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J. Kaylor, R. Radu, A. Miu, G. Travis; Retinoid Processing in Müller Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3521.
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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. A strong candidate for ARAT is acyl CoA:diacylglyerol acyltransferase type-1 (DGAT1). 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. We measured expression of several visual cycle mRNA’s by quantitative real-time PCR. We determined the levels of visual retinoids synthesized in vivo by incubation of the Müller cells with all-trans-ROL added to the medium. The media and cells were separated for analysis. We also used cell homogenates 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 expressed the mRNA’s for several retinoid-processing proteins including cellular retinaldehyde binding protein (CRALBP), DGAT1, and RGR-opsin. Addition of all-trans-ROL to the culture medium resulted in the synthesis of all-trans-retinyl palmitate (all-trans-RP) and all-trans-retinaldehyde by the Müller cells. Addition of 11-cis-ROL to the medium resulted in the synthesis of 11-cis-RP and 11-cis-retinaldehyde by the cells. In the presence of palm CoA, Müller cell homogenates synthesized all-trans-RP and 11-cis-RP from all-trans-ROL and 11-cis-ROL substrates, respectively. In contrast to RPE homogenates, we observed very low palm CoA-independent synthesis of retinyl esters by Müller cell homogenates. Significantly, we observed synthesis of 11-cis-RP from all-trans-ROL and palm CoA substrates by Müller homogenates in the dark, indicating the presence of isomerase-2 activity in these cells.
Cultured chicken Müller cells contain robust ARAT activity, which is probably mediated by DGAT1. In contrast, lecithin:retinol acyl-transferase (LRAT) activity is virtually undetectable in these cells. The Müller cells also contain isomerase-2 activity, indicated by palm CoA-dependent synthesis of 11-cis-RP from all-trans-ROL.
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