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Y. Horibe, K. Shinomiya, S. Fujii, O. Katsuta, T. Ikuse, K. Kawazu; Immunohistochemical Demonstration of Monocarboxylate Transporters in Cultured Rabbit Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2906.
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© ARVO (1962-2015); The Authors (2016-present)
Cell culture models of the ocular barriers (cornea, conjunctiva, blood–retinal barrier) are considered to be promising tools for studying the drug uptake and transport into ocular tissues. We examined the localization of monocarboxylate transporters (MCTs) in cultured rabbit corneal epithelial cells by using an immunohistochemistry.
Primary cultures of rabbit corneal epithelial cells (RCECs) were obtained from Kurabo Industries, Japan and were grown using Dulbecco’s modified Eagle medium/nutrient mixture F–12 at pH 7.4, which was supplemented with 5 % FBS, 10 ng/mL epidermal growth factor, 0.1 µg/mL cholera toxin, 5 µg/mL insulin, 0.5 µg/mL hydrocortisone, and antibiotics. The RCECs were seeded at a density of 4 x 104 cells/cm2 on Transwell–COL inserts and cultured at 37°C under 95% air and 5% CO2 by 7 days after seeding. The RCECs were fixed in 2 or 4 % paraformaldehyde in phosphate buffer (0.1 mol/L, pH 7.4) for 4 to 24 hours at 4°C. Samples were embedded in optimal cutting temperature compound and 4–µm–thick serial sections were cut using a freezing microtome. A Streptavidin–Biotin–peroxidase method was applied for immunohistochemistry. The sections were incubated with primary antibodies against goat polyclonal MCT1, MCT2, MCT3, and MCT4, respectively, and then were incubated with secondary antibody, biotin conjugated against goat IgG, and horse–radish peroxidase conjugated streptavidin. These antibodies were purchased from Santa Cruz Biotechnology, Inc., USA. Staining was visualized using 3–3’–diaminobenzidin.
Immunohistochemically, MCT2 was revealed showing the strongest positive–reactivity for the RCECs. MCT2–positive reactivity was localized all the layers of the RCECs with a fine granular pattern. Especially, the surface cells showed marked reactivity and higher positive rate for the anti–MCT2 antibody. Other antibodies, anti–MCT1, –MCT3 and –MCT4 were also localized in some cells of the surface layer of the RCECs. On the other hand, most of the small–sized round cells in the basal layer showed negative reactivity against the antibodies.
Data obtained from this study suggests that MCTs are localized in the RCECs on the both cellular membrane and cytoplasm. Particularly, the surface cells showed strongly positive–immunoreactivity. Therefore, it is considered that the RCECs would be useful tools for studying the drug uptake and transport via MCTs in the cornea.
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