Similarities and Differences in Photochemistry of Type I and Type II Rhodopsins.

The diversity of the retinal-containing proteins (rhodopsins) in nature is extremely large. Fundamental similarity of the structure and photochemical properties unites them into one family. However, there is still a debate about the origin of retinal-containing proteins: divergent or convergent evolution? In this review, based on the results of our own and literature data, a comparative analysis of the similarities and differences in the photoconversion of the rhodopsin of types I and II is carried out. The results of experimental studies of the forward and reverse photoreactions of the bacteriorhodopsin (type I) and visual rhodopsin (type II) rhodopsins in the femto- and picosecond time scale, photo-reversible reaction of the octopus rhodopsin (type II), photovoltaic reactions, as well as quantum chemical calculations of the forward photoreactions of bacteriorhodopsin and visual rhodopsin are presented. The issue of probable convergent evolution of type I and type II rhodopsins is discussed.

Short-Term and Long-Term Effects after Exposure to Ionizing Radiation and Visible Light on Retina and Retinal Pigment Epithelium of Mouse Eye.

A comparative in vivo study of the effects of ionizing radiation (accelerated protons) and visible light (400-700 nm) on the retina and retinal pigment epithelium (RPE) of the mouse eye was carried out. Using the methods of fluorescence spectroscopy and high-performance liquid chromatography (HPLC), we analyzed the relative composition of retinoids in chloroform extracts obtained from the retinas and RPEs immediately after exposure of animals to various types of radiation and 4.5 months after they were exposed and maintained under standard conditions throughout the period. The fluorescent properties of chloroform extracts were shown to change upon exposure to various types of radiation. This fact indicates the accumulation of retinoid oxidation and degradation products in the retina and RPE. The data from fluorescence and HPLC analyses of retinoids indicate that when exposed to ionizing radiation, retinoid oxidation processes similar to photooxidation occur. Both ionizing radiation and high-intensity visible light have been shown to be characterized by long-term effects. The action of any type of radiation is assumed to activate the mechanism of enhanced reactive oxygen species production, resulting in a long-term damaging effect.

Lipofuscin-Mediated Photic Stress Induces a Dark Toxic Effect on ARPE-19 Cells.

Lipofuscin granules from retinal pigment epithelium (RPE) cells contain bisretinoid fluorophores, which are photosensitizers and are phototoxic to cells. In the presence of oxygen, bisretinoids are oxidized to form various products, containing aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that bisretinoid oxidation and degradation products have both hydrophilic and amphiphilic properties, allowing their diffusion through the lipofuscin granule membrane into the RPE cell cytoplasm, and are thiobarbituric acid (TBA)-active. The purpose of the present study was to determine if these products exhibit a toxic effect to the RPE cell also in the absence of light. The experiments were performed using the lipofuscin-fed ARPE-19 cell culture. The RPE cell viability analysis was performed with the use of flow cytofluorimetry and laser scanning confocal microscopy. The results obtained indicated that the cell viability of the lipofuscin-fed ARPE-19 sample was clearly reduced not immediately after visible light irradiation for 18 h, but after 4 days maintaining in the dark. Consequently, we could conclude that bisretinoid oxidation products have a damaging effect on the RPE cell in the dark and can be considered as an aggravating factor in age-related macular degeneration progression.

Water-Soluble Products of Photooxidative Destruction of the Bisretinoid A2E Cause Proteins Modification in the Dark.

Aging of the retina is accompanied by a sharp increase in the content of lipofuscin granules and bisretinoid A2E in the cells of the retinal pigment epithelium (RPE) of the human eye. It is known that A2E can have a toxic effect on RPE cells. However, the specific mechanisms of the toxic effect of A2E are poorly understood. We investigated the effect of the products of photooxidative destruction of A2E on the modification of bovine serum albumin (BSA) and hemoglobin from bovine erythrocytes. A2E was irradiated with a blue light-emitting diode (LED) source (450 nm) or full visible light (400-700 nm) of a halogen lamp, and the resulting water-soluble products of photooxidative destruction were investigated for the content of carbonyl compounds by mass spectrometry and reaction with thiobarbituric acid. It has been shown that water-soluble products formed during A2E photooxidation and containing carbonyl compounds cause modification of serum albumin and hemoglobin, measured by an increase in fluorescence intensity at 440-455 nm. The antiglycation agent aminoguanidine inhibited the process of modification of proteins. It is assumed that water-soluble carbonyl products formed as a result of A2E photodestruction led to the formation of modified proteins, activation of the inflammation process, and, as a consequence, to the progression of various senile eye pathologies.

Lipofuscin Granule Bisretinoid Oxidation in the Human Retinal Pigment Epithelium forms Cytotoxic Carbonyls.

Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.

Dark-adaptation in the eyes of a lake and a sea population of opossum shrimp (Mysis relicta): retinoid isomer dynamics, rhodopsin regeneration, and recovery of light sensitivity.

We have studied dark-adaptation at three levels in the eyes of the crustacean Mysis relicta over 2-3 weeks after exposing initially dark-adapted animals to strong white light: regeneration of 11-cis retinal through the retinoid cycle (by HPLC), restoration of native rhodopsin in photoreceptor membranes (by MSP), and recovery of eye photosensitivity (by ERG). We compare two model populations ("Sea", Sp, and "Lake", Lp) inhabiting, respectively, a low light and an extremely dark environment. 11-cis retinal reached 60-70% of the pre-exposure levels after 2 weeks in darkness in both populations. The only significant Lp/Sp difference in the retinoid cycle was that Lp had much higher levels of retinol, both basal and light-released. In Sp, rhodopsin restoration and eye photoresponse recovery parallelled 11-cis retinal regeneration. In Lp, however, even after 3 weeks only ca. 25% of the rhabdoms studied had incorporated new rhodopsin, and eye photosensitivity showed only incipient recovery from severe depression. The absorbance spectra of the majority of the Lp rhabdoms stayed constant around 490-500 nm, consistent with metarhodopsin II dominance. We conclude that sensitivity recovery of Sp eyes was rate-limited by the regeneration of 11-cis retinal, whilst that of Lp eyes was limited by inertia in photoreceptor membrane turnover.

Fluorescence characteristics of lipofuscin fluorophores from human retinal pigment epithelium.

Lipofuscin granules accumulate in the retinal pigment epithelium (RPE) with age, especially in patients with visual diseases, including progressive age-related macular degeneration (AMD). Bisretinoids and their photooxidation and photodegradation products are major sources of lipofuscin granule fluorescence. The present study focused on examining the fluorescence decay characteristics of bisretinoid photooxidation and photodegradation products to evaluate the connection between fluorescence lifetime and spectral characteristics of target fluorophore groups. The primary objective of the study was to apply experimental spectral analysis results of lipofuscin granule fluorescence properties to interpretation of fluorescence lifetime imaging ophthalmoscopy data. Fluorescence analysis of the lipofuscin granule fluorophores in RPE collected from cadaver eyes was performed. The fluorescence lifetimes were measured by picosecond-resolved time correlated single photon counting technique. A global analytical method was applied to analyze data sets. The photooxidation and photodegradation products of bisretinoids exhibited a longer fluorescence lifetime (average value approximately 6 ns) and a shorter wavelength maximum (530-580 nm). Further, these products significantly contributed (more than 30%), to total fluorescence compared to the other fluorophores in lipofuscin granules. Thus, the contribution of oxidized lipofuscin bisretinoids to autofluorescence decay kinetics is an important characteristic for fluorescence lifetime imaging microscopy data analysis. The higher average fluorescence lifetime in AMD eyes was likely due to the higher abundance of oxidized bisretinoids compared with non-oxidized bisretinoids. Because higher level of oxidized bisretinoids is indicative of pathological processes in the retina and RPE, the present findings have the potential to improve fluorescence lifetime imaging approaches for early diagnosis of degenerative processes in the retina and RPE.

Lutein and its oxidized forms in eye structures throughout prenatal human development.

The presence of carotenoids in the vitreous body, retina, lens, retinal pigment epithelium together with choroid (hereinafter RPE), and ciliary body and iris together with choroidal stroma (hereinafter CBI) was studied throughout the second trimester of prenatal development of the human eye. It has been found that the vitreous body, retina, and RPE contain lutein and its oxidized forms. Zeaxanthin was not found in the tissues studied. The presence of lutein in the vitreous body is transient and no longer detected after 28 weeks of gestation. Lutein was not detected in the lens and CBI, but its oxidized forms were found. The presence of carotenoids in different tissues of the eye in the course of normal eye development and the antioxidant role of carotenoids are discussed.

Eye spectral sensitivity in fresh- and brackish-water populations of three glacial-relict Mysis species (Crustacea): physiology and genetics of differential tuning.

Absorbance spectra of single rhabdoms were studied by microspectrophotometry (MSP) and spectral sensitivities of whole eyes by electroretinography (ERG) in three glacial-relict species of opossum shrimps (Mysis). Among eight populations from Fennoscandian fresh-water lakes (L) and seven populations from the brackish-water Baltic Sea (S), L spectra were systematically red-shifted by 20-30 nm compared with S spectra, save for one L and one S population. The difference holds across species and bears no consistent adaptive relation to the current light environments. In the most extensively studied L-S pair, two populations of M. relicta (L(p) and S(p)) separated for less than 10,000 years, no differences translating into amino acid substitutions have been found in the opsin genes, and the chromophore of the visual pigments as analyzed by HPLC is pure A1. However, MSP experiments with spectrally selective bleaching show the presence of two rhodopsins (λ(max) ≈ 525-530 nm, MWS, and 565-570 nm, LWS) expressed in different proportions. ERG recordings of responses to "red" and "blue" light linearly polarized at orthogonal angles indicate segregation of the pigments into different cells differing in polarization sensitivity. We propose that the pattern of development of LWS and MWS photoreceptors is governed by an ontogenetic switch responsive to some environmental signal(s) other than light that generally differ(s) between lakes and sea, and that this reaction norm is conserved from a common ancestor of all three species.

Time-of-flight secondary ion mass spectrometry to assess spatial distribution of A2E and its oxidized forms within lipofuscin granules isolated from human retinal pigment epithelium.

Lipofuscin granules accumulate in the cells of retinal pigment epithelium with age, particularly in patients with hereditary diseases. These granules are heterogeneous, being composed of mixtures of proteins and lipids, including more than 21 different fluorescent compounds. Bisretinoids and their photo-oxidation and photodegradation products represent the main source of lipofuscin fluorescence and exhibit phototoxic properties. This study used time-of-flight secondary ion mass spectrometry (ToF-SIMS) with in-depth probing to assess the depth distribution of N-retinylidene-N-retinylethanolamine (A2E) and its singly and doubly oxidized forms (A2E-ox and A2E-2ox, respectively) within lipofuscin granules and in their surface layer (lipid membrane). ToF-SIMS showed that A2E and its oxidized forms were uniformly distributed throughout lipofuscin granules but were not present at the membrane surface layer. This finding is important for understanding the process involved in the formation of lipofuscin granules and in their toxicity.

Changes in spectral properties and composition of lipofuscin fluorophores from human-retinal-pigment epithelium with age and pathology.

Fundus autofluorescence mostly originates from bisretinoid fluorophores in lipofuscin granules, which accumulate in retinal-pigment-epithelium cells with age. The dynamics of accumulation, photo-oxidation, and photodegradation of bisretinoids during aging or in the presence of pathology have been insufficiently investigated. Changes in spectral properties and composition of human lipofuscin-granule fluorophores with age and pathology have now been investigated by a high-performance liquid chromatography method using spectrophotometric and fluorescent detectors connected in series. It was found that: (i) N-retinylidene-N-retinylethanolamine (A2E) fluorescence intensity is not predominant in the chloroform extract of human-cadaver-eye retinal pigment epithelium studied; bisretinoid photo-oxidation and photodegradation products have much higher fluorescent properties; (ii) the relative emission maximum in the fluorescence spectrum of suspended retinal-pigment-epithelium cells obtained from an individual human-cadaver eye without pathology is irrespective of donor age and falls within the range 575 ± 15 nm; in two cadaver eyes with signs of age-related macular degeneration, emission maxima were shifted by 23-36 nm towards the shortwave region; and (iii) the ratio of bisretinoid photo-oxidation and photodegradation products to unoxidized bisretinoids in the chloroform extract of cadaver-eye retinal pigment epithelium increases with donor age, from 0.69 ± 0.03 to 1.32 ± 0.04. The differences in fluorescence properties between chloroform extracts obtained from cadaver eyes with and without signs of age-related macular degeneration could be used to increase the potential of fundus autofluorescence imaging as a noninvasive diagnostic method.

Femtosecond laser spectroscopy of the rhodopsin photochromic reaction: a concept for ultrafast optical molecular switch creation (ultrafast reversible photoreaction of rhodopsin).

Ultrafast reverse photoreaction of visual pigment rhodopsin in the femtosecond time range at room temperature is demonstrated. Femtosecond two-pump probe experiments with a time resolution of 25 fs have been performed. The first рump pulse at 500 nm initiated cis-trans photoisomerization of rhodopsin chromophore, 11-cis retinal, which resulted in the formation of the primary ground-state photoproduct within a mere 200 fs. The second pump pulse at 620 nm with a varying delay of 200 to 3750 fs relative to the first рump pulse, initiated the reverse phototransition of the primary photoproduct to rhodopsin. The results of this photoconversion have been observed on the differential spectra obtained after the action of two pump pulses at a time delay of 100 ps. It was found that optical density decreased at 560 nm in the spectral region of bathorhodopsin absorption and increased at 480 nm, where rhodopsin absorbs. Rhodopsin photoswitching efficiency shows oscillations as a function of the time delay between two рump pulses. The quantum yield of reverse photoreaction initiated by the second pump pulse falls within the range 15%±1%. The molecular mechanism of the ultrafast reversible photoreaction of visual pigment rhodopsin may be used as a concept for the development of an ultrafast optical molecular switch.

Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

Ionized Radiation-Mediated Retinoid Oxidation in the Retina and Retinal Pigment Epithelium of the Murine Eye.

The present study evaluated the effects of proton and gamma-ray ionizing radiation on the mouse eye. The aim of this work was to analyze radiation-mediated retinoid oxidation in the retina and retinal pigment epithelium (RPE). The findings from this analysis can be used to develop a noninvasive method for rapid assessment of the effects of ionizing radiation. Comparative fluorescence and chromatographic analyses of retinoids before and after irradiations were performed. The fluorescent properties of chloroform extracts from irradiated mouse retina and RPE exhibited an increase in fluorescence intensity in the short-wave region of the spectrum (λ < 550 nm). This change is due to increased retinal and RPE retinoid oxidation and degradation products after radiation exposure. Comparative analyses of radiation effects demonstrated that the effect of proton exposure on the retina and RPE was higher than that of gamma-ray exposure. The present study revealed a new approach to assessing the level of radiation exposure in ocular tissues.

Comparative Femtosecond Spectroscopy of Primary Photoreactions of Exiguobacterium sibiricum Rhodopsin and Halobacterium salinarum Bacteriorhodopsin.

The primary stages of the Exiguobacterium sibiricum rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400-900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bRPM) and in recombinant form (bRrec). The primary intermediates of the ESR photocycle were similar to intermediates I, J, and K in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product J, and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bRPM and 0.74 ps in bRrec. The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.

Small-angle neutron and X-ray scattering analysis of the supramolecular organization of rhodopsin in photoreceptor membrane.

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.

Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium.

PURPOSE: The aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE). METHODS: RPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method. RESULTS: Comparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530-580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530-580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes. CONCLUSION: Because photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.

Femtosecond spectroscopic study of photochromic reactions of bacteriorhodopsin and visual rhodopsin.

Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20-25fs. The dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3-5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process.

Lake and sea populations of Mysis relicta (Crustacea, Mysida) with different visual-pigment absorbance spectra use the same A1 chromophore.

Glacial-relict species of the genus Mysis (opossum shrimps) inhabiting both fresh-water lakes and brackish sea waters in northern Europe show a consistent lake/sea dichotomy in eye spectral sensitivity. The absorbance peak (λmax) recorded by microspectrophotometry in isolated rhabdoms is invariably 20-30 nm red-shifted in "lake" compared with "sea" populations. The dichotomy holds across species, major opsin lineages and light environments. Chromophore exchange from A1 to A2 (retinal → 3,4-didehydroretinal) is a well-known mechanism for red-shifting visual pigments depending on environmental conditions or stages of life history, present not only in fishes and amphibians, but in some crustaceans as well. We tested the hypothesis that the lake/sea dichotomy in Mysis is due to the use of different chromophores, focussing on two populations of M. relicta from, respectively, a Finnish lake and the Baltic Sea. They are genetically very similar, having been separated for less than 10 kyr, and their rhabdoms show a typical lake/sea difference in λmax (554 nm vs. 529 nm). Gene sequencing has revealed no differences translating into amino acid substitutions in the transmembrane parts of their opsins. We determined the chromophore identity (A1 or A2) in the eyes of these two populations by HPLC, using as standards pure chromophores A1 and A2 as well as extracts from bovine (A1) and goldfish (A2) retinas. We found that the visual-pigment chromophore in both populations is A1 exclusively. Thus the spectral difference between these two populations of M. relicta is not due to the use of different chromophores. We argue that this conclusion is likely to hold for all populations of M. relicta as well as its European sibling species.