dose) for 42 d (once a day), and these changes are also prevented by the co-administration of 200 mg/kg Mg2+. In conclusion, the co-administration of Mg2+ suppresses the ulcerogenic response to IMC in the jejunal and ileal mucosa of AA rats, probably by preventing the elevation of iNOS and NO levels in the region.