Strong association of unesterified [H-3]docosahexaenoic acid and [H-3-docosahexaenoyl]phosphatidate to rhodopsin during in vivo labeling of frog retinal rod outer segments

Docosahexaenoic acid (DHA, 22:6n-3), the most prevalent fatty acid in phosp holipids of rod outer segments (ROS), is essential for visual transduction and daily renewal of ROS membranes. We investigated the association of [H-3 ]DHA-lipids to rhodopsin in ROS from frogs (Rana pipiens) after in vitro (4 hrs) and in vivo (1 day and 32 days) labeling. Lipids from lyophilized ROS were sequentially extracted with hexane (neutral lipids), chloroform:metha nol (phospholipids) and acidified chloroform:methanol (acidic phospholipids ). After in vitro labeling, free [H-3]DHA was easily extracted with hexane (66% of total ROS free DHA), implying a weak association with proteins (rho dopsin). In contrast, after in vive labeling free [H-3]DHA was mainly recov ered in the acidic solvent extract (89-99%). Of all phospholipids, [H-3-DHA ]phosphatidic acid (PA) displayed the highest binding to rhodopsin after bo th in vitro (43% in acidic extract) and in vive (>70%) labeling suggesting a possible modulatory role of free DHA and DHA-PA in visual transduction.