Visual sensitivity and interphotoreceptor retinoid binding protein in the mouse: regulation by vitamin A.

Interphotoreceptor retinoid binding protein (IRBP) is a retinoid and fatty acid binding glycoprotein secreted by rod and cone photoreceptors in all vertebrates. IRBP is believed to serve as a carrier for retinoids in the bleaching and regeneration cycle of rhodopsin. IRBP protein has been found to be decreased in vitamin A-deprived rats; it is rapidly recovered after retinol repletion. To understand the mechanism for this recovery, we determined whether vitamin A affects transcription and translation of the IRBP gene. Wild-type and transgenic mice harboring the IRBP promoter-CAT reporter fusion gene were maintained on a retinol-deficient diet supplemented with retinoic acid (-A) or on a control diet (+A) for up to 60 wk postweaning. Some of the -A mice were given retinol repletion for 7 days (-A+A). Electroretinography analysis revealed alterations in waveform and a 2 log unit decrease in b-wave sensitivity in the -A mice over a broad range of stimulus wavelengths. Retinol repletion effected a full recovery. Immunochemistry showed a significant decrease in the immunogold-labeled IRBP between the retinal pigment epithelium and the outer segments of the -A mice compared with +A and -A+A mice. Northern blots showed no differences in the amounts of IRBP or CAT mRNA between these three treatment groups. These results suggest that the regulation of IRBP by retinol is not transcriptional.

Control of Drosophila opsin gene expression by carotenoids and retinoic acid: northern and western analyses.

In the fly, thorough retinoid deprivation is possible, to optimize investigation of the effects of vitamin A metabolites and retinoic acid (RA) on visual development. Retinoids had been found to control fly opsin gene transcription, though this finding was contested. Northern blots on Drosophila heads showed that mRNA of Rh1 (the predominant rhodopsin) was high in vitamin A replete controls, very low in deprived flies, and increased upon feeding carrot juice to deprived flies as early as 1 hr. Expression of the ribosomal protein 49 [rp49] gene (the control) was equal both in deprivation and in replacement. Recovery of Rh1 protein upon such carotenoid replacement followed, barely detectable on Western blots at 4 hr but conspicuous by 8 hr. Alternative chromophore deprivation with yeast-glucose food yielded flies with opsin mRNA on Northerns but not rhodopsin, as demonstrated by Western blots, spectrophotometry and the electroretinogram (ERG). Rh1's mRNA but not Rh1 protein resulted from rearing flies from egg to adult on the otherwise deprivational medium supplemented with RA or beef brain-heart infusion. By comparing results from these different media it was concluded that: [1] deprivation and replacement affect opsin gene transcription; and [2] contradictory conclusions were from chromophore deprivation which does not eliminate all retinoid dependent factors which could affect the opsin promoter. Preliminary evidence shows that carotenoid deprivation decreases two proteins relevant to visual function: [1] phospholipase C (PLC); and [2] Drosophila retinoid binding protein (DRBP).

Electrophysiological sensitivity of carotenoid deficient and replaced Drosophila.

R1-6 dominated electroretinographic (ERG) spectral sensitivities were determined as a function of days posteclosion from carotenoid deprived and replaced white-eyed Drosophila. The sensitivity of flies deprived from egg to adult waxed (about 1.5 log units by day 3), and then waned gradually from 3-11 days (over 2 log units by day 11). Carotenoid replacement (feeding nothing but carrot juice) effected recovery to near the replete controls' level in about 1 day throughout (tested at 0, 4, and 11 days). The normal yellow cornmeal-agar-molasses-brewers yeast fly food (in our laboratory, supplemented with beta-carotene) renders a slower recovery (requiring 7-9 days) since it is a medium designed largely for larval growth. Placing replete adults on deprivational medium did not create a deprivational syndrome in over 11 days. At 3-7 days, deprived flies reared and maintained in constant darkness had substantially enhanced sensitivity, beyond the 1.5 log unit increment already described for cyclic light rearing conditions. All spectral analyses are consistent with the ultraviolet (UV) sensitization of the blue (480 nm) rhodopsin by a replacement-dependent retinoid including two unexpected findings: (1) sensitivity recovery with carrot juice was so fast that the UV peak was already high at 6 h; and (2) the waxing of the deprived fly's sensitivity in dark rearing was so great that the UV peak was present at 4-7 days.

Photoreceptor-specific efficiencies of beta-carotene, zeaxanthin and lutein for photopigment formation deduced from receptor mutant Drosophila melanogaster.

Drosophila rearing media had only beta-carotene, zeaxanthin or lutein as precursors for photopigment chromophores. Zeaxanthin and lutein are potentially optimum sources of the 3-hydroxylated retinoids of visual and accessory photopigments. Mutants made the electroretinogram in white (w) eyes selective for compound eye photoreceptors R1-6, R7 and R8: R1-6 dominates w's electroretinogram; R7/8 generates w;ora's (ora = outer rhabdomeres absent); R8 generates w sev;- ora's (sev = sevenless). Microspectrophotometry revealed R1-6's visual pigment. In w, all 3 carotenoids yielded monotonic dose-responses for sensitivity or visual pigment. An ultraviolet sensitivity peak from R1-6's sensitizing pigment was present at high but not low doses. In w;ora, all 3 carotenoids gave similar spectra dominated by R7's high ultraviolet sensitivity. For w sev;ora, all spectra were the shape expected for R8, peaking around 510 nm. The sensitivity dose-response was at its ceiling except for low doses in w;ora and zero supplementation in w sev;ora. Hence, without R1-6, most of our dose range mediated maximal visual pigment formation. In Drosophila, beta-carotene, zeaxanthin and lutein mediate the formation of all major photopigments in R1-6, R7 and R8.