Specificity of ARAT Activity for 11–cis Retinol in Primary Chicken Muller Cells

Abstract

Purpose: : Recently a novel retinoid cycle has been identified in the cone–dominated chicken retina. This novel retinoid cycle accumulates 11–cis retinyl esters upon light adaptation. The purpose of this study is to study the formation of 11–cis retinyl ester in primary Muller cells from the cone–dominated chicken retina.

Methods: : Primary Muller cells were freshly explanted from chicken retinas and cultured to confluence. All–trans retinol or 11–cis retinol were introduced into the culture media (10 µM with 500 µg fatty acid free BSA/ml of media) and incubated for 24 hours. Cells were then washed, collected, and homogenized, before retinoids were extracted and analyzed by HPLC. For in–vitro membrane studies, Muller cells were harvested and membrane fractions were prepared. 40µg of membrane proteins were incubated with all–trans or 11–cis retinol in the presence or absence of CRALBP and palmitoyl–CoA. Retinoids were extracted and analyzed by normal phase HPLC.

Results: : Esterification of exogenous 11–cis retinol by primary Muller cells (0.294 nmole/1x10⁶ cells) was 4 times greater than esterification of all–trans retinol (0.065 nmole/1x10⁶ cells). In the presence of palmitoyl CoA and CRALBP, Muller cell membrane catalyzed the synthesis of 11–cis retinyl ester at a rate (0.166 nmole/min/mg) which was 24 fold higher than that for all–trans retinyl ester synthesis (0.007 nmole/min/mg). In the absence of CRALBP, 11–cis retinyl ester synthesis was significantly reduced (to 0.023 nmole/min/mg). In the absence of palmitoyl CoA, retinyl esters were not synthesized.

Conclusions: : Primary cultured chicken Muller cells selectively incorporate and esterify exogenous 11–cis retinol over all–trans retinol. Likewise, Muller cell membranes also esterify 11–cis retinol more efficiently than all–trans retinol. Because this esterification activity is CoA dependent, an 11–cis ARAT activity is localized to chicken Muller cells. CRALBP increased this ARAT activity by facilitating the delivery of 11–cis retinol. Our data suggest that Muller cell is the location in the chicken retina where 11–cis retinyl ester is formed in the cone visual cycle.

Keywords: Muller cells • enzymes/enzyme inhibitors • retina