Vitamin A and the role of the pigment epithelium during bleaching and regeneration of rhodopsin in the frog eye

Abstract

The distribution of 11- cis and all- trans isomers of retinol and retinyl ester was investigated in the frog eye during light-and dark-adaptation (LA and DA). It was also established that freshly isolated rod outer segments (ROS) could regenerate their rhodopsin almost completely from added 11- cis retinol, provided that NADP was also present. The DA frog eye stores vitamin A equivalent to 120–230 mol% of the rhodopsin in the ROS. About 96% is in the RPE and is concentrated mainly in the oil-droplets (which can be separated by centrifugal flotation), the remaining 4% being split roughly equally between ROS and the rest of the retina. The oil-droplet vitamin A is virtually 100% esterified. Vitamin A occurs mainly as ester elsewhere, with the notable exception of the ROS, where the alcohol predominates. As much as 60% of this retinol can be 11- cis, and probably constitutes a pool of prosthetic groups for rhodopsin biosynthesis in the dark. In frogs dark-adapted (DA) for more than a day, more than half of the oil-droplet retinyl ester can be 11- cis. Since ROS lack an esterifying capability, all- trans retinol accumulates during LA of the isolated retina. If contact with the retinal pigment epithelium (RPE) is maintained, however, at least 87% of this retinol leaves the ROS, is esterified in the RPE and accumulates as all- trans retinyl ester in the oil-droplets. The 11- cis isomer is gradually consumed during LA and DA. The form in which vitamin A returns to the ROS has not been established, but some lines of evidence suggest that it is as the ester, mainly in the all- trans configuration. Isomerization to 11- cis probably takes place in the ROS, and during prolonged DA this appears to “leak” back to the RPE and cause a slow rise in the proportion of 11- cis over 20–30 hr in darkness (it should be noted that this effect cannot be accounted for by an influx of prosthetic groups from phagocytized ROS particles). Since the isolated retina has only a limited regenerative capacity, it is suggested that the ROS isomerase is specific for retinyl ester, which under normal circumstances can only be provided by the RPE.