Strong association of unesterified [3H]docosahexaenoic acid and [3H-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 phospholipids of rod outer segments (ROS), is essential for visual transduction and daily renewal of ROS membranes. We investigated the association of [3H]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:methanol (phospholipids) and acidified chloroform:methanol (acidic phospholipids). After in vitro labeling, free [3H]DHA was easily extracted with hexane (66% of total ROS free DHA), implying a weak association with proteins (rhodopsin). In contrast, after in vivo labeling free [3H]DHA was mainly recovered in the acidic solvent extract (89-99%). Of all phospholipids, [3H-DHA]phosphatidic acid (PA) displayed the highest binding to rhodopsin after both in vitro (43% in acidic extract) and in vivo (>70%) labeling suggesting a possible modulatory role of free DHA and DHA-PA in visual transduction.