ISOPRENOID LIPID-METABOLISM IN THE RETINA - DYNAMICS OF SQUALENE AND CHOLESTEROL INCORPORATION AND TURNOVER IN FROG ROD OUTER SEGMENT MEMBRANES

Frogs were injected intravitreally with [H-3]acetate, and the formatio n of [H-3]-labeled squalene and cholesterol in the retina and their in corporation into rod outer segment (ROS) membranes were evaluated bioc hemically over a 60-day time course. ROS [H-3]squalene specific activi ty was maximal by 1-3 days, then declined with a half-time of similar to 20-30 days. In contrast, the specific activity of ROS [H-3]choleste rol initially increased to a level substantially less than that of [H- 3]squalene, and then remained constant. Thus, ROS squalene appears to turn over without obligatory conversion to, or coturnover with, ROS ch olesterol. When [H-3]acetate was injected into one eye, radiolabel in non-saponifiable lipids of the contralateral retina represented < 1% o f those recovered from the ipsilateral retina; hence, systemic contrib utions to de novo synthesis were obviated. Long-term (greater than or equal to 8 hr) in vitro incubations of isolated retinas with [H-3]acet ate resulted in incorporation of [H-3]-labeled sterols and squalene in to ROS, at levels comparable to those observed in ROS from companion i ncubated eyecup preparations and from retinas 8 hr after intravitreal injection of [H-3]acetate. These results demonstrate that the in vitro system faithfully reflects the in vivo biosynthetic capacity with res pect to isoprenoid lipid metabolism, and suggest that de novo synthesi s within the neural retina is responsible for generating most, if not all, of the [H-3]squalene and [H-3]cholesterol formed under the given conditions. Treatment of retinas in vitro with brefeldin A or energy p oisons blocked transport of newly synthesized opsin, but not squalene, to the ROS. Furthermore, frogs maintained at 8 degrees C exhibited ma rked suppression of incorporation of newly synthesized protein into th e ROS, while [H-3]squalene incorporation was only minimally reduced, c ompared with frogs maintained at 22 degrees C. These results are consi stent with prior findings that suggest that lipids are transported to the ROS by a mechanism distinct and independent from that employed for intracellular trafficking of opsin and other ROS-destined membrane pr oteins.