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K.G. Shadrach, P. Senanayake, J.G. Hollyfield; Synthesis and Turnover of Hyaluronin and Chondroitins (OS, 6S, 4S) in the IPM, Retina and RPE/Choroid . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2971.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Chondroitin sulfates and hyaluronin are glycosaminoglycans (GAGS) present within the interphotoreceptor matrix (IPM). Hyaluronan is a free GAG, not covalently linked to a core protein, while chondroitins are present in proteoglycans covalently linked to the core protein. In this study, we determine the rate of synthesis and turnover of these GAGS in the IPM, retina and RPE/choroid of the mouse. Methods: Intraocular injections of 3H–glucosamine and 35S–sulfate (40 µCi/eye in a volume of 2–3 µl/eye) were administered to adult C57Bl/6J mice. Retinas and retinal pigment epithelium/choroid were recovered 1, 2, 4, and 8 days post–injection using 10 mice per recovery time. Immediately after sacrificing, the retinas were isolated and the vitreous removed under microscopic dissection from the inner retinal surface. The retinas were then rinsed with 1X PBS and extracted with TBS, pH 8.0. For FACE analysis, complex carbohydrates of retina, RPE/choroid and IPM were isolated and frozen. The tissues and retinal rinse/extracts were digested with proteinase K followed by ethanol precipitation of the complex carbohydrates. The precipitate fractions were digested with hyaluronidase SD and chondroitinase ABC to liberate disaccharides from the contained chondroitin and hyaluronan glycosaminoglycans (GAGs). The reducing end of the resulting saccharides were fluorotagged with AMAC and separated by electrophoresis.The fluorescent bands were digitized and the intensities quantified. Specific bands were cut from the gel for scintillation counting. Results: Radiolabeled OS, 4S and 6S disaccharides of chondroitin were present 10–12 fold higher levels than were labeled HA disaccharides. In the IPM, HA specific activity was highest at day 1, with a calculated half–life at 40 hrs or approximately 1.5 days. Both OS and 6S chondroitins peaked and/or sustained until a later time, having a half–life of 160 hrs or approximately one week. 4S specific activity had the quickest turnover time, with peak activity before day 1 and a half–life of 24 hrs. Each tissue type demonstrated a specific signature of HA and chondroitin specific activity peaks. Conclusions: The mouse retina synthesizes hyaluronin and chondroitin sulfates, which can be extracted with procedures that remove the IPM. The differences in synthesis and turnover of these GAGS may enable us to further define the possible assembly and turnover patterns of this matrix.
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