Anthocyanins have been suggested to improve visual functions. This study examined the effect of four anthocyanins in black currant fruits on the regeneration of rhodopsin using frog rod outer segment (ROS) membranes. Cyanidin 3-glycosides, glucoside and rutinoside, stimulated the regeneration, but the corresponding delphinidins showed no significant effect. The formation of a regeneration intermediate was suggested to be accelerated by cyanidin 3-rutinoside. Their effects on the cGMP-phosphodiesterase activity in the ROS membranes were also investigated but found to be negligible. It was concluded that the major effect of anthocyanins in rod photoreceptors is on the regeneration of rhodopsin.

Vertebrate cone photoreceptors are known to show lower light sensitivity and briefer photoresponses than rod photoreceptors. To understand the molecular mechanisms characterizing cone photoresponses, we compared some of the reactions in the phototransduction cascade between rods and cones. For this purpose, rods and cones were obtained in quantities large enough to do biochemical studies. The cells were purified from the retina of carp (Cyprinus carpio) with a stepwise Percoll gradient. The purified rod fraction contained almost no other kinds of cells besides rods, and the purified cone fraction contained a mixture of red-, green-, and blue-sensitive cones in the ratio 3:Ϸ1:Ϸ1. We prepared membrane preparations from the rod and the cone fraction, and in these membranes, we measured activation efficiencies of the reactions in the phototransduction cascade. The results showed that the signal amplification is lower in the cone membranes, which accounts for the lower light sensitivity in cones. Furthermore, we measured the time courses of visual pigment phosphorylation. The result showed that the phosphorylation is much faster in the cone membranes, which also explains the lower light sensitivity and, in addition, the briefer photoresponse in cones.

The visual transduction processes in rod and cone photoreceptor cells begin with photon absorption by the different types of visual pigments. Cone visual pigments exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (meta II) than does the rod visual pigment, rhodopsin, as expected, due to the functional difference between rod and cone photoreceptor cells. To identify the amino acid residue(s) responsible for the difference in molecular properties between rod and cone visual pigments, we selected three amino acid positions (64, 122, and 150), where cone visual pigments have amino acid residues electrically different from those of rhodopsin, and prepared mutants of rhodopsin and chicken greensensitive cone visual pigment. The results showed that the replacement of Glu-122 of rhodopsin by the residue containing green-or red-sensitive cone pigment converted rhodopsin's rates of regeneration and meta II decay into those of the respective cone pigments, whereas the introduction of Glu-122 into green-sensitive cone visual pigment changed the rates of these processes into rates similar to those of rhodopsin. Furthermore, exchange of the residue at position 122 between rhodopsin and chicken green-sensitive cone pigment interchanges their efficiencies in activating retinal G protein transducin. Thus, the amino acid residue at position 122 is a functional determinant of rod and cone visual pigments.

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Cone photoreceptors show briefer photoresponses than rod photoreceptors. Our previous study showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. Here, we measured the early time course of this rapid phosphorylation with good time resolution and directly compared it with the photoresponse time course in cones. At the time of photoresponse recovery, almost two phosphates were incorporated into a bleached cone pigment molecule, which indicated that the visual pigment phosphorylation coincides with the photoresponse recovery. The rapid phosphorylation in cones is attributed to very high activity of visual pigment kinase [G protein-coupled receptor kinase (GRK) 7] in cones. Because of this high activity, cone pigment is readily phosphorylated at very high bleach levels, which probably explains why cone photoresponses recover quickly even after a very bright light and do not saturate under intense background light. The high GRK7 activity is brought about by high content of a highly potent enzyme. The expression level of GRK7 was 10 times higher than that of rod kinase (GRK1), and the specific activity of a single GRK7 molecule was Ϸ10 times higher than that of GRK1. The specific activity of GRK7 is the highest among the GRKs so far known. Our result seems to explain the response characteristics of cone photoreceptors in many aspects, including the nonsaturation of the cone responses during daylight vision.rod ͉ photoreceptors ͉ retina ͉ phototransduction O ur visual system consists of two components: rods and cones (1, 2). These photoreceptors differ in their light sensitivity so that rods mediate twilight vision, and cones mediate daylight vision. Rods and cones are distinguished not only in their light sensitivity, but also in other response characteristics. The photoresponse time course is much briefer in cones, which improves the time resolution of our daylight vision greatly. Rods are saturated with bright background light and do not respond to more intense light (3). In contrast, cones are not saturated and respond to very bright light (4, 5). The molecular mechanisms underlying in the differences of these response characteristics are not yet known. In previous biochemical studies on the cone phototransduction mechanism, only slight quantitative differences were known in the transduction components between rods and cones (6-8).In our previous study, we obtained a large quantity of isolated cones (enough to perform biochemistry) and showed that transducin activation and cGMP phosphodiesterase activation, the reactions involved in the generation of a photoresponse, are less efficient in cones (9). These findings reasonably explained the lower light sensitivity in cones. Another remarkable difference was found in the phosphorylation of light-activated visual pigment. When light-activated, visual pigment is phosphorylated by a class of kinase known as rhodopsin kinase (rod kinase or G protein-coupled receptor kinase (GRK) 1) in...

Through low-temperature spectroscopy and G-protein (transducin) activating experiments, we have investigated molecular properties of chicken blue, the cone visual pigment present in chicken blue-sensitive cones, and compared them with those of the other cone visual pigments, chicken green and chicken red (iodopsin), and rod visual pigment rhodopsin. Irradiation of chicken blue at -196 degrees C results in formation of a batho intermediate which then converts to BL, lumi, meta I, meta II, and meta III intermediates with the transition temperatures of -160, -110, -40, -20, and -10 degrees C. Batho intermediate exhibits an unique absorption spectrum having vibrational fine structure, suggesting that the chromophore of batho intermediate is in a C6-C7 conformation more restricted than those of chicken blue and its isopigment. As reflected by the difference in maxima of the original pigments, the absorption maxima of batho, BL, and lumi intermediates of chicken blue are located at wavelengths considerably shorter than those of the respective intermediates of chicken green, red and rhodopsin, but the maxima of meta I, meta II, and meta III are similar to those of the other visual pigments. These facts indicate that during the lumi-to-meta I transition, retinal chromophore changes its original position relative to the amino acid residues which regulate the maxima of original pigments through electrostatic interactions. Using time-resolved low-temperature spectroscopy, the decay rates of meta II and meta III intermediates of chicken blue are estimated to be similar to those of chicken red and green, but considerably faster than those of rhodopsin. Efficiency in activating transducin by the irradiated chicken blue is greatly diminished as the time before its addition to the reaction mixture containing transducin and GTP increases, while that by irradiated rhodopsin is not. The time profile is almost identical with those observed in chicken red and green. Thus, the faster decay of enzymatically active state is common in cone visual pigments, independent of their spectral sensitivity.

Cones show briefer light responses than rods and do not saturate even under very bright light. Using purified rod and cone homogenates, we measured the activity of guanylate cyclase (GC), an enzyme responsible for cGMP synthesis and therefore recovery of a light response. The basal GC activity was 36 times higher in cones than in rods: It was mainly caused by higher expression levels of GC in cones (GC-C) than in rods (GC-R). With identification and quantification of GC-activating protein (GCAP) subtypes expressed in rods and cones together with determination of kinetic parameters of GC activation in the presence and absence of GCAP, we estimated the in situ GC activity in rods and cones at low and high Ca 2؉ concentrations. It was revealed that the GC activity would be >10 times higher in cones than in rods in both the dark-adapted and the light-adapted states. Electrophysiological estimation of the GC activity measured in the truncated preparations of rod and cone outer segments gave consistent results. Our estimation of the in situ GC activity reasonably explained the rapid recovery and nonsaturating behavior of cone light responses.Ca 2ϩ ͉ guanylate cyclase-activating protein ͉ guanylate cyclase ͉ phototransduction ͉ rods I n the vertebrate retina, there are 2 types of photoreceptors, rods and cones. They differ in several aspects. Rods show higher sensitivity to light than cones. Because of this difference, rods mediate twilight vision and cones mediate daylight vision. The range of light intensity where cones show light adaptation is much wider than that of rods, and cones essentially do not saturate even under very bright light (1). Time course of a flashlight response of a cone is much faster than that of a rod, which increases the time resolution of our daylight vision (2).The molecular mechanism of generation and termination of a light response is well documented in rods (3, 4): Rods use cGMP as the second messenger that mediates photon absorption by a visual pigment to the closure of the cGMP-gated cation channel. The cGMP level in the outer segment (OS) decreases as a result of hydrolysis by cGMP phosphodiesterase (PDE) that is activated in the light. The level of cGMP is restored after a light by guanylate cyclase (GC), a synthesizing enzyme of cGMP from GTP. Those studies were made mainly in rods. Although it is known that a similar mechanism is present in cones (3-5), our knowledge of cones is limited. In previous studies, however, we have shown that some of the reactions essential for characterizing light responses are quantitatively different between rods and cones (6-8).In rods and cones, the cytoplasmic Ca 2ϩ concentration decreases in the light, and GC is activated (9) by a family of Ca 2ϩ -binding proteins, GC-activating proteins (GCAPs) (10, 11). Because cGMP directly determines the electrical activity of rods and cones, the regulation of its synthesis is very critical for the function of rods and cones. Electrophysiological analysis using GCAP-deficient mouse rods showed that this family o...

After bleaching of visual pigment in vertebrate photoreceptors, all-trans retinal is reduced to all-trans retinol by retinol dehydrogenases (RDHs). We investigated this reaction in purified carp rods and cones, and we found that the reducing activity toward alltrans retinal in the outer segment (OS) of cones is >30 times higher than that of rods. The high activity of RDHs was attributed to high content of RDH8 in cones. In the inner segment (IS) in both rods and cones, RDH8L2 and RDH13 were found to be the major enzymes among RDH family proteins. We further found a previously undescribed and effective pathway to convert 11-cis retinol to 11-cis retinal in cones: this oxidative conversion did not require NADP ؉ and instead was coupled with reduction of all-trans retinal to all-trans retinol. The activity was >50 times effective than the oxidizing activity of RDHs that require NADP ؉ . These highly effective reactions of removal of all-trans retinal by RDH8 and production of 11-cis retinal by the coupling reaction are probably the underlying mechanisms that ensure effective visual pigment regeneration in cones that function under much brighter light conditions than rods.11-cis retinal ͉ 11-cis retinol ͉ all-trans retinal ͉ retinol dehydrogenase ͉ visual cycle L ight detection in vertebrates is mediated by rods and cones. Both rods and cones consist of two parts, the outer segment (OS) and the inner segment (IS). The OS contains the machinery responsible for conversion of a light signal to an electrical signal. The light-absorbing molecule, the visual pigment, is present in the OS and is composed of a chromophore, 11-cis retinal, and a protein moiety, opsin. Light isomerizes 11-cis retinal to all-trans retinal, which induces a conformational change in opsin to lead to activation of an enzymatic cascade to evoke a light response (1, 2).All-trans retinal, the product formed after light absorption, then dissociates from opsin and is reduced to all-trans retinol. This form of retinol is transported to the retinal pigment epithelium (RPE), isomerized, and oxidized to 11-cis retinal with multistep reactions. The newly synthesized 11-cis retinal is sent back to photoreceptors to regenerate visual pigment. This pathway of retinal processing (visual cycle) has been mainly studied in rods or rod-dominant retinas (3, 4). Because cones function under much brighter light conditions, the visual cycle for cones could differ from that for rods in a qualitative and/or quantitative way, as is seen in the phototransduction mechanism in rods and cones (5, 6).The reduction of all-trans retinal to all-trans retinol is attained by RDHs within a photoreceptor cell. This reduction could be the rate-limiting step of the visual cycle (7), and the rate of the reduction has been reported to be 10-40 times higher in cones than in rods under in vivo conditions (8). The supply of 11-cis retinal to regenerate visual pigment could be faster in cones. Several lines of evidence suggest that cone pigment is regenerated in an RPE-independent manner. Fo...