Action spectra for the photoconsumption of oxygen by human ocular lipofuscin and lipofuscin extracts

The action spectra for the photoconsumption of oxygen by lipofuscin isolated from human retinal pigment epithelium cells and liposomal suspensions containing extracts of lipofuscin are reported. The lipofuscin and lipofuscin extract action spectra are similar, demonstrating the phototoxic constituents of lipofuscin are present in the lipofuscin solvent extract. 2-[2,6-Dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E), present in both intact granules and the solvent extract, has been invoked as an important contributor to the phototoxicity of lipofuscin. The action spectrum for oxygen photoconsumption by A2E follows its absorption spectrum but does not resemble the action spectrum for photoconsumption of oxygen by lipofuscin granules or lipofuscin extract. These results combined with recently reported experimental studies on the aerobic photoreactivity of A2E indicate that it is not a major contributor to the phototoxicity of lipofuscin.     

Retinal pigment epithelium lipofuscin proteomics

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.     

A study on the killing by light of photosensitized cells of halobacterium salinarium

Summary The present study aimed at a demonstration of a protecting effect of carotenoids against photokilling of Halobacterium salinarium in the presence of exogenous photosensitizer. In comparative experiments with a colorless mutant and the carotenoid-containing wild type the use of toluidine blue as photosensitizer gave erratic results. It seems probable that the erratic results were due to the varying ability of cells from one culture to another to reduce toluidine blue to a leuco form which is inactive as photosensitizer. The use of phenosafranine as photosensitizer gave consistent results; this dye has a lower oxidation-reduction potential than toluidine blue and is less easily reduced to a leuco form by the cells. When aerated cultures were exposed to light in the presence of phenosafranine the colorless cells were killed; the carotenoid-containing cells were not so affected. It can therefore be concluded that carotenoids protect the cells against photokilling under these conditions.The killing of the colorless cells was closely parallelled by an extensive lysis of the cells; the structure of the carotenoid-containing cells was barely affected by the exposure. There are reasons to believe that the carotenoids are located in or on the cell envelope. It thus seems likely that the cell envelope is a site of the photochemically induced damage.     

Effect of nitric oxide on viscosity of nerve cell membranes

The influence of nitric oxide on the microviscosity of nerve cell membranes was investigated by resonance Raman (RR) spectroscopy. Changes in membrane viscosity were estimated from the resonance Raman-spectra of carotenoids localized in the axon plasmatic membrane and membranes of subcellular vesicles (cytosomes). For the nerve fibre, the extracellular addition of nitric oxide donor, sodium nitroprusside (0.5 mM), caused an increase in the 1526 cm(-1) band relative half-width and the modification of 1160 cm-1 band structure. Moreover, sodium nitroprusside led to an increase in the I1526/I1160 ratio by 13% in 25 min and a decrease in this ratio by 10% in 50 min. In the case of cytosomes, sodium nitroprusside (0.5 mM) resulted in the reduction of the I1526/I1160 ratio by 8% in 25 and 50 min. It was shown that the neuron rhythmic activity correlated with the I1526/I1160 ratio and cytosome membrane microviscosity. We suppose that nitric oxide causes a conformational transition of carotenoids in the axon plasmatic membrane and the membranes of cytosomes. This process can be due to nitric oxide-induced changes of the membrane microviscosity or potential.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

Opposite effect of lipofuscin granules and melanosomes from human retinal pigment epithelium of eye on photooxidation of cardiolipin

The influence of lipofuscin granules and melanosomes from human retinal pigment epithelium on the light-induced photooxidation of cardiolipin liposomes and the generation of superoxide radicals was studied. Lipofuscin granules were able to stimulate, while melanosomes inhibited, the cardiolipin photooxidation. The visible light irradiation of both melanosomes and lipofuscin granules generated superoxide radicals with mean rates of 1.5 nmole/min/10(7) and 38 nmole/min/10(7) granules, accordingly. However, melanosomes but not lipofuscin granules reacted readily with superoxide radicals. Moreover, the rate constant of degradation of superoxide radicals in the presence of melanosomes was about five orders of magnitude higher than the rate constant of its photogeneration. Therefore, we propose that melanosomes in retinal pigment epithelium cells have a photoprotective role whereas lipofuscin granules may stimulate photodestructive reactions.     

Comparison of extracellular ATP and UTP signalling in rat renal mesangial cells. No indications for the involvement of separate purino- and pyrimidino-ceptors.

The visible c.d. spectrum of wild-type Rhodospirillum rubrum shows positive bands [Dratz, Schultz & Sauer (1966) Brookhaven Symp. Biol. 19, 303-318] that are largely due to the B880 antenna pigments, bacteriochlorophyll a and carotenoids. The bacteriochlorophyll c.d. band was absent from the spectrum of R. rubrum G9, a mutant unable to synthesize coloured carotenoids, and could be partly restored by adding extracted carotenoids to freeze-dried membrane vesicles isolated from that mutant. Therefore it seems to arise from either bacteriochlorophyll-carotenoid interactions or bacteriochlorophyll-protein interactions that are induced by the carotenoid. The more complex carotenoid c.d. band had different shapes in native and reconstituted carotenoid-containing membranes. Such differences suggest that the optical activity of the carotenoid in the B880 antenna arises from both non-degenerate and degenerate interactions.     

Lipofuscin-mediated photic stress inhibits phagocytic activity of ARPE-19 cells; effect of donors’ age and antioxidants

The risk of chronic oxidative stress in the retinal pigment epithelium (RPE) increases with age due to accumulation of the photoreactive age pigment lipofuscin (LFG). Here, we asked whether sublethal and weakly lethal photic stress, induced by irradiation of ARPE-19 cells containing phagocytised LFG, affected the cell specific phagocytic activity, which is critically important for proper functioning and survival of the retina, and if natural antioxidants could modify the observed outcomes. ARPE-19 cells preloaded with LFG isolated from human donors of different age or containing LFG enriched with zeaxanthin and α-tocopherol (LFG-A), were irradiated with blue light. Phagocytosis of fluorescein-5-isothiocyanate (FITC)-labelled photoreceptor outer segments was determined by flow cytometry. Photoreactivity of LFG and LFG-A was analysed by measuring photoconsumption of oxygen and photogeneration of singlet oxygen mediated by the granules. LFG-mediated photic stress in ARPE-19 cells induced significant inhibition of their specific phagocytosis. The inhibitory effect increased with age of LFG donors and was reduced by enrichment of the granules with antioxidants. Oxygen consumption and generation of singlet oxygen induced by the photoexcited LFG increased with donor’s age and was partially quenched by antioxidants. Although the phototoxic potential of lipofuscin increased with age, natural antioxidants reduced photoreactivity of LFG and their efficiency to induce oxidative stress. This study has demonstrated, for the first time, that mild oxidative stress, mediated by the age pigment lipofuscin, impairs specific phagocytic activity of RPE, and that natural antioxidants can protect this important cellular function by reducing lipofuscin photoreactivity.     

Antioxidant effects of carotenoids in a model pigment-protein complex

The effect of carotenoids on stability of model photosynthetic pigment-protein complexes subjected to chemical oxidation with hydrogen peroxide or potassium ferricyanide was investigated. The oxidation of carotenoid-less and carotenoid-containing complexes was conducted in the presence or absence of ascorbic acid. The progress of the reactions was monitored by use of absorption and fluorescence spectroscopy. Our results show that carotenoids may significantly enhance the stability of photosynthetic complexes against oxidation and their protective (antioxidant) effect depends on the type of the oxidant.     

Loss of melanin by eye retinal pigment epithelium cells is associated with its oxidative destruction in melanolipofuscin granules

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.     

Effects of prolonged ACTH-stimulation on adrenocortical accumulation of lipofuscin granules in aged rats

Subcellular deposition of lipofuscin granules is a marker of aging. Human and rodent adrenal cortices accumulate lipofuscin granules with age, but the mechanism that leads to the accumulation is not known. The ultrastructural appearance of lipofuscin granules resembles that of secondary lysosomes. Since adrenocortical subcellular events are predominantly influenced by ACTH action, we therefore studied the effect of prolonged ACTH-stimulation on adrenocortical accumulation of secondary lysosome-like granules, designated herein as lipofuscin granules. Using aged Fischer 344 male rats as a model, we found that a 7 day ACTH stimulation exerts a reducing effect on adrenocortical lipofuscin accumulation. Thus, adrenocortical accumulation of lipofuscin granules with age in vivo may not be an irreversible process.     

Degradation of mitochondria to lipofuscin upon heating and illumination

In the present work, it has been shown that isolated mitochondria can undergo transformation to lipofuscin granules without any additional factors (oxygen saturation or prooxidants). The process occurs spontaneously and slowly at low temperature and rapidly upon heating (thermolipofuscin) or UV irradiation (photolipofuscin). The main contribution to the formation of mitochondrial lipofuscin is made by denatured proteins. The formation of thermolipofuscin depends on lipid peroxidation, while the presence of lipids is not required for photolipofuscin formation. It has been shown that the use of a detergent that is able to degrade mitochondria is necessary to measure the lipofuscin content properly.     

Retinyl palmitate and the blue-light-induced phototoxicity of human ocular lipofuscin

Lipofuscin accumulation in the retinal pigment epithelium is associated with the onset of age-related macular degeneration. Lipofuscin is phototoxic and affects cellular function through the photochemical generation of reactive oxygen intermediates. Mass spectral analysis of solvent extracts of human retinal lipofuscin granules reveals the presence of retinyl palmitate, the substrate for the enzymatic regeneration of 11-cis-retinal. Retinyl palmitate has an appreciable binding constant for phosphatidylcholine liposomes, and based on the glycophospholipids present in lipofuscin, retinal palmitate likely accumulates within the lipid content of the granule. Photochemical oxidation of retinal palmitate generates anhydroretinol, an intracellular signaling retinoid in the signal transduction cascade from the plasma membrane that causes apoptosis by generating reactive oxygen intermediates. These data are used to propose a model for the phototoxicity of lipofuscin.     

Ultrastructure of testicular macrophages in aging mice

Testicular macrophages of aging mice were studied by TEM. Testicular macrophages retained with Leydig cells the close morphological relationships observed in the adult young animals, but digitations were not found. Lipofuscin granules like those of the Leydig cells from aging mice were observed in the cytoplasm. These organelles were generally absent in the testicular macrophages of young adult mice. Testicular macrophages did not display phagocytosis of the lipofuscin granules. In addition, the latter were not found in the intercellular spaces. These observations indicated that lipofuscin granules were formed, at least in a great part, within testicular macrophages as a consequence of metabolic changes occurring with age. Fine lamellar organization was seen in the lipofuscin granules of both Leydig cells and testicular macrophages. Frequently, lipofuscin granules originated from secondary lysosomes containing lipidic vacuoles only. Together with accumulation of the lipofuscin granules, changes of testicular macrophage fine morphology were observed. Endoplasmic reticulum and Golgi apparatus became poorly developed, and coated vesicles were rarely found. Fewer mitochondria were encountered, but their ultrastructure was not altered. These results suggest that in testicular macrophages lipofuscin accumulation is associated with a functional involution.     

Isolation of lipofuscin from the bovine myocardium

The paper describes the procedure of lipofuscin isolation from bovine myocardium with the use of 15-45% (weight/volume) linear gradient of sucrose density and differential centrifugation. Two coloured populations of lipofuscin granules with different density have been distinguished. Electron microscopic studies of the isolated lipofuscin have revealed the homogeneity of fractions and good maintenance of the granules. The electron microscopic, absorptive and luminescent spectral properties of lipofuscin preparations correspond to its properties in situ. The isolation of lipofuscin preparations suggests the possibility of studying the chemical composition and intramolecular structure of the granules.     

Changes in Pollen Autofluorescence Induced by Ozone

The changes in the autofluorescence spectra of intact Philadelphus grandiflorus, Epiphyllum hybridum, and Plantago major pollen have been observed with microspectrofluorometric method after 100-h exposure to ozone (3 h per day for 5 d per week in total dose 5.0 cm³ m^-3). The fluorescence maximum at 530 - 550 nm disappeared in carotenoid-containing pollen of Philadelphus grandiflorus and Epiphyllum hybridum and new maximum at 475 - 480 nm arose that correlated with lipofuscin-like substances observed in the extracts from the pollen grains. The carotenoid-less pollen of Plantago major showed only the increase of the maximum at 470 nm, and no lipofuscin in the extracts. It is supposed that main indicator of the pollen damage by ozone in carotenoid-containing microspores are the lipofuscin-like compounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants.

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or alpha-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and alpha-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.     

Carotenoid limitation of sexual coloration along an environmental gradient in guppies

Carotenoids produce most of the brilliant orange and yellow colours seen in animals, but animals cannot synthesize these pigments and must rely on dietary sources. The idea that carotenoids make good signals because they are a scarce limiting resource was proposed two decades ago and has become the leading hypothesis for the role of carotenoids in animal communication. To our knowledge, until now, however, there has been no direct evidence that carotenoids are a limiting resource in nature. We showed that carotenoid availability in the wild limits the expression of sexual coloration in guppies (Poecilia reticulata), a species in which females prefer males with brighter orange carotenoid-containing spots. Further, the degree of carotenoid limitation varies geographically along a replicated environmental gradient (rainforest canopy cover), which opens new avenues for testing signal evolution theory.     

Quantification of Ceroid and Lipofuscin in Skeletal Muscle

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).     

Electron microscopic study on the thyroid gland of the salamander Hynobius nebulosus in the breeding season

Summary The thyroid gland of adult salamanders, Hynobius nebulosus, in the breeding season was studied by electron microscopy. The follicular cells are different in cell height and fine structures; the taller cells with many cell organelles and granules and the lower cells with a few cell organelles and granules are both present in the same follicle. In the cytoplasm, three types of membrane-bounded granules, namely, cytosomes, colloid droplets, and vacuolar bodies and circular membrane complexes occur. The vacuolar bodies are subdivided into two types; the ordinary type having loosely distributed particles and the specific type containing tubules and/or closely packed filaments, crystalloid structures, except for the particles. The chromophobe colloids within the Bensley-cells correspond to extremely large, ordinary type vacuolar bodies, while the Langendorff-colloid cells possess increased numbers of granular cisternae of endoplasmic reticulum and a ribosome-rich, dense cytoplasmic matrix but not extremely large colloid. The intracytoplasmic circular membrane complexes appear in the Golgi area of cytosome-rich cells. It is suggested that they originate from the Golgi apparatus which was activated to produce many cytosomes. Intranuclear inclusions consisting of microtubules and filaments and tight junctions between two adjacent lateral plasma membranes are occasionally encountered.