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Y. Kanan, A. Kasus-Jacobi, G. Moiseyev, J.-X. Ma, M. R. Al-Ubaidi; 661W Cells Possess Cone Specific Retinoid Cycle Enzymes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3055.
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© ARVO (1962-2015); The Authors (2016-present)
Studies of the retinoid cycle in cones, even in cone dominated retina, are complicated by the presence of other retinal cell types. In this study, the retinoid cycle is investigated in the cone photoreceptor cell line 661W.
Cells, incubated with 10 µM 9-cis retinal, were exposed to light for 30 min and left in the dark overnight. HPLC was used for identification of retinoids and western blots and RT-PCR were done to investigate the presence of various retinoid cycle components. Retinol dehydrogenase (RDH) activity was determined in microsomal fractions of cells incubated with all-trans retinal (ATR)
HPLC analysis showed the presence of ATR, all-trans retinol (ATol) and retinyl-ester (RE). Conversion of ATR to ATol suggests the presence of a RDH. RT-PCR generated products for RDH 5, 11, 12, 13, 14 and retSDR1. The presence of RDH11 and RDH5 was further confirmed by western analysis. The preferred activity of the detected RDHs is reduction of ATR in presence of NADPH with an apparent Km value of 2.3 µM, and a Vmax of 72 pmol/min/mg protein. The reverse reaction was also catalyzed in the presence of NADP with at least 10-fold lower efficiency. The presence of RE points to the existence of a RE synthase. However, western analysis and addition of palmityl CoA suggested that the activity is unique and not due to Lecithin:retinol acyltransferase or acyl CoA:retinol acyltransferase. 661W cells do not possess RPE65 and cannot convert RE to 11-cis retinol but are capable of oxidizing 11-cis retinol to 11-cis retinal.
Similar to data from cone dominated retinas, 661W expresses RDHs and very low levels of ester synthase and exhibit RDHs activity, both for the reduction of ATR and for the oxidation of 11-cis retinol. This differentiates them from rod cells which do not have the latter activity. Finally, data demonstrates that 661W is a model system for studies involving cone retinoid cycle and other aspects of cone photoreceptors biochemistry.
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