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J. Kaylor, A. Miu, G. Travis; Retinoid Processing in Müller Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2998.
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© ARVO (1962-2015); The Authors (2016-present)
The visual cycle for the regeneration of rhodopsin is well defined in RPE cells. However, several lines of evidence suggest that cones may regenerate visual pigments by a mechanism independent of the RPE that might involve Müller cells. Prior work from our laboratory indicated the existence of a retinoid isomerase (distinct from Rpe65) in chicken and ground-squirrel retinas that catalyzes the direct conversion of all-trans-retinol (all-trans-ROL) to 11-cis-retinol (11-cis-ROL). This ‘isomerase-2’ activity is coupled to a palmitoyl coenzyme A (palm CoA) dependent retinyl-ester synthase (ARAT), to yield 11-cis-retinyl palmitate (11-cis-RP) as a final product. We are working to understand the role of Müller cells in the processing of visual retinoids.
Primary Müller cell cultures were prepared from chicken retinas. Rpe65 expression was checked by Western with a chicken Rpe65 specific antibody. We determined the levels of visual retinoids synthesized in vivo by incubation of the Müller cells with all-trans-ROL and palm CoA added to the medium. Cell homogenates were also used as an enzyme source to assay in vitro the activities of isomerase-2 and ARAT in Müller cells. Retinoids in samples were extracted with hexane and analyzed by liquid chromatography.
Cultured chicken Müller cells do not express Rpe65 based on Western analysis. Addition of all-trans-ROL and palm CoA to the culture medium resulted in the synthesis of 11-cis-ROL, all-trans-retinyl palmitate (all-trans-RP) and all-trans-retinaldehyde inside the Müller cells. Multiple 11-cis-retinyl esters were present in the media outside the cell. In the presence of palm CoA, Müller cell homogenates synthesized all-trans-RP and 11-cis-RP from all-trans-ROL substrate.
Cultured chicken Müller cells contain robust ARAT activity. The Müller cells also contain isomerase-2 activity, indicated by palm CoA-dependent synthesis of 11-cis-ROL and 11-cis-RP from all-trans-ROL.
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