RESUMEN
Purpose: Accumulating evidence suggests that dopamine, the major catecholamine in the vertebrate retina, may modulate cAMP-mediated signaling in photoreceptors to optimize vision in the light/dark cycle. The main putative mechanism of dopamine-induced adaptation changes in photoreceptors is activation of D2-like receptors (D2R), which leads to a decrease of the intracellular cAMP level and reduction of protein kinase A (PKA) activity. However, the mechanisms by which dopamine exerts its regulating effect on the phototransduction cascade remain largely unknown. The aim of the present study was to investigate the effects of dopamine and dopamine receptor agonists on rod photoresponses. Methods: The experiments were performed on solitary rods of the Rana ridibunda frog. Photoreceptor currents were recorded using a suction pipette technique. The effects of dopamine (0.1-50 µM) and selective dopamine receptor agonists-D1R agonist SKF-38393 (0.1-50 µM), D2R agonist quinpirole (2.5-50 µM), and D1-D2 receptor heterodimer agonist SKF-83959 (50 µM)-were examined. Results: We found that, when applied to the rod inner segments (RISs), dopamine and dopamine receptor agonists had no effect on photoresponses. In contrast, the rods responded to dopamine and all agonists applied to their outer segments by decreasing sensitivity to light. At the highest tested concentration (50 µM), the most prominent effect on light sensitivity was induced by D1R agonist SKF-38393, while dopamine, D2R agonist quinpirole, and D1-D2 receptor heterodimer agonist SKF-83959 produced somewhat lower and approximately equal effects. Moreover, SKF-38393 reduced sensitivity at all tested concentrations starting from the smallest one (0.1 µM), whereas dopamine and quinpirole started their action from the higher concentrations of 2.5 µM and 50 µM, respectively. In addition, dopamine, SKF-38393, and quinpirole, on average, did not change the intracellular calcium level as judged from the "exchange current", while SKF-83959 increased it by ~1.3 times. Conclusions: Dopamine induces a decrease in rod sensitivity, mostly by reducing the activation rate of the cascade, and to a much lesser extent, speeding up the turning off of the cascade. The sign of the reaction to all tested drugs, lack of selectivity of dopamine and dopamine receptor agonist action, and analysis of factors that determine sensitivity of photoreceptors suggest that, in rod outer segments (ROSs), dopamine action is mediated by D1-D2 receptor heterodimers but not D1R or D2R alone. This work supports the assumption made earlier by other authors that dopamine exercises its regulatory effect via at least two independent mechanisms, which are cAMP and Ca2+ mediated.
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Agonistas de Dopamina/farmacología , Dopamina/farmacología , Fototransducción/efectos de los fármacos , Ranidae/fisiología , Receptores de Dopamina D1/agonistas , Células Fotorreceptoras Retinianas Bastones/fisiología , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Animales , Calcio/metabolismo , Cinética , Luz , Fototransducción/efectos de la radiación , Receptores de Dopamina D1/metabolismo , Segmento Externo de la Célula en Bastón/efectos de los fármacos , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Factores de TiempoRESUMEN
The aim of the present research was to evaluate if the endocannabinoid system (enzymes and receptors) could be modulated by light in rod outer segment (ROS) from bovine retina. First, we analyzed endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in purified ROS obtained from dark-adapted (DROS) or light-adapted (LROS) retinas. To this end, diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL), and lysophosphatidate phosphohydrolase (LPAP) enzymatic activities were analyzed using radioactive substrates. The protein content of these enzymes and of the receptors to which cannabinoids bind was determined by immunoblotting under light stimulus. Our results indicate that whereas DAGL and MAGL activities were stimulated in retinas exposed to light, no changes were observed in LPAP activity. Interestingly, the protein content of the main enzymes involved in 2-AG metabolism, phospholipase C ß1 (PLCß1), and DAGLα (synthesis), and MAGL (hydrolysis), was also modified by light. PLCß1 content was increased, while that of lipases was decreased. On the other hand, light produced an increase in the cannabinoid receptors CB1 and CB2 and a decrease in GPR55 protein levels. Taken together, our results indicate that the endocannabinoid system (enzymes and receptors) depends on the illumination state of the retina, suggesting that proteins related to phototransduction phenomena could be involved in the effects observed.
Asunto(s)
Endocannabinoides/metabolismo , Luz , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Bovinos , Lipoproteína Lipasa/metabolismo , Modelos Biológicos , Monoacilglicerol Lipasas/metabolismo , Fosfolipasa C beta/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
Mutations in the Joubert syndrome-associated small GTPase ARL13B are linked to photoreceptor impairment and vision loss. To determine the role of ARL13B in the development, function, and maintenance of ciliated photoreceptors, we generated a pan-retina knock-out (Six3-Cre) and a rod photoreceptor-specific inducible conditional knock-out (Pde6g-CreERT2) of ARL13B using murine models. Embryonic deletion of ARL13B led to defects in retinal development with reduced cell proliferation. In the absence of ARL13B, photoreceptors failed to develop outer segment (OS) membranous discs and axonemes, resulting in loss of function and rapid degeneration. Additionally, the majority of photoreceptor basal bodies did not dock properly at the apical edge of the inner segments. The removal of ARL13B in adult rod photoreceptor cells after maturation of OS resulted in loss of photoresponse and vesiculation in the OS. Before changes in photoresponse, removal of ARL13B led to mislocalization of rhodopsin, prenylated phosphodiesterase-6 (PDE6), and intraflagellar transport protein-88 (IFT88). Our findings show that ARL13B is required at multiple stages of retinogenesis, including early postnatal proliferation of retinal progenitor cells, development of photoreceptor cilia, and morphogenesis of photoreceptor OS discs regardless of sex. Last, our results establish a need for ARL13B in photoreceptor maintenance and protein trafficking.SIGNIFICANCE STATEMENT The normal development of photoreceptor cilia is essential to create functional, organized outer segments with stacked membrane discs that house the phototransduction proteins necessary for sight. Our study identifies a complex role for ARL13B, a small GTPase linked to Joubert syndrome and visual impairment, at various stages of photoreceptor development. Loss of ARL13B led to defects in retinal proliferation, altered placement of basal bodies crucial for components of the cilium (transition zone) to emanate, and absence of photoreceptor-stacked discs. These defects led to extinguished visual response and dysregulated protein trafficking. Our findings show the complex role ARL13B plays in photoreceptor development, viability, and function. Our study accounts for the severe retinal impairment observed in ARL13B-linked Joubert syndrome patients.
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Factores de Ribosilacion-ADP/fisiología , Retina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Factores de Ribosilacion-ADP/deficiencia , Factores de Ribosilacion-ADP/genética , Envejecimiento/metabolismo , Animales , Axonema/metabolismo , Axonema/ultraestructura , Cilios/metabolismo , Cilios/ultraestructura , Proteínas del Ojo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos C57BL , Biogénesis de Organelos , Transporte de Proteínas/fisiología , Retina/anomalías , Retina/embriología , Retina/crecimiento & desarrollo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Rodopsinas Sensoriales/metabolismoRESUMEN
Oxidative stress is a primary risk factor for both inflammatory and degenerative retinopathies. Our previous data on blue light-irradiated retinas demonstrated an oxidative stress higher in the rod outer segment (OS) than in the inner limb, leading to impairment of the rod OS extra-mitochondrial aerobic metabolism. Here the oxidative metabolism and Reactive Oxygen Intermediates (ROI) production was evaluated in purified bovine rod OS in function of exposure to different illumination conditions. A dose response was observed to varying light intensities and duration in terms of both ROI production and ATP synthesis. Pretreatment with resveratrol, inhibitor of F1Fo-ATP synthase, or metformin, inhibitor of the respiratory complex I, significantly diminished the ROI production. Metformin also diminished the rod OS Complex I activity and reduced the maximal OS response to light in ATP production. Data show for the first time the relationship existing in the rod OS between its -aerobic- metabolism, light absorption, and ROI production. A beneficial effect was exerted by metformin and resveratrol, in modulating the ROI production in the illuminated rod OS, suggestive of their beneficial action also in vivo. Data shed new light on preventative interventions for cone loss secondary to rod damage due to oxidative stress.
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Luz/efectos adversos , Estrés Oxidativo/fisiología , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Antioxidantes/farmacología , Bovinos , Radicales Libres , Estrés Oxidativo/efectos de los fármacos , Segmento Externo de la Célula en Bastón/efectos de los fármacosRESUMEN
The G protein-coupled receptor (GPCR) signaling pathways mediating information exchange across the cell membrane are central to a variety of biological processes and therapeutic strategies, but visualizing the molecular-level details of this exchange has been difficult for all but a few GPCR-G protein complexes. A study by Gao et al. now reports new strategies and tools to obtain receptor complexes in a near-native state, revealing insights into the gross conformational features of rhodopsin-transducin interactions and setting the stage for future studies.
Asunto(s)
Proteínas del Ojo/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Modelos Moleculares , Rodopsina/metabolismo , Transducina/metabolismo , Animales , Proteínas del Ojo/química , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/química , Humanos , Dominios y Motivos de Interacción de Proteínas/efectos de la radiación , Multimerización de Proteína/efectos de la radiación , Rodopsina/química , Segmento Externo de la Célula en Bastón/enzimología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Transducina/químicaRESUMEN
The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (i.e. raftophilicity) upon light-dependent binding with the cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and that Rh is posttranslationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, using biochemical assays, we found that Rh*-Gt complexes in the detergent-resistant membrane are partially resistant to cholesterol depletion by methyl-ß-cyclodextrin and that the Rh-to-Gt stoichiometry in this methyl-ß-cyclodextrin-resistant complex is 2:1. Next, we found that IgG-mediated Rh-Rh cross-linking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Rh depalmitoylation via reduction of thioester linkages blocked the translocation of IgG-cross-linked Rh to the detergent-resistant membrane, highlighting that the two palmitoyl moieties are important for the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs such as Rh can acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.
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Lipoilación , Microdominios de Membrana/metabolismo , Modelos Moleculares , Procesamiento Proteico-Postraduccional , Rana catesbeiana/fisiología , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Proteínas Anfibias/química , Proteínas Anfibias/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Cisteína/química , Cistina/química , Adaptación a la Oscuridad , Dimerización , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Luz , Lipoilación/efectos de la radiación , Microdominios de Membrana/química , Microdominios de Membrana/efectos de la radiación , Oxidación-Reducción , Conformación Proteica/efectos de la radiación , Multimerización de Proteína/efectos de la radiación , Procesamiento Proteico-Postraduccional/efectos de la radiación , Estabilidad Proteica/efectos de la radiación , Rodopsina/química , Segmento Externo de la Célula en Bastón/química , Segmento Externo de la Célula en Bastón/efectos de la radiación , Transducina/química , Transducina/metabolismoRESUMEN
The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT-GTP and ß1γ1 subunit complex. Structural information for the Rho*-GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and ß1γ1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the ß2-adrenergic receptor-GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.
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Proteínas del Ojo/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Modelos Moleculares , Rodopsina/metabolismo , Transducina/metabolismo , Animales , Bovinos , Cristalografía por Rayos X , Detergentes/química , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas del Ojo/aislamiento & purificación , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/aislamiento & purificación , Luz , Microscopía Electrónica , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Conformación Proteica/efectos de la radiación , Multimerización de Proteína/efectos de la radiación , Estabilidad Proteica/efectos de la radiación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Retina/enzimología , Retina/metabolismo , Retina/efectos de la radiación , Rodopsina/química , Rodopsina/aislamiento & purificación , Segmento Externo de la Célula en Bastón/enzimología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Dispersión del Ángulo Pequeño , Solubilidad , Transducina/química , Transducina/genética , Transducina/aislamiento & purificación , Difracción de Rayos XRESUMEN
The accumulation of lipofuscin in the cells of the retinal pigment epithelium (RPE) is thought to play an important role in the development and progression of degenerative diseases of the retina. The bulk of RPE lipofuscin originates in reactions of the rhodopsin chromophore, retinal, with components of the photoreceptor outer segment. The 11-cis retinal isomer is generated in the RPE and supplied to rod photoreceptor outer segments where it is incorporated as the chromophore of rhodopsin. It is photoisomerized during light detection to all-trans and subsequently released by photoactivated rhodopsin as all-trans retinal, which is removed through reduction to all-trans retinol in a reaction requiring metabolic input in the form of NADPH. Both 11-cis and all-trans retinal can form lipofuscin precursor fluorophores in rod photoreceptor outer segments. Increased accumulation of lipofuscin has been suggested to result from excess formation of lipofuscin precursors due to buildup of all-trans retinal released by light exposure. In connection with this suggestion, the Abca4 transporter protein, an outer segment protein defects in which result in recessive Stargardt disease, has been proposed to promote the removal of all-trans retinal by facilitating its availability for reduction. To examine this possibility, we have measured the outer segment levels of all-trans retinal, all-trans retinol, and of lipofuscin precursors after bleaching by imaging the fluorescence of single rod photoreceptors isolated from wild type and Abca4-/- mice. We found that all-trans retinol and all-trans retinal levels increased after bleaching in both wild type and Abca4-/- rods. At all times after bleaching, there was no significant difference in all-trans retinal levels between the two strains. All-trans retinol levels were not significantly different between the two strains at early times, but were lower in Abca4-/- rods at times longer than 20 min after bleaching. Bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels and increased formation of lipofuscin precursors in both wild type and Abca4-/- rods. The results show that conditions that result in buildup of all-trans retinal levels result in increased generation of lipofuscin precursors in both wild type and Abca4-/- rods. The results are consistent with the proposal that Abca4 facilitates the reduction of all-trans retinal to retinol; absence of Abca4 however does not appear to be associated with higher all-trans retinal levels compared to wild type.
Asunto(s)
Lipofuscina/metabolismo , Degeneración Retiniana/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Células Cultivadas , ADN/genética , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Mutación , Imagen Óptica , Degeneración Retiniana/patología , Segmento Externo de la Célula en Bastón/efectos de la radiación , Rayos UltravioletaRESUMEN
Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation.
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Luz , Retina/efectos de la radiación , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Anuros , Ratones , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Fototropismo , Imagen de Lapso de Tiempo , Tomografía de Coherencia ÓpticaRESUMEN
PURPOSE: We previously reported a transgenic Xenopus laevis model of retinitis pigmentosa in which tadpoles express the bovine form of P23H rhodopsin (bP23H) in rod photoreceptors. In this model, retinal degeneration was dependent on light exposure. Here, we investigated ultrastructural changes that occurred in the rod photoreceptors of these retinas when exposed to light. METHODS: Tadpoles expressing bP23H in rods were transferred from constant darkness to a 12-hour light:12-hour dark (12L:12D) regimen. For comparison, transgenic tadpoles expressing an inducible form of caspase 9 (iCasp9) were reared in a 12L:12D regimen, and retinal degeneration was induced by administration of the drug AP20187. Tadpoles were euthanized at various time points, and eyes were processed for confocal light and transmission electron microscopy. RESULTS: We observed defects in outer and inner segments of rods expressing bP23H that were aggravated by light exposure. Rod outer segments exhibited vesiculations throughout and were rapidly phagocytosed by the retinal pigment epithelium. In rod inner segments, we observed autophagic compartments adjacent to the endoplasmic reticulum and extensive vesiculation at later time points. These defects were not found in rods expressing iCasp9, which completely degenerated within 36 hours after drug administration. CONCLUSIONS: Our results indicate that ultrastructural defects in outer and inner segment membranes of bP23H expressing rods differ from those observed in drug-induced apoptosis. We suggest that light-induced retinal degeneration caused by P23H rhodopsin occurs via cell death with autophagy, which may represent an attempt to eliminate the mutant rhodopsin and/or damaged cellular compartments from the secretory pathway.
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Autofagia/efectos de la radiación , Luz/efectos adversos , Traumatismos Experimentales por Radiación/fisiopatología , Segmento Interno de las Células Fotorreceptoras Retinianas/efectos de la radiación , Retinitis Pigmentosa/fisiopatología , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Animales Modificados Genéticamente , Caspasa 9/metabolismo , Modelos Animales de Enfermedad , Fotoperiodo , Segmento Interno de las Células Fotorreceptoras Retinianas/ultraestructura , Células Fotorreceptoras Retinianas Bastones , Retinitis Pigmentosa/inducido químicamente , Retinitis Pigmentosa/patología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/ultraestructura , Tacrolimus/análogos & derivados , Xenopus laevisRESUMEN
Most vertebrate retinas contain two types of photoreceptor cells, rods and cones, which show different photoresponses to mediate scotopic and photopic vision, respectively. These cells contain different types of visual pigments, rhodopsin and cone visual pigments, respectively, but little is known about the molecular properties of cone visual pigments under physiological conditions, making it difficult to link the molecular properties of rhodopsin and cone visual pigments with the differences in photoresponse between rods and cones. Here we prepared bovine and mouse rhodopsin (bvRh and mRh) and chicken and mouse green-sensitive cone visual pigments (cG and mG) embedded in nanodiscs and applied time-resolved fluorescence spectroscopy to compare their Gt activation efficiencies. Rhodopsin exhibited greater Gt activation efficiencies than cone visual pigments. Especially, the Gt activation efficiency of mRh was about 2.5-fold greater than that of mG at 37 °C, which is consistent with our previous electrophysiological data of knock-in mice. Although the active state (Meta-II) was in equilibrium with inactive states (Meta-I and Meta-III), quantitative determination of Meta-II in the equilibrium showed that the Gt activation efficiency per Meta-II of bvRh was also greater than those of cG and mG. These results indicated that efficient Gt activation by rhodopsin, resulting from an optimized active state of rhodopsin, is one of the causes of the high amplification efficiency of rods.
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Células Fotorreceptoras Retinianas Conos/metabolismo , Pigmentos Retinianos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Transducina/metabolismo , Animales , Bovinos , Pollos , Células HEK293 , Humanos , Cinética , Luz , Ratones , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Análisis EspectralRESUMEN
Photostasis is a phenomenon where the photoreceptor outer segment (OS) length and its rhodopsin content vary depending on environmental lighting. When light is reduced for extended periods, it is argued that OS lengthen and its rhodopsin concentration rises to increase photon capture in darker environment. Increases in OS length may occur because the retinal pigment epithelium (RPE) cells reduce OS consumption in prolonged darkness. But sample sizes in assessing changes in OS length have been small, and results highly varied with no statistical analysis ever offered. Further, animals used were often albinos, which have abnormal RPE cells. Here we keep pigmented and albino mice for 21 days in darkness and compare OS length with those in a normal 12:12 light/dark environment. We measured approximately 1300 OS but found no statistically significant difference in their lengths between light and dark groups in either pigmentation phenotype, although there was a small trend in the data favoring OS extension in the dark. Given that earlier studies were undertaken on limited samples with no statistical analysis, our data pose serious questions for the notion of mammalian photostasis in terms of significant OS plasticity.
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Luz , Rodopsina/fisiología , Segmento Externo de la Célula en Bastón/fisiología , Envejecimiento/fisiología , Animales , Adaptación a la Oscuridad/fisiología , Ambiente , Ojo/crecimiento & desarrollo , Enucleación del Ojo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Disco Óptico/fisiología , Tetróxido de Osmio , Epitelio Pigmentado Ocular/fisiología , Pigmentación , Retina/fisiología , Rodopsina/metabolismo , Rodopsina/efectos de la radiación , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Fijación del TejidoRESUMEN
PURPOSE: In a previous paper, we showed that chemiluminescence from radical recombination (initiated by lipid peroxidation and propagated by polyunsaturated fatty acids [PUFA]) has a bleaching effect comparable to that caused by light on the rhodopsin of retinal rod outer segment (RdOS) prepared from bovine eyes. Photons generated by radical recombination were suggested to be the origin of phosphenes perceived as light flashes by the human eye. Irradiation with (12)C carbon ions was used in this study to stimulate radical production, propagation and recombination leading to photoluminescence. MATERIALS AND METHODS: (12)C radiation bleached RdOS rhodopsin, but structural damage increasing with the radiation dose was also observed. For this reason, only the effects on rhodopsin at doses producing next to negligible biodamage and permitting regeneration have been considered as bleaching effects. RESULTS: (12)C irradiation bleached RdOS rhodopsin, but increasing structural damage with radiation dose was also observed. For the measure of bleaching and to reveal dose response effects on rhodopsin that were able to be regenerated only results from doses producing nearly negligible biodamage have been considered. CONCLUSIONS: Recombination of radicals appears responsible for the release of photons with subsequent bleaching of rhodopsin. This effect could have an important role in the generation of the anomalous visual effects (phosphenes) experienced by patients during hadrotherapy or by astronauts in space.
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Carbono/efectos adversos , Medio Ambiente Extraterrestre , Luz/efectos adversos , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Bovinos , Humanos , Protección Radiológica , Segmento Externo de la Célula en Bastón/metabolismoRESUMEN
The present study shows the selective light-dependent distribution of 1,2-diacylglycerol kinase epsilon (DAGKÉ) in photoreceptor cells from bovine and albino rat retina. Immunofluorescence microscopy in isolated rod outer segments from bleached bovine retinas (BBROS) revealed a higher DAGKÉ signal than that found in rod outer segments from dark-adapted bovine retinas (BDROS). The light-dependent outer segment localization of DAGKÉ was also observed by immunohistochemistry in retinas from albino rats. DAGK activity, measured in terms of phosphatidic acid formation from a) [(3)H]DAG and ATP in the presence of EGTA and R59022, a type I DAGK inhibitor, or b) [γ-(32)P]ATP and 1-stearoyl, 2-arachidonoylglycerol (SAG), was found to be significantly higher in BBROS than in BDROS. Higher light-dependent DAGK activity (condition b) was also found when ROS were isolated from dark-adapted rat retinas exposed to light. Western blot analysis of isolated ROS proteins from bovine and rat retinas confirmed that illumination increases DAGKÉ content in the outer segments of these two species. Light-dependent DAGKÉ localization in the outer segment was not observed when U73122, a phospholipase C inhibitor, was present prior to the exposure of rat eyecups (in situ model) to light. Furthermore, no increased PA synthesis from [(3)H]DAG and ATP was observed in the presence of neomycin prior to the exposure of bovine eyecups to light. Interestingly, when BBROS were pre-phosphorylated with ATP in the presence of 1,2-dioctanoyl sn-glycerol (di-C8) or phorbol dibutyrate (PDBu) as PKC activation conditions, higher DAGK activity was observed than in dephosphorylated controls. Taken together, our findings suggest that the selective distribution of DAGKÉ in photoreceptor cells is a light-dependent mechanism that promotes increased SAG removal and synthesis of 1-stearoyl, 2-arachidonoyl phosphatidic acid in the sensorial portion of this cell, thus demonstrating a novel mechanism of light-regulated DAGK activity in the photoreceptors of two vertebrate species.
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Diacilglicerol Quinasa/metabolismo , Estimulación Luminosa , Segmento Externo de la Célula en Bastón/enzimología , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Western Blotting , Bovinos , Adaptación a la Oscuridad , Diacilglicerol Quinasa/antagonistas & inhibidores , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Luz , Ácidos Fosfatidicos/metabolismo , Pirimidinonas/farmacología , Pirrolidinonas/farmacología , Ratas , Ratas Wistar , Segmento Externo de la Célula en Bastón/efectos de los fármacos , Tiazoles/farmacologíaRESUMEN
The mechanisms that control the natural rate of lipofuscin accumulation in the retinal pigment epithelial (RPE) cell and its stability over time are not well understood. Similarly, the contributions of retinoids, phospholipids and oxidation to the rate of accumulation of lipofuscin are uncertain. The experiments in this study were conducted to explore the individual contribution of rod outer segments (ROS) components to lipofuscin formation and its accumulation and stability over time. During the period of 14 days incubation of ROS, lipofuscin-like autofluorescence (LLAF) determined at two wavelengths (530 and 585 nm) by fluorescence-activated cell sorting (FACS) was measured from RPE cells. The autofluorescence increased in an exponential manner with a strong linear component between days 1 and 7. The magnitude of the increase was larger in cells incubated with 4-hydroxynonenal (HNE-ROS) compared with cells incubated with either bleached or unbleached ROS, but with a different spectral profile. A small (10-15%) decrease in LLAF was observed after stopping the ROS feeding for 14 days. The phagocytosis rate of HNE-ROS was higher than that of either bleached or unbleached ROS during the first 24 h of supplementation. Among the different ROS components, the increase of LLAF was highest in cells incubated with all-trans-retinal. Surprisingly, incubation with 11-cis-retinal and 9-cis-retinal also resulted in strong LLAF increase, comparable to the increase induced by all-trans-retinal. Supplementation with liposomes containing phosphatidylethanolamine (22: 6-PE) and phosphatidylcholine (18:1-PC) also increased LLAF, while incubation with opsin had little effect. Cells incubated with retinoids demonstrated strong dose-dependence in LLAF increase, and the magnitude of the increase was 2-3 times higher at 585 nm compared to 530 nm, while cells incubated with liposomes showed little dose-dependence and similar increase at both wavelengths. Very little difference in LLAF was noted between cells incubated with either unbleached or bleached ROS under any conditions. In summary, results from this study suggest that supplementation with various ROS components can lead to an increase in LLAF, although the autofluorescence generated by the different classes of components has distinct spectral profiles, where the autofluorescence induced by retinoids results in a spectral profile closest to the one observed from human lipofuscin. Future fluorescence characterization of LLAF in vitro would benefit from an analysis of multiple wavelengths to better match the spectral characteristics of lipofuscin in vivo.
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Lipofuscina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/farmacología , Segmento Externo de la Célula en Bastón/efectos de los fármacos , Aldehídos/farmacología , Animales , Bovinos , Línea Celular , Células Cultivadas , Diterpenos , Citometría de Flujo , Humanos , Liposomas , Microscopía Confocal , Fagocitosis/fisiología , Fosfatidilcolinas/farmacología , Fosfatidiletanolaminas/farmacología , Retinaldehído/farmacología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Tretinoina/farmacologíaRESUMEN
OBJECTIVE: Changes in fundus autofluorescence (AF) are observed in various retinal disorders. Lipofuscin accumulation within the retinal pigment epithelium (RPE) is a source of fundus AF (FAF); however, the causes of short-term increases in FAF observed in inflammatory conditions or after laser treatment are unknown. Here, we describe an RPE cell culture model that is useful for investigations of FAF. METHODS: ARPE-19 cells were cultured in 2-well chamber slides. Cells were exposed to isolated rabbit photoreceptor outer segments (POS) to mimic in vivo phagocytic activity. The AF of RPE cells exposed to POS was measured before and after focal coagulation of the cultures. AF was measured over a period of 4 weeks. Cell lysates were examined by two-dimensional (2D) gel electrophoresis and mass spectrometry analysis. RESULTS: The exposure of ARPE cells to POS did not lead to increased AF; however, after coagulation, cells exposed to POS showed a statistically significant increase in AF (p < 0.05). 2D electrophoresis of the cell lysates revealed changes in 3 proteins. One of these proteins, identified by mass spectrometry as ezrin-radixin-moesin-binding phosphoprotein 50, was reduced in the coagulated cell population. CONCLUSIONS: We have established an in vitro model of RPE cells in culture that can be used to evaluate the development of AF and changes in cellular proteins that accompany laser photocoagulation.
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Fluorescencia , Coagulación con Láser , Epitelio Pigmentado de la Retina/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Células Cultivadas , Modelos Biológicos , Conejos , Segmento Externo de la Célula en Bastón/fisiologíaRESUMEN
Vigabatrin (VGB) is a commonly prescribed antiepileptic drug designed to inhibit GABA-transaminase, effectively halting seizures. Unfortunately, VGB treatment is also associated with the highest frequencies of peripheral visual field constriction of any of the antiepileptic drugs and the mechanisms that lead to these visual field defects are uncertain. Recent studies have demonstrated light exposure exacerbates vigabatrin-induced retinal toxicity. We further assessed this relationship by examining the effects of vigabatrin treatment on the retinal structures of mice with genetically altered photoreception. In keeping with previous studies, we detected increased toxicity in mice exposed to continuous light. To study whether cone or rod photoreceptor function was involved in the pathway to toxicity, we tested mice with mutations in the cone-specific Gnat2 or rod-specific Pde6g genes, and found the mutations significantly reduced VGB toxicity. Our results confirm light is a significant enhancer of vigabatrin toxicity and that a portion of this is mediated, directly or indirectly, by phototransduction signaling in rod and cone photoreceptors.
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Anticonvulsivantes/toxicidad , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Segmento Externo de la Célula en Bastón/efectos de los fármacos , Vigabatrin/toxicidad , Visión Ocular/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Luz , Ratones , Ratones Transgénicos , Retina/anomalías , Retina/efectos de los fármacos , Retina/fisiopatología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Segmento Externo de la Célula en Bastón/fisiología , Segmento Externo de la Célula en Bastón/efectos de la radiaciónRESUMEN
Rod outer segments of photoreceptors are characterized by rhodopsin, a membrane protein surrounded by phospholipids containing a very high concentration of polyunsaturated fatty acids. These fatty acids can propagate free radicals, initiated by peroxidation, whose recombination is eventually associated with light emission as chemiluminescence. The results reported here indicate that this effect produces an isomerization of the retinal (bleaching effect) of the rhodopsin, similar to that induced by light in normal vision. In vitro experiments on detergent-suspended rod outer segments (RdOS) from bovine eyes, using an enzymatic source of radicals, xanthine/xanthine oxidase, were carried out. The results indicate that the proposed mechanism is likely, because they can show the bleaching of rhodopsin in RdOS, owing to its extraordinary sensitivity. Thus this mechanism is, also, a possible explanation for anomalous visual effects such as light flashes (phosphene-like) perceived by humans. The functionality of the rhodopsin in the RdOS was first tested by visible light. Rhodopsin reactivation after bleaching was obtained by adding cis-retinal to the suspension, demonstrating the reversibility of the bleaching process. A special experimental system was developed to observe the bleaching from luminescence by radical recombination, avoiding physical contact between the rod outer segment suspension and the radicals to prevent radical-induced damage and modifications of the delicate structure of the rod outer segment.
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Ácidos Grasos Insaturados/metabolismo , Radicales Libres/metabolismo , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Bovinos , Ácidos Grasos Insaturados/química , Depuradores de Radicales Libres/farmacología , Radicales Libres/química , Glutatión/farmacología , Isomerismo , Peroxidación de Lípido , Luminiscencia , Fotoblanqueo , Rodopsina/química , Segmento Externo de la Célula en Bastón/metabolismo , Xantina/química , Xantina Oxidasa/químicaRESUMEN
Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTRCNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%-70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.
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Secuencia Conservada , Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Evolución Molecular , Femenino , Proteína Adaptadora GRB7/genética , Células HEK293 , Humanos , Luz , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/efectos de la radiación , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Segmento Externo de la Célula en Bastón/efectos de la radiación , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The visual pigment, rhodopsin, consists of opsin protein with 11-cis retinal chromophore, covalently bound. Light activates rhodopsin by isomerizing the chromophore to the all-trans conformation. The activated rhodopsin sets in motion a biochemical cascade that evokes an electrical response by the photoreceptor. All-trans retinal is eventually released from the opsin and reduced to vitamin A. Rod and cone photoreceptors contain vast amounts of rhodopsin, so after exposure to bright light, the concentration of vitamin A can reach relatively high levels within their outer segments. Since a retinal analog, ß-ionone, is capable of activating some types of visual pigments, we tested whether vitamin A might produce a similar effect. In single-cell recordings from isolated dark-adapted salamander green-sensitive rods, exogenously applied vitamin A decreased circulating current and flash sensitivity and accelerated flash response kinetics. These changes resembled those produced by exposure of rods to steady light. Microspectrophotometric measurements showed that vitamin A accumulated in the outer segments and binding of vitamin A to rhodopsin was confirmed in in vitro assays. In addition, vitamin A improved the sensitivity of photoreceptors to ultraviolet (UV) light. Apparently, the energy of a UV photon absorbed by vitamin A transferred by a radiationless process to the 11-cis retinal chromophore of rhodopsin, which subsequently isomerized. Therefore, our results suggest that vitamin A binds to rhodopsin at an allosteric binding site distinct from the chromophore binding pocket for 11-cis retinal to activate the rhodopsin, and that it serves as a sensitizing chromophore for UV light.