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Retinal rods and cones underlie scotopic and photopic vision, respectively. Their pigments exhibit spontaneous isomerizations (quantal noise) in darkness due to intrinsic thermal energy. This quantal noise, albeit exceedingly low in rods, dictates the light threshold for scotopic vision. The same quantal noise in cones, however, is too low to explain the much higher diurnal light threshold. Separately, a dark continuous noise is present in rods, long accepted to originate from an intrinsic random activation of the cyclic guanosine monophosphate (cGMP)-phosphodiesterase enzyme mediating phototransduction downstream of the pigment. Here, we report the surprising finding that most of this rod dark continuous noise actually originates from rhodopsin itself. Importantly, we found the same continuous noise with a much higher magnitude from cone pigments. The rod and cone continuous noises are apparently both associated with a hitherto unrecognized "metastable" pigment conformational state physiologically resembling that in apo-opsin (opsin devoid of chromophore) and is intermittently active for very brief moments. The cone holopigment's high continuous noise is expected to act as an intrinsic equivalent light and adapt the cone dramatically, accounting for a major part of the light-sensitivity difference between rods and cones in darkness., Competing Interests: Competing interests statement:The authors declare no competing interest.

Changes in visual pigments were studied in two marine fish species, the masked greenling Hexagrammos octogrammus and the prickleback Pholidapus dybowskii. A microspectrophotometric (MSP) analysis showed that the rods and cones of the fish collected from the natural marine environment in summer or kept in a tank at a high illumination level predominantly contained porphyropsins based on chromophore A2. As a result, λ max of the double cones significantly shifted to longer wavelengths, reaching 625 and 609 nm, respectively. After several weeks of dark adaptation, the spectra of all the photoreceptor types shifted to shorter wavelengths, as the A1:A2 ratio switched to A1. The MSP data from the fish kept under controlled light conditions were confirmed by chromatography (HPLC), which showed that the changes in the chromophore ratio were reversible and independent of the water temperature. After the preliminary deep dark adaptation, the first noticeable shift in the pigment ratio from A1 to A2 occurred within two weeks of exposure to bright light. A novel finding in this study was a reverse polarity of A1/A2 changes, unlike the case in most other fish species, where A2 chromophore predominated after the dark exposure. This demonstration of the unusual phenomenon of visual pigment transformation suggests a modification or a new way for the activation of specific biochemical mechanisms of A1:A2 conversion at both high and low illumination levels., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A2E-laden ARPE-19 cells were exposed to a blue light to induce cytotoxicity, in order to investigate the protective effects of various tinted ophthalmic lenses against photo-induced cytotoxicity in human retinal pigment epithelial (RPE) cells laden with A2E, known to be among the etiologies of age-related macular degeneration (AMD). Different-colored tinted lenses with varying levels of tint and different filtering characteristics, such as polarized, blue-cut, and photochromatic lenses, were placed over the cells, and the protective efficacies thereof were evaluated by lactate dehydrogenase assay. When tinted lenses were placed over ARPE-19 cells, there were different reductions in cytotoxicity according to the colors and tint levels. The level of protection afforded by brown-tinted lenses was 6.9, 36.1, and 49% with a tint level of 15, 50, and 80%, respectively. For gray-tinted lenses, the protective effect was 16.3, 35, and 43.4% for the corresponding degree of tint, respectively. In the case of blue-tinted lenses, a protective effect of 20% was observed with 80% tinted lenses, but 15 and 50% tinted lenses provided no significant protection. In addition, photochromic lenses showed a protective effect but blue-cut lenses and polarized lenses provided no significant protection. Tinted lenses significantly reduced cytotoxicity in RPE cells irradiated with blue light. The protection was more efficient in lenses with a brown or gray tint than in blue-tinted lenses. Tinted glasses may provide significant protection against potential blue-light-induced photochemical and photo-oxidative damage in RPE cells., (© 2016 S. Karger AG, Basel.)

Retinal damage can occur in normal ambient lighting conditions. Infants are particularly vulnerable to retinal damage, and thousands of preterm infants sustain vision damage each year. The size of the ocular fundus affects retinal light absorption, but there is a lack of understanding of this effect for infants. In this work, retinal light absorption is simulated for different ocular fundus sizes, wavelengths, and pigment concentrations by using the Monte Carlo method. The results indicate that the neural retina light absorption per volume for infants can be two or more times that for adults.

Signal reception and production form the basis of animal visual communication, and are largely constrained by environmental light. However, the role of environmental light in producing variation in either signal reception or production has not been fully investigated. To chart the effect of environmental light on visual sensitivity and body colouration throughout ontogeny, we measured spectral sensitivity, lens transmission and body pattern reflectance from juvenile and adult Nile tilapia held under two environmental light treatments. Spectral sensitivity in juveniles reared under a broad-spectrum light treatment and a red-shifted light treatment differed mostly at short wavelengths, where the irradiance of the two light treatments differed the most. In contrast, adults held under the same two light treatments did not differ in spectral sensitivity. Lens transmission in both juveniles and adults did not differ significantly between environmental light treatments, indicating that differences in spectral sensitivity of juveniles originated in the retina. Juveniles and adults held under the two environmental light treatments differed in spectral reflectance, and adults transferred to a third, white light treatment differed in spectral reflectance from their counterparts held under the two original treatments. These results demonstrate that environmental light plays a crucial role in shaping signal reception in juveniles and signal production throughout ontogeny, reinforcing the notion that environmental light has the capacity to influence animal communication, and suggesting that the characteristics of environmental light should be considered in models of ecological speciation.

Purpose: Prolonged periods of constant lighting are known to perturb circadian clock function at the molecular, physiological, and behavioral levels. However, the effects of ambient lighting regimes on clock gene expression and clock outputs in retinal photoreceptors--rods, cones and intrinsically photosensitive retinal ganglion cells--are only poorly understood., Methods: Cone-rich diurnal rodents (Muridae: Arvicanthis ansorgei) were maintained under and entrained to a 12 h:12 h light-dark cycle (LD; light: ~300 lux). Three groups were then examined: control (continued maintenance on LD); animals exposed to a 36 h dark period before sampling over an additional 24 h period of darkness (DD); and animals exposed to a 36 h light period before sampling over an additional 24 h period of light (~300 lux, LL). Animals were killed every 3 or 4 h over 24 h, their retinas dissected, and RNA extracted. Oligonucleotide primers were designed for the Arvicanthis clock genes Per1, Per2, Cry1, Cry2, and Bmal1, and for transcripts specific for rods (rhodopsin), cones (short- and mid-wavelength sensitive cone opsin, cone arrestin, arylalkylamine N-acetyltransferase) and intrinsically photosensitive retinal ganglion cells (melanopsin). Gene expression was analyzed by real-time PCR., Results: In LD, expression of all genes except cone arrestin was rhythmic and coordinated, with acrophases of most genes at or shortly following the time of lights on (defined as zeitgeber time 0). Arylalkylamine N-acetyltransferase showed maximal expression at zeitgeber time 20. In DD conditions the respective profiles showed similar phase profiles, but were mostly attenuated in amplitude, or in the case of melanopsin, did not retain rhythmic expression. In LL, however, the expression profiles of all clock genes and most putative output genes were greatly altered, with either abolition of daily variation (mid-wavelength cone opsin) or peak expression shifted by 4-10 h., Conclusions: These data are the first to provide detailed measures of retinal clock gene and putative clock output gene expression in a diurnal mammal, and show the highly disruptive effects of inappropriate (nocturnal) lighting on circadian and photoreceptor gene regulation.

Purpose: To study the bleaching dynamics of individual foveal cone photoreceptors using an adaptive optics ophthalmoscope., Methods: After dark adaptation, cones were progressively bleached and imaged by a series of flashes of 545-nm to 570-nm light at 12 Hz. Intensity measurements were made within the foveal avascular zone (FAZ) to avoid confounding signals from the inner retinal blood supply. Over 1300 cones in this region were identified and tracked through the imaging sequences. A single subject was used who demonstrated the necessary steady fixation, wide FAZ, and resolvability of cones close to the foveal center., Results: The mean intensity of all cones was well-described by first-order kinetics. Individual cones showed marked differences from the mean, both in rate of bleach and amount of photopigment; there was an inverse correlation between these two parameters. A subset of the cones showed large oscillations in intensity consistent with interference from light scattered within the cone outer segment. These cones also bleached more quickly, implying that rapid bleaching induces greater amounts of scatter., Conclusions: Neighboring cones in the fovea display high variability in their optical properties.

In the vertebrate retina, rods mediate twilight vision and cones mediate daylight vision. Their photoresponse characteristics are different. The light-sensitivity of a cone is 10(2)-10(3) times lower than that of a rod. In addition, the photoresponse time course is much faster in cones. The mechanism characterizing cone photoresponses has not been known mainly because of the difficulty in isolating cones in large quantities to perform biochemistry. Recently, we developed a method to purify cones from carp retina using a density gradient, which made it possible to analyze the differences in the molecular mechanism of phototransduction between rods and cones. The results showed that signal amplification in cones is less effective, which explains the lower light-sensitivity of cones. The results also showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. The rapid phosphorylation in cones is attributed to a very high total kinase activity in cones. Because of this high activity, cone pigment is readily phosphorylated even at very high bleaching levels, which probably explains why cone photoresponses recover quickly. Based on these findings, the molecular mechanisms of the differences in the photoresponse characteristics between rods and cones are outlined.

Due to our own preference for bright light, we tend to forget that many insects are active in very dim light. Nocturnal insects possess in general superposition compound eyes. This eye design is truly optimized for dim light as photons can be gathered through large apertures comprised of hundreds of lenses. In apposition eyes, on the other hand, the aperture consists of a single lens resulting in a poor photon catch and unreliable vision in dim light. Apposition eyes are therefore typically found in day-active insects. Some nocturnal insects have nevertheless managed the transition to a strictly nocturnal lifestyle while retaining their highly unsuitable apposition eye design. Large lenses and wide photoreceptors enhance the sensitivity of nocturnal apposition eyes. However, as the gain of these optical adaptations is limited and not sufficient for vision in dim light, additional neural adaptations in the form of spatial and temporal summation are necessary.

We propose a new luminosity function, V*(lambda), that improves upon the original CIE 1924 V(lambda) function and its modification by D. B. Judd (1951) and J. J. Vos (1978), while being consistent with a linear combination of the A. Stockman & L. T. Sharpe (2000) long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) cone fundamentals. It is based on experimentally determined 25 Hz, 2 degrees diameter, heterochromatic (minimum) flicker photometric data obtained from 40 observers (35 males, 5 females) of known genotype, 22 with the serine variant L(ser180), 16 with the alanine L(ala180) variant, and 2 with both variants of the L-cone photopigment. The matches, from 425 to 675 nm in 5-nm steps, were made on a 3 log troland xenon white (correlated color temperature of 5586 K but tritanopically metameric with CIE D65 standard daylight for the Stockman and Sharpe L- and M-cone fundamentals in quantal units) adapting field of 16 degrees angular subtense, relative to a 560-nm standard. Both the reference standard and test lights were kept near flicker threshold so that, in the region of the targets, the total retinal illuminance averaged 3.19 log trolands. The advantages of the new function are as follows: it forms a consistent set with the new proposed CIE cone fundamentals (which are the Stockman & Sharpe 2000 cone fundamentals); it is based solely on flicker photometry, which is the standard method for defining luminance; it corresponds to a central 2 degrees viewing field, for which the basic laws of brightness matching are valid for flicker photometry; its composition of the serine/alanine L-cone pigment polymorphism (58:42) closely matches the reported incidence in the normal population (56:44; Stockman & Sharpe, 1999); and it specifies luminance for a reproducible, standard daylight condition. V*(lambda) is defined as 1.55L(lambda) + M(lambda), where L(lambda) and M(lambda) are the Stockman & Sharpe L- & M-cone (quantal) fundamentals. It is extrapolated to wavelengths shorter than 425 nm and longer than 675 nm using the Stockman & Sharpe cone fundamentals.

The ability to increase the synthesis or vary the distribution of pigment in response to light is an important feature of many pigment cells. Unlike other light-sensitive pigment cells, erythrophores of Nile tilapia change the direction of pigment migration depending on the peak wavelength of incident light: light near 365, 400 or 600 nm induces pigment aggregation, while dispersion occurs in response to light at 500 nm. How these phenomena are achieved is currently unknown. In the present study, the phototransduction involved in the pigment dispersion caused by light at 500 nm or the aggregation by light at 600 nm was examined, using pertussis toxin, cholera toxin, blockers of ion channels, various chemicals affecting serial steps of signaling pathways and membrane-permeable cAMP analog. The results show that light-induced bidirectional movements in tilapia erythrophores may be controlled by cytosolic cAMP levels via Gi- or Gs-type G proteins. In addition, RT-PCR demonstrated for the first time the expression of mRNAs encoding red and green opsins in tilapia fins, only where erythrophores exist. Here, we suggest that multiple cone-type visual pigments may be present in the erythrophores, and that unique cascades in which such opsins couple to Gi or Gs-type G proteins are involved in the photoresponses in these pigment cells. Thus, tilapia erythrophore system seems to be a nice model for understanding the photoresponses of cells other than visual cells.

Spontaneous activation of rhodopsin without light absorption occurs at a much lower rate in rod photoreceptors and insect rhabdoms than in cones. The difference lies in the pigment molecules themselves, and has implications for the design of visual photoreceptors.

Purpose: Biochemical research has demonstrated that lutein and zeaxanthin, the two macular carotenoids, are bleachable pigments. Further, evidence suggests that exposure to UV light can degrade plasma carotenoid levels in vivo. The present study investigated the effects of exposure to normal levels of light on the levels of lutein and zeaxanthin in the retina., Methods: The optical density of macular pigment (MPOD) was measured in two male subjects under four different light-adaptation conditions for 20 days. Heterochromatic flicker photometry was used to measure MPOD at 0.5 degrees eccentricity., Results: The four conditions of light adaptation did not significantly affect MPOD. As in previous studies, however, a significant day-to-day difference was observed for both subjects., Conclusions: The results suggest that lutein and zeaxanthin levels in the eye are unaffected by light and oxidation throughout the day. This justifies current research methods in which MPOD measures are made regardless of the time of day. However, significant between-day variance indicates that multiple MPOD measures may be necessary to evaluate lutein and zeaxanthin levels in the retina accurately.

Background: The mammalian eye shows marked adaptations to time of day. Some of these modifications are not acute responses to short-term light exposure but rely upon assessments of the photic environment made over several hours. In the past, all attempts at a mechanistic understanding have assumed that these adaptations originate with light detection by one or other of the classical photoreceptor cells (rods or cones). However, previous work has demonstrated that the mammalian eye contains non-rod, non-cone photoreceptors. This study aimed to determine whether such photoreceptors contribute to retinal adaptation., Results: In the human retina, second-order processing of signals originating in cones takes significantly longer at night than during the day. Long-term light exposure at night is capable of reversing this effect. Here, we employed the cone ERG as a tool to examine the properties of the irradiance measurement pathway driving this reversal. Our findings indicate that this pathway (1) integrates irradiance measures over time periods ranging from at least 15 to 120 min; (2) responds to relatively bright light, having a dynamic range almost entirely outside the sensitivity of rods; (3) acts on the cone pathway primarily through a local retinal mechanism; and (4) detects light via an opsin:vitamin A photopigment (lambda(max) approximately 483 nm)., Conclusions: A photopigment with a spectral sensitivity profile quite different from those of the classical rod and cone opsins but matching the standard profile of an opsin:vitamin A-based pigment drives adaptations of the human primary cone visual pathway according to time of day.

Rapid color changes of lower vertebrates are caused by the motile activities of pigment cells (chromatophores) present in the skin tissue. Chromatophore motility is generally regulated by neural and/or by endocrine systems. However, in some cases, light also induces pigment aggregation or dispersion directly, which suggests the existence of visual pigments in chromatophores. In fact, some opsins, including melanopsin, have been identified. This article reviews light-sensitive chromatophores of lower vertebrates. Photoreceptive molecules (visual pigments) and signal transduction of light via a GTP-binding protein (G protein) are also discussed.

Purpose: The basic assumption of fundus reflection densitometry is that changes in reflectance are solely determined by photolysis and regenerating visual pigments. This study was undertaken to investigate small but systematic deviations from this rule., Methods: Spectral reflectance changes (450-740 nm) of the fovea were measured during light and dark adaptation over a period of 66 minutes in five healthy subjects. The directional properties of the fundus reflection were examined with a custom-built scanning laser ophthalmoscope (SLO) at 514, 633, and 790 nm. The same instrument was also used to find the spatial distribution of the reflectance changes., Results: In addition to fast changes consistent with visual pigment, slower reflectance changes (lasting 10-20 minutes) were observed at all wavelengths including 740 nm. Because visual pigment does not absorb at 740 nm, a second mechanism must be involved. Factor analysis generated two factors (i.e., spectral curves) that explained more than 97% of the variations in the time course of the spectral reflectance. Total reflectance was modeled by means of an existing model for fundus reflection, and it was found that the first factor strongly resembled the rapid changes in absorption of the cone pigments. The second factor seems linked to slow changes in cone reflectance. Measurements with the SLO showed a clear increase in directionally dependent reflectance from 6 to 30 minutes in the dark. This was observed only in the central 6 degrees of the retina., Conclusions: The characteristics of the slow reflectance changes all point to the cone photoreceptors as the origin. Most likely, alterations in the index of refraction between the interphotoreceptor matrix and photoreceptors lie at the base of this hitherto unknown phenomenon.

Photocurrents from isolated rods of adults and sub-adults of three species of amphibians, Rana pipiens, Ambystoma tigrinum, and Xenopus laevis, were measured with suction pipette electrodes. The intensity for a half-maximal response was 0.91+/-0.48 photons microm(-2) flash(-1) (mean +/- S.D., 10-ms flashes) for Rana, 0.92+/-0.44 for Ambystoma, and 6.14+/-1.33 for Xenopus. The mean number of photoisomerizations at half-saturation was 22+/-12 for Rana, 50+/-24 for Ambystoma, and 221+/-48 for Xenopus. The photocurrent per photoisomerization is several times smaller in Xenopus rods than in the other two species. Spectral sensitivity was measured from 277-737 nm with light polarized both parallel and perpendicular to the planes of the membrane disks. Dichroism fell in the near UV and was absent in the region of absorption by tryptophan and tyrosine. Maximum sensitivity of Rana was at 503.9+/-2.6 nm (n = 86), and of Ambystoma, 505.8+/-1.8 nm (n = 24). Animals from these same batches that were sampled by HPLC had no 3-dehydroretinal (retinal2). Xenopus containing about 94% retinal2 and 6% retinal1 had lambda(max) at 519.3+/-2.7 nm (n = 11). Spectral position of the beta-band, estimated by the method of Stavenga et al. (1993), appears to be at longer wavelengths in amphibian photoreceptors than in other vertebrates. Fits of log sensitivity to a normalized-frequency template that tracks the long-wavelength tail of the alpha-band (Lamb, 1995) show that the rod pigments of Rana and Ambystoma are slightly narrower than those found in the photoreceptors of fish and mammals.

Photocurrents of cones in the retinas of a small fish, Danio aequipinnatus (Cyprinidae) were recorded with suction pipette electrodes. Spectral sensitivity was measured between 277 and 697 nm. Four spectral classes of cone were found, with lambdamax at 560, 480, 408, and 358 nm. For the latter, we provide the first complete characterization of spectral sensitivity of a vertebrate ultraviolet (UV) photoreceptor. All cones responded with similar kinetics, except for a subset of the 560-nm cones, which were distinctly faster. The alpha-bands of the three cones absorbing maximally in the visible have the same bandwidth when log sensitivity is plotted versus normalized frequency, and in this respect they are indistinguishable from primate cones ("Mansfield's rule"). An eighth-degree polynomial in lambdamax/lambda based on this combined data set (fish, primate) is presented as a template that is likely to have predictive value in describing cone spectra from other vertebrates. The alpha-band of the UV cone, however, is somewhat narrower than predicted by this function, is similar to other UV visual pigments, and an eighth-degree polynomial that describes its shape is also presented. These measurements also provide information on the beta-band (i.e. cis peak region), difficult to obtain by microspectrophotometry. The beta-band of cone pigments is found at longer wavelengths as the alpha-band shifts toward the red. A secondary rise in cone sensitivity around 280 nm indicates that photons absorbed by aromatic amino acids in the opsin (gamma-band) excite the transduction cascade, but the quantum efficiency is not as high as when absorption occurs in the retinal-protein chromophore.

The effects of stray light upon retinal pigment densitometric measurements were evaluated by adding specified amounts of stray light to fundus reflections. To allow for interpolations and extrapolations, a computer simulation was devised and validated against the empirical data. The results demonstrate that measured density is greatly decreased by increases in stray light but the time constant of pigment regeneration is little affected.

The radial and longitudinal distribution of retinular screening pigment in the lateral eye of the horseshoe crab Limulus polyphemus was quantified under a variety of experimental conditions. Pigment position was characterized by the center and width of the radial distribution at four levels in the ommatidium. Under diurnal lighting, intact animals show movement of pigment granules from the periphery of the retinular cell at night towards the junction of the arhabdomeral and rhabdomeral segments of the retinular cell in the day. In constant darkness, intact animals exhibit the same circadian rhythm in pigment migration. Animals with bilaterally cut optic nerves do not receive circadian efferent input from the brain and show little pigment movement in diurnal lighting. In all of these cases, pigment was either aggregated in a band just peripheral to the rays of the rhabdom or dispersed to the periphery of the retinular cell. When dark-adapted animals are exposed to a sudden large light increment, pigment moves inward between the rays of the rhabdom. During the day, this inward response begins immediately and reverses as the ommatidial aperture begins to close. At night, the onset of the inward movement is delayed, but then occurs more rapidly than during the day. No significant longitudinal movement of photoreceptor screening pigment was detected under any of these experimental conditions. Two opposing mechanisms control the movement of screening pigment in these cells. Release of neurotransmitters from the circadian efferents causes outward movement; large increments of light cause inward movement. In the absence of sudden changes in light intensity, circadian efferent input, not cyclic lighting, appears to be the major determinant of screening pigment position. A sudden and large increment of light triggers the rapid inward movement which appears to be a protective mechanism optimized for daytime performance.

Light-induced conformational changes occurring at the cytosolic surface of rhodopsin were investigated by performing limited digestions of native and illuminated visual pigment with thermolysin, Arg-C endoproteinase, papain and proteinase K. A higher susceptibility of the extradiscal regions of the bleached pigment to the proteases were observed together with altered capacities of the digested bleached rhodopsins to activate the cGMP phosphodiesterase. The overall results strongly suggest that light induces conformational changes not only in the C-terminal end but also in the second and the third extradiscal loop of rhodopsin.

The position of the distal retinal pigments was measured in the eyes of Palaemon which had been exposed to various light intensities (0-2000 lux) for periods up to 7 days. Electron microscopical observations of sinus glands from these animals revealed that long-term illumination (1000 lux, 7 days) resulted in almost complete degranulation of the Type 2 axon terminals, reflecting intense discharge of the granules. On the other hand, the Type 2 terminals of dark-adapted prawns showed no signs of hormone release, such as exocytosis. The results indicate that the light-adapting hormone is liberated from Type 2 terminals. The Type C cell somata in the medulla terminalis, containing granules of similar size to those in the Type 2 terminals, may be the site of synthesis of this hormone.

1. The biosynthesis of melatonin was investigated in retinaextracts of bovine eyes in dependence of light adaptation of the animals. S-adenososyl (Methyl-14C) methionin and acetylserotonin were used as substrates. A higher melatonin-biosynthesis was found in the retina from animals which were adapted to lower illumination. 2. The effect of triamterene on the biosynthesis of melatonin in the retina was examined. An increase of melatonin was found in the presence of the diureticum, which is a pteridine compound, when retinas of animals which were kept under low illumination were investigated. The role of melatoninsynthesis in dark adaptation is discussed and it is pointed to a possible danger of blinding in patients which used triamterene. Furthermore it is suggested that triamterene also increases the melatoninsynthesis in the pineal gland and that this may possibly contribute to an understanding of the effect of the diurecticum.

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4-dihydoxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half-maximal effect at approximately 2 nM; clonidine, at 1 microM. Dopamine-induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by D1 or alpha-adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a D1 dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine-induced dispersion was inhibited by the alpha 2-adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and alpha 2-adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark-adaptive pigment aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Albino rats were born and raised in 12 hr light: 12 hr dark regimes of illuminances varying from 3- to 800 lx. At 15 weeks of age, the animals were killed and determinations were made of the following: dark-adapted and steady-state rhodopsin levels; rod outer-segment length and photoreceptor-cell density; retinal topography of rhodopsin absorbance, and regeneration rate of visual pigment in vivo. It was found that there is a four-fold drop in the dark-adapted rhodopsin level of animals raised in 400-lx cyclic light compared with those raised in 3 lx. This difference can be accounted for by differences in rod outer-segment length and transverse absorbance of frozen retinal sections. Further, this change in rhodopsin content, coupled with variations in the visual pigment regeneration rate, allows the rat to control the amount of pigment in its retina at steady-state bleach. In this way, the rat can regulate the number of photons its retina catches each day. Animals raised in cyclic illuminances differing by more than two orders of magnitude catch very nearly equal number of photons (1.10 +/- 0.2 X 10(16) per eye) during the light period. A reduction in the number of photoreceptor cells also occurs with increasing illuminance, and these changes are more pronounced in the inferior region of the retina. This is not typical of the type of light-induced retinal damage caused by acute exposures.

Intense ultraviolet (UV) and blue stimulation decreases visual pigment concentration and increases long wavelength fluorescent emission in R1-6 photoreceptors in the white eyed fruit fly Drosophila melanogaster. We used microspectrophotometry to show that the threshold for visual pigment decrease is about 1 log unit lower for UV than for blue light (18.7 vs approximately 19.9 log quanta/cm2 respectively). UV and blue stimuli about 0.2 log units brighter had been shown to cause structural degeneration. Above the threshold for structural damage, visual pigment is decreased permanently while below this level, a recovery of visual pigment was achieved within several hours. Microspectrofluorometric data are partially consistent with the hypothesis that the visual pigment is converted into a fluorescent product which had been named M'. M' had been proposed to be a new form of metarhodopsin which absorbs chiefly in the yellow and which has a fluorescent emission in the red; long wavelength stimulation had been reported to regenerate the native visual pigment from M'. Our data suggest that the situation is significantly more complex than this simple model. For instance, we report that long wavelength stimulation regenerates only a small fraction of the visual pigment which had been decreased by UV or blue stimulation. Furthermore, several lines of evidence suggest that other fluorescent products are also created by intense UV and blue stimulation. We were particularly interested in the lower damage threshold for UV light because of the hypothesis that UV visual sensitivity is mediated by a sensitizing pigment which absorbs UV light and transfers its energy to the blue absorbing rhodopsin. Our data suggest that the UV light decreases the rhodopsin without preferentially decreasing the sensitizing pigment.

The absorption of light by rhodopsin leads to the formation of an activated intermediate (R*) capable of catalysing the exchange of GTP for GDP in a retinal guanine-nucleotide-binding regulatory protein (transducin). The ability of R* to function as a catalyst is terminated by the rhodopsin kinase. The 10 nonactive-site lysine residues of rhodopsin can be reductively dimethylated to form permethylated rhodopsin (PMRh). This derivative is phosphorylated to the same extent as rhodopsin after photolysis. The monomethylation of the active-site lysine residue of PMRh yields active-site-methylated rhodopsin (AMRh). It had previously been shown, by using AMRh, that the formation of R* and its spectroscopic signature metarhodopsin II requires the photochemically induced deprotonation of the active-site Schiff base [Longstaff, Calhoon & Rando (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4209-4213]. Here it is demonstrated that active-site Schiff-base deprotonation is also mandatory in the formation of the form of photolyzed rhodopsin that is susceptible to phosphorylation by rhodopsin kinase. In terms of the spectroscopically defined rhodopsin intermediates, this means that only metarhodopsin II and possibly metarhodopsin III are the actual substrates for rhodopsin kinase.

A protein kinase has been extracted from bovine rod outer segments by a mild procedure. The enzyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of histones, phosvitin, or casein. We propose the name "opsin kinase" for the enzyme, which is not affected by cyclic nucleotides but which is inhibited by theophylline. Preparations of purified rod outer segments, however, appear to contain only low concentration of opsin phosphatase activity.

ATP quenches light-dependent phosphodiesterase (PDE) activation in rod outer segments presumably due to rhodopsin phosphorylation. Here we compared the efficiency of phosphorylated and non-phosphorylated rhodopsins as PDE activators in a reconstituted cell-free system. It is shown that the ability of phosphorylated membranes to activate this enzyme is suppressed compared with non-phosphorylated ones.

The rate of thermal denaturation of bovine and rat opsin in the photoreceptor membranes was studied within a wide temperature range (between 37 and 70 degrees C). It was found that the rate of thermal denaturation of opsin at a physiological temperature (37 degrees C) might be commensurable or even exceed the known rate of rhodopsin renewal produced by photoreceptor disk formation and shedding. Lipid peroxidation caused an increase in the rate of opsin denaturation at a physiological temperature. It is assumed that accumulation of denatured opsin in the photoreceptor membranes during raised illumination together with lipid peroxidation induction may be one of the mechanisms leading to vision deterioration under raised illumination.

Levels of rhodopsin in the photoreceptors of Long-Evans rats were reduced by approximately 55% through dietary deprivation of vitamin A. The interaction of visual pigment with G-protein was examined in receptor outer segment (ROS) membranes obtained from these animals. A binding assay was used to quantitate affinity of the visual pigment in unbleached and bleached ROS membranes for the alpha and beta subunits of exogenous G-protein. Extents of binding were similar to those observed for ROS membranes of rats raised on a normal (vitamin A-supplemented) diet. The results are consistent with a normal capacity for G-activation by photoactivated rhodopsin (R*) in vitamin A-deprived animals. They further indicate that "free opsin" arising in vitamin A deficiency, unlike R*, has relatively low affinity for G-protein.

Seven monoclonal antibodies to the alpha subunit (G alpha) of the frog photoreceptor guanyl nucleotide-binding protein (transducin or G-protein) have been characterized as to their effect on G-protein function, and this has been correlated in the accompanying paper (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) with the antibody-binding sites on G alpha tryptic fragments. Antibodies 4A, 7A, 7B, 7C, and 7D are members of a class of antibodies that block G-protein activation by light and therefore also block activation of the cGMP phosphodiesterase. All these blocking antibodies also block the interaction of G-protein with rhodopsin as measured by the light-scattering "binding signal," and as measured by the stabilization of meta-rhodopsin II by bound G-protein (extra-meta-rhodopsin II). The antibodies (or Fab fragments) also solubilize G alpha beta gamma from the membrane in the dark under isosmotic conditions and thus interfere with G alpha interaction with the membrane. Antibody 4A also blocks the extra-meta-rhodopsin II generated by G-protein-rhodopsin interaction in detergent solubilized membranes. Thus, even in the absence of phospholipids, antibody 4A blocks G-protein-rhodopsin interaction. Therefore, we suggest that the antibodies recognize a region of G alpha involved with binding to rhodopsin. An alternative hypothesis is that this antigenic site is a region of interaction between the alpha and beta gamma subunits, disruption of this interaction leading to removal of both the alpha and beta gamma subunits from the membrane and blocking interaction with rhodopsin. This does not seem to be the case because the antibodies immunoprecipitate the alpha beta gamma complex, and not just the alpha subunit. Other antibodies, 4C and 4H, do not block phosphodiesterase activation, the light-scattering signal, extra-meta-rhodopsin II formation, or interaction with the membrane in the dark and therefore recognize other sites on G alpha.

Arrestin (also named '48 kDa protein' or 'S-antigen') is a soluble protein involved in controlling light-dependent cGMP phosphodiesterase activity in retinal rods, and is also known for its ability to induce autoimmune uveitis of the eye. We report a rapid and simple purification method based on the property of arrestin to bind specifically and reversibly to illuminated and phosphorylated rhodopsin [(1984) FEBS Lett. 176, 473-478]. This method does not require column chromatography and yields about 2-4 mg purified arrestin from 15 bovine retinas. Pure arrestin can be resolved by isoelectric focusing into at least 10 distinct bands, all of which stain with a monoclonal antibody specific for S-antigen.

Illumination of rod outer segments with bright visible light results in the oxidation of both protein and lipid components of the photoreceptor membrane. The oxidation degree depends on the intensity and time of illumination. The inhibitors of free radical processes completely inhibit lipid oxidation and somewhat decrease protein oxidation. Photooxidation systems of lipids and rhodopsin also react differently to oxygen content in the incubation medium. Retinal is the photosensitizer of the oxidation of the photoreceptor membrane components.

Phosphorylation of squid photoreceptor membrane components by Mg-[gamma-32P]ATP is regulated by light. Illumination of squid photoreceptors (Loligo opalescens or Loligo pealei) resulted in phosphorylation of rhodopsin and a 55 000-dalton protein. Rhodopsin phosphorylation was increased 15-20-fold by light, to an average of 0.9-1.8 phosphates/metarhodopsin. The linear dependence of rhodopsin phosphorylation on photoconversion of rhodopsin to metarhodopsin suggests that metarhodopsin is a light-activated substrate for phosphorylation. Phospholipids also were phosphorylated by [gamma-32P]ATP. In the dark, 32P was incorporated into phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid. Illuminated membranes showed increased 32P incorporation into phosphatidic acid and decreased incorporation into the phosphorylated phosphoinositides. These results suggest, for the first time, the participation of a light-activated phospholipase C in squid photoreceptors.

Rhodopsin (opsin), lipid, and fatty acids were measured in rod outer segments (ROS's) of rats maintained for at least 2 weeks in continuous darkness or in 12 hours per day cyclic light. Average rhodopsin per eye was 1.8 nmol. for the 5 ft.-c. cyclic light groups compared to 2.4 nmol. for the dark groups of the same age. The phospholipid/opsin ratio was significantly higher after cyclic light maintenance, suggesting that slow adaptive processes control the opsin density of the ROS membranes. Estimates indicate that ROS length also depends on the long-term light environment. ROS lipid and fatty acid composition were not consistently different in dark and light groups.

We have previously described [H, Kühn et al. (1981) Proc. Natl Acad. Sci. USA, 78, 6873-6877] a light-induced scattering change ('binding signal') associated with a stoichiometric binding between photoexcited rhodopsin and a peripheral membrane protein, the GTP-binding protein, in bovine rod outer segment suspensions. We have attempted here to identify the rhodopsin intermediate R* which is responsible for this interaction, by studying its dependence on pH, temperature and ionic strength. The results strongly suggest that the active state is metarhodopsin II (M II). 1. The initial phase of the binding signal is slightly slower than the formation of metarhodopsin II (2-37 degrees C, pH 5.5-9). 2. The kinetics of the decay of the active rhodopsin state are similar to those of the metarhodopsin II leads to metarhodopsin III transition (37 degrees C, pH 7.3). 3. All conditions which lead to light-induced binding of the GTP-binding protein to R* also lead to the formation of M II. At 2 degrees C, pH 8.3, in particular where no M II is formed in the absence of GTP-binding protein, binding signals and light-induced attachment of the GTP-binding protein to the membrane are still observed. Consistently, addition of GTP-binding protein to a suspension of extracted membranes bleached at 2 degrees C (pH 8.3) shifts the metarhodopsin I in equilibrium metarhodopsin II equilibrium towards metarhodopsin II. The shift is reversed by GTP, which dissociates the rhodopsin--GTP-binding protein complex. 4. At low ionic strength, where the GTP-binding protein is soluble in the dark (instead of being associated to the membrane as in the above experiments) M II still induces the binding whereas M I does not, indicating a much lower affinity of the GTP-binding protein for MI.

In the subretinal space of albino mice, macrophages appear from the time of eye opening and increase in number for 6 months; thereafter they decline with age. Dark rearing retards the accumulation of these cells, and exposure to constant light results in a rapid increase. Observations suggest that macrophages appear as a response to visual cell decay in albino mice and supplement the phagocytic activity of the pigment epithelium.