Bovine Retina Research Articles

The interaction network of rhodopsin involving the heterotrimeric G-protein transducin and the monomeric GTPase Rac1 is determined by distinct binding processes.

The monomeric G-protein Rac1, a member of the family of Rho/Rac/Cdc42 GTPases, is involved in light-induced photoreceptor degeneration, but its specific role remains elusive. In particular, reports on Rac1 interacting with the visual pigment rhodopsin are puzzling and need a more quantitative examination. We probed the presence of Rac1 in rod outer segments by immunohistochemical staining of bovine retinae and western blot analysis of isolated rod outer segments. Rac1 was present throughout the whole retina except in the outer and inner nuclear layers, but was strongly expressed in photoreceptor cells. Rac1 was distributed in three different fractions of rod outer segments: one fraction was soluble in detergents, a second fraction cosegregated with lipid rafts, and a third fraction was associated with lipid bilayer free axonemal/cytoskeletal structures. We also investigated the interaction between rhodopsin and Rac1 by using surface plasmon resonance spectroscopy under dark and light conditions. Biophysical interaction studies revealed that Rac1 could interact with rhodopsin, but in a light-independent manner, and kinetic analysis indicated that binding of Rac1 occurred with lower affinity and speed than the association of transducin and rhodopsin. Thus, in dark-adapted rod cells, Rac1 cannot compete with transducin for binding to rhodopsin, and signalling can proceed normally. Instead, the concentration of transducin has to drop significantly so that Rac1 can bind to rhodopsin; in the outer segment, this occurs only under intense illumination, when transducin is translocated to the inner segment.

Role of the non-enzymatic metabolite of eicosapentaenoic acid, 5-epi-5-F3t-isoprostane in the regulation of [ (3)H]D-aspartate release in isolated bovine retina.

We have evidence that F2-isoprostanes (F2-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F3-IsoPs are generated in mammals, in vivo, their pharmacological actions on neurotransmitter release remain unknown. In this study, we investigated the effect of 5-epi-5-F3t-IsoP on K(+)-evoked [(3)H]D-aspartate release in isolated bovine retina using the superfusion method. Furthermore, we examined the role of arachidonic acid metabolites in the regulation of the neurotransmitter release by this novel IsoP. In the concentration range, 0.01nM-0.1µM, 5-epi-5-F3t-IsoP inhibited K(+)-evoked [(3)H]D-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9% at 0.1µM (IC30=1nM). The prostanoid receptor antagonists, AH 6809 (EP1-3/DP; 10µM), SC 51322 (EP1; 10µM) and SC 19220 (EP1; 1µM) partially reversed 5-epi-5-F3t-IsoP-mediated inhibition of K(+)-induced [(3)H]D-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3μM) unmasked a biphasic action of 5-epi-5-F3t-IsoP that was inhibitory at lower (0.1-10pM) and stimulatory at higher concentrations (≥0.1nM). The prostanoid pathway antagonists, BAY-u3405 (10μM; TP/DP-receptors), SQ 29548 (10μM; TP-receptor) and ozagrel (10μM; Tx-synthase inhibitor) abolished the stimulatory action of the 5-epi-5-F3t-IsoP (0.1μM) on neurotransmitter release. In conclusion, 5-epi-5-F3t-IsoP attenuates K(+)-induced [(3)H]D-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP1-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5-epi-5-F3t-IsoP that is inhibitory at low concentrations and stimulatory at higher concentrations. Products of the thromboxane synthase pathway may partially account for the stimulatory action of this F3-IsoP on isolated bovine retina.

Argonaute high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation reveals a snapshot of miRNA gene regulation in the mammalian retina.

Mounting evidence points to roles for miRNA gene regulation in promoting development, function, and cell survival in the mammalian retina. However, little is known regarding which retinal genes are targets of miRNAs. Here, we employed a systematic, nonbiased, biochemical approach to identify targets of miRNA gene regulation in the bovine retina, a common model species for vision research. Using Argonaute high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation analysis, we identified 348 high-confidence miRNA target sites within 261 genes. This list was enriched in rod and cone photoreceptor genes and included 28 retinal disease genes, providing further evidence of a role of miRNAs in the pathology of blinding diseases.

Testing the effects of the dye acid violet-17 on retinal function for an intraocular application in vitreo-retinal surgery.

To facilitate epiretinal or inner limiting membrane peeling, dyes like Indocyanine Green (ICG) as well as Trypan Blue (TB) were used so far. However, toxic effects on the retina were described for both dyes. The aim of our study was to investigate the effects of a novel vital dye Acid violet-17 (AV-17) on retinal histology and function to assess a possible application in vitreo-retinal surgery. AV-17 was dissolved in a solvent with heavy water. An electroretinogram was recorded on perfused bovine retina. After reaching stable b-wave amplitudes, AV-17 (0.125-0.5 mg/ml) or the solvent was applied epiretinally for 30-300 seconds. The b-wave amplitudes were recorded before, during, and after treatment. Cultures of bovine retina were incubated for 30 or 300 seconds with the dye or solvent and processed for live/dead staining, immunohistochemistry, and immunoblotting. Reductions of the b-wave amplitudes were observed directly after the exposure to AV-17, which were rapidly and completely reversible within the recovery period for all exposure times at the concentrations of 0.125 and 0.25 mg/ml as opposed to the partial recovery after exposure to 0.5 mg/ml. A high degree of damage in the ganglion cell layer (GCL) and glial reactivity were detected at the concentrations of 0.25 and 0.5 mg/ml but not after exposure to lower concentrations or the solvent. Application of AV-17 at a concentration of up to 0.125 mg/ml was well tolerated in terms of retinal function, survival in the GCL, and glial reactivity whereas higher concentrations are not recommended.

Differential synaptic distribution of the scaffold proteins Cask and Caskin1 in the bovine retina.

Scaffold proteins organize pre- and postsynaptic compartments and align pre- and postsynaptic events. Cask is a multi-domain scaffold protein essential for brain synaptic functions. Caskin1 is a recently discovered, brain-specific Cask-interacting multi-domain protein of unknown function. In the present study, we determined the localization of these scaffold proteins in the bovine retina. The retina contains tonically active ribbon synapses and conventional synapses. We found Cask highly enriched in virtually all retinal synapses. Cask was localized in close vicinity to the active zone protein RIM1/2 in ribbon and conventional synapses. Caskin1 is also enriched in retinal synapses but is present only in a subset of Cask-positive synapses. These findings suggest that Cask plays an important role in all retinal synapses. In contrast, Caskin1 appears to execute more specialized functions in distinct sets of retinal synapses, possibly for neuronal pathway formation and stabilization of distinct synaptic contacts.

Examination of VLC-PUFA-deficient photoreceptor terminals.

Juvenile-onset autosomal dominant Stargardt-like macular dystrophy (STGD3) is caused by mutations in ELOVL4 (elongation of very long fatty acids-4), an elongase necessary for the biosynthesis of very long chain fatty acids (VLC-FAs ≥ C26). Photoreceptors are enriched with VLC polyunsaturated fatty acids (VLC-PUFAs), which are necessary for long-term survival of rod photoreceptors. The purpose of these studies was to determine the effect of deletion of VLC-PUFAs on rod synaptic function in retinas of mice conditionally depleted (KO) of Elovl4. Retina function was assessed in wild-type (WT) and KO by electroretinography. Outer plexiform structure was evaluated by immunofluorescence and transmission electron microscopy. Single-cell recordings measured rod ion channel operation and rod bipolar glutamate signaling. Sucrose gradient centrifugation was used to isolate synaptosomes from bovine retina. Proteins and lipids were analyzed by Western blotting and tandem mass spectroscopy, respectively. Inner retinal responses (b-wave, oscillatory potentials, and scotopic threshold responses) of the ERG were decreased in the KO mice compared to controls. However the rod ion channel operation and bipolar glutamate responses were comparable between groups. Biochemical analysis revealed that conventional and ribbon synapses have VLC-PUFAs. Ultrastructural analysis showed that the outer plexiform layer was disorganized and the diameter of vesicles in rod terminals was smaller in the KO mice. Very long chain PUFAs affect rod function by contributing to synaptic vesicle size, which may alter the dynamics of synaptic transmission, ultimately resulting in a loss of neuronal connectivity and death of rod photoreceptors.

Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein.

The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system.

A novel light-dependent activation of DAGK and PKC in bovine photoreceptor nuclei

In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacylglycerol kinase (EC 2.7.1.107, DAGK) and the phosphorylated protein kinase C alpha (pPKCα) in a nuclear fraction of photoreceptor cells from bovine retinas. A nuclear fraction enriched in small nuclei from photoreceptor cells (PNF), was obtained when a modified nuclear isolation protocol developed by our laboratory was used. We measured and compared DAGK activity as phosphatidic acid (PA) formation in PNF obtained from retinas exposed to light and in retinas kept in darkness using [γ-32P]ATP or [3H]DAG. In the absence of exogenous substrates and detergents, no changes in DAGK activity were observed. However, when DAGK activity assays were performed in the presence of exogenous substrates, such as stearoyl arachidonoyl glycerol (SAG) or dioleoyl glycerol (DOG), and different detergents (used to make different DAGK isoforms evident), we observed significant light effects on DAGK activity, suggesting the presence of several DAGK isoforms in PNF. Under conditions favoring DAGKζ activity (DOG, Triton X-100, dioleoyl phosphatidylserine and R59022) we observed an increase in PA formation in PNF from retinas exposed to light with respect to those exposed to darkness. In contrast, under conditions favoring DAGKɛ (SAG, octylglucoside and R59022) we observed a decrease in its activity. These results suggest different physiological roles of the above-mentioned DAGK isoforms. Western blot analysis showed that whereas light stimulation of bovine retinas increases DAGKζ nuclear content, it decreases DAGKɛ and DAGKβ content in PNF. The role of PIP2-phospholipase C in light-stimulated DAGK activity was demonstrated using U73122. Light was also observed to induce enhanced pPKCα content in PNF. The selective distribution of DAGKζ and ɛ in PNF could be a light-dependent mechanism that in vertebrate retina promotes selective DAG removal and PKC regulation.

Mercury-induced dark-state instability and photobleaching alterations of the visual g-protein coupled receptor rhodopsin.

Mercuric compounds were previously shown to affect the visual phototransduction cascade, and this could result in vision impairment. We have analyzed the effect of mercuric chloride on the structure and stability of the dim light vision photoreceptor rhodopsin. For this purpose, we have used both native rhodopsin immunopurified from bovine retinas and a recombinant mutant rhodopsin carrying several Cys to Ser substitutions in order to investigate the potential binding site of mercury on the receptor. Our results show that mercuric chloride dramatically reduces the stability of dark-state rhodopsin and alters the molecular features of the photoactived conformation obtained upon illumination by eliciting the formation of an altered photointermediate. The thermal bleaching kinetics of native and mutant rhodopsin is markedly accelerated by mercury in a concentration-dependent manner, and its chromophore regeneration ability is severely reduced without significantly affecting its G-protein activation capacity. Furthermore, fluorescence spectroscopic measurements on the retinal release process, ensuing illumination, suggest that mercury impairs complete retinal release from the receptor binding pocket. Our results provide further support for the capacity of mercury as a hazardous metal ion with reported deleterious effect on vision and provide a molecular explanation for such an effect at the rhodopsin photoreceptor level. We suggest that mercury could alter vision by acting in a specific manner on the molecular components of the retinoid cycle, particularly by modifying the ability of the visual photoreceptor protein rhodopsin to be regenerated and to be normally photoactivated by light.

Binding of rhodopsin and rhodopsin analogues to transducin, rhodopsin kinase and arrestin-1.

To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. Rod outer segments (ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtained by extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-((3)H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of (γ-(32)P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a light-dependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.

Investigating retinal toxicity of tempol in a model of isolated and perfused bovine retina.

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl) is a membrane-permeable superoxide dismutase and potentially neuroprotective substance. This study evaluates the retinal tolerance of 0.5mM, 1mM, 2mM, and 5mM tempol measured by the electroretinogram (ERG) of an isolated and perfused retina whole mount. For functionality testing, bovine retinas were prepared and perfused with an oxygen-saturated standard solution, and the ERG was recorded until stable b-wave amplitudes were reached. Tempol concentrations of 0.5mM, 1mM, 2mM, and 5mM were tested for 45minutes. To investigate the effects on photoreceptor function, 1mM aspartate was added to suppress the b-wave and obtain isolated a-waves. ERG amplitudes were monitored for 100minutes. While no toxic effects for concentrations of 0.5mM and 1mM tempol could be detected, concentrations of 2mM tempol and higher caused statistically significant negative effects on the b-wave amplitude (-38%, p = 0.02 for 2mM; -54%, p =0.02 for 5mM). The a-wave amplitude remained stable even at higher concentrations. Although the photoreceptors seem to have a tolerance to high concentrations of tempol, higher intravitreal concentrations than 1mM should be considered critical.

Interaction between hydrogen sulfide and nitric oxide in isolated bovine retina (1060.2)

Hydrogen sulfide (H2S) and nitric oxide (NO) are gaseous mediators that are endogenously produced in mammalian ocular tissues. We tested the hypothesis that NO can regulate the basal production of H2S in isolated bovine neural retina. Methods: H2S and NO concentrations in bovine retina was measured using well‐established colorimetric assays. Retina homogenates were incubated at 37°C for different time periods for determination of basal endogenous production of H2S and NO. In studies examining the effects of the NO donor (SNP) and NO synthase inhibitor (L‐NAME) on basal levels of H2S production, retina tissues were homogenized in the presence of the respective compounds before assays were performed. Results: L‐NAME (300 nM ‐ 500 µM) caused a concentration‐dependent decrease (p < 0.05) in the basal endogenous production of H2S reaching a maximum at 500 µM. On the other hand, SNP (10 ‐ 100 µM) enhanced significantly (p < 0.05) the endogenous H2S production. In the concentration range, 10 µM to 300 µM, L‐cysteine attenuated basal endogenous production of NO reaching a maximum at 300 µM. Conclusions: We conclude that NO can interact with the pathway/s involved in the production of H2S in the retina. Furthermore, endogenously produced H2S can regulate basal NO concentrations. Further studies are needed to understand the pharmacological/physiological basis of the interaction between these two gases in the retina.

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.

The Concentration-Dependent Effects of Indocyanine Green on Retinal Function in the Electrophysiological ex vivo Model of Isolated Perfused Vertebrate Retina

Background: Dye solutions such as indocyanine green (ICG) are used for the staining of intraocular structures. The aim of the presented study was to investigate the effects of ICG on bovine retinal function using different concentrations of ICG. Methods: Bovine retina preparations were perfused with a standard solution and the electroretinogram was recorded. The nutrient solution was substituted by an ICG solution at varying concentrations for 45 min. Afterwards the preparations were reperfused with standard solution for at least 85 min. Results: Significant reductions in b-wave amplitude were found for concentrations of 0.0025% (p = 0.0099) and 0.025% (p = 0.0378). For the concentration of 0.025%, the b-wave amplitude remained significantly decreased (p = 0.0082) after the observation period, but a full recovery of the b-wave was observed for the concentration of 0.0025% (p = 0.1917). Conclusion: Intraocular application of sufficient ICG concentrations for internal limiting membrane staining seems not possible without interfering with retinal function.

Structural and Functional Analysis of the Native Peripherin-ROM1 Complex Isolated from Photoreceptor Cells

Peripherin and its homologue ROM1 are retina-specific members of the tetraspanin family of integral membrane proteins required for morphogenesis and maintenance of photoreceptor outer segments, regions that collect light stimuli. Over 100 pathogenic mutations in peripherin cause inherited rod- and cone-related dystrophies in humans. Peripherin and ROM1 interact in vivo and are predicted to form a core heterotetrameric complex capable of creating higher order oligomers. However, structural analysis of tetraspanin proteins has been hampered by their resistance to crystallization. Here we present a simplified methodology for high yield purification of peripherin-ROM1 from bovine retinas that permitted its biochemical and biophysical characterization. Using size exclusion chromatography and blue native gel electrophoresis, we confirmed that the core native peripherin-ROM1 complex exists as a tetramer. Peripherin, but not ROM1, is glycosylated and we examined the glycosylation site and glycan composition of ROM1 by liquid chromatographic tandem mass spectrometry. Mass spectrometry was used to analyze the native complex in detergent micelles, demonstrating its tetrameric state. Our electron microscopy-generated structure solved to 18 Å displayed the tetramer as an elongated structure with an apparent 2-fold symmetry. Finally, we demonstrated that peripherin-ROM1 tetramers induce membrane curvature when reconstituted in lipid vesicles. These results provide critical insights into this key retinal component with a poorly defined function.

Electrophysiological toxicity testing of VEGF Trap‐Eye in an isolated perfused vertebrate retina organ culture model

Age-related macular degeneration (AMD) is the leading cause of visual impairment in Western nations. Since the discovery of the importance of vascular endothelial growth factor (VEGF) in the pathogenesis of neovascular AMD, anti-VEGF agents including pegaptanib, ranibizumab and bevacizumab provide a treatment option to improve vision in affected persons. VEGF Trap-Eye (Aflibercept) is a new agent available for the treatment of exudative AMD. The molecule is a receptor decoy with a longer half-life and a higher affinity to VEGF compared with ranibizumab or bevacizumab. The presented study has been designed to evaluate the short-term toxic effects of VEGF Trap-Eye on retinal function during and after direct exposure to the drug. Isolated bovine retinas were perfused with an oxygen-saturated nutrient solution, and the electroretinogram (ERG) was recorded using silver/silver chloride electrodes. A total of 0.5mg or 2mg VEGF Trap-Eye was added to the nutrient solution and retinas were exposed for 45min, followed by a washout period of 100min. The percentage of a- and b-wave reduction at the end of the washout was compared with the baseline values. Additionally, retinal whole mount cultures were exposed for 24hr to VEGF Trap-Eye, and the amount of apoptotic cells were determined using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labelling (TUNEL) assay. During simulation of intraocular application, no significant reduction in the a-wave amplitude for 0.5mg (2.70%, p=0.37) and 2mg (3.84%, p=0.37) VEGF Trap-Eye and b-wave amplitude for 0.5mg (19.68%, p=0.17) and 2mg (24.1%, p=0.06) VEGF Trap-Eye was observed at the end of the washout. However, there were significant changes in a-wave and b-wave amplitudes directly after exposure to 0.5mg VEGF Trap-Eye (18.4%, p=0.004 and 43.1%, p=0.006, respectively). The presented data suggest that intraocular application of up to 2mg VEGF Trap-Eye does not induce irreversible toxic retinal damage. However, short-term results showed a negative effect directly after the application for 0.5mg and 2mg VEGF Trap-Eye.

Effects of Quensyl on the ERG a‐wave amplitude from the isolated superfused vertebrate retina

AbstractPurpose Long‐term therapy with quensyl is known to cause neurodegenerative changes in the retina. In our present research we study acute toxic effects of quensyl on the a‐wave response of electroretinogram (ERG) of isolated superfused bovine retinas.Methods Isolated bovine retinae were mounted in a temperature‐controlled recording chamber. After light stimulation electric field potentials were recorded as a transretinal potential using Ag/AgCl‐electrodes. Isolated bovine retinas were perfused with phosphate buffered saline (PBS) containing 1mM L‐aspartate to block further synaptic transmission in order to record the effects of quensyl on photoreceptors. We tested the low and high light intensities of 100 mlux and 10 lux. After reaching a stable ERG amplitude, quensyl (190 µM, 570 µM or 1,9 mM) was added to the perfusing solution. After 90 min quensyl was washed out for 90 min with PBS containing 1mM L‐aspartate. Changes in a‐wave amplitude were calculated and plotted.Results 190 µM quensyl showed a 1,3 fold stimulation of the a‐wave amplitude at 100 mlux, the effect at 10 lux was not significant. 570 µM quensyl reduced the a‐wave amplitude by 3‐folds independent of light‐intensity. The inhibition was good reversible by washing with PBS containing 1mM L‐aspartate only at low light intensity (2 folds), there was almost no recovery at 10 lux. 1,9 mM quensyl showed a massive depression of a‐wave amplitude (6 to 7 folds) and no wash out effect over 90 minutes at both light intensities.Conclusion Quensyl has a toxic effect on photoreceptors, even with slight increase of concentration showing a huge progression of inhibition and reduction of recovery. An exact dosage of quensyl is of great importance to avoid an irreversible neuronal damage.

New findings in ATP supply in rod outer segments: Insights for retinopathies

The rod outer segment (OS) is the specialised organelle where phototransduction takes place. Our previous proteomic and biochemical analyses on purified rod disks showed the functional expression of the respiratory chain complexes I-IV and F1 Fo -ATP synthase in OS disks, as well as active soluble tricarboxylic acid cycle enzymes. Here, we focussed our study on the whole OS that contains the cytosol and plasma membrane and disks as native flattened saccules, unlike spherical osmotically intact disks. OS were purified from bovine retinas and characterised for purity. Oximetry, ATP synthesis and cytochrome c oxidase (COX) assays were performed. The presence of COX and F₁F₀-ATP synthase (ATP synthase) was assessed by semi-quantitative Western blotting, immunofluorescence or confocal laser scanning microscopy on whole bovine retinas and bovine retinal sections and by immunogold transmission electron microscopy (TEM) of purified OS or bovine retinal sections. Both ATP synthase and COX are catalytically active in OS. These are able to consume oxygen (O₂) in the presence of pyruvate and malate. CLSM analyses showed that rhodopsin autofluorescence and MitoTracker Deep Red 633 fluorescence co-localise on rod OS. Data are confirmed by co-localisation studies of ATP synthase with Rh in rod OS by immunofluorescence and TEM in bovine retinal sections. Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O₂. The ability of OS to manipulate O₂ may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.

Diacylglycerol kinase epsilon in bovine and rat photoreceptor cells. Light-dependent distribution in photoreceptor cells

The present study shows the selective light-dependent distribution of 1,2-diacylglycerol kinase epsilon (DAGKɛ) in photoreceptor cells from bovine and albino rat retina. Immunofluorescence microscopy in isolated rod outer segments from bleached bovine retinas (BBROS) revealed a higher DAGKɛ signal than that found in rod outer segments from dark-adapted bovine retinas (BDROS). The light-dependent outer segment localization of DAGKɛ was also observed by immunohistochemistry in retinas from albino rats. DAGK activity, measured in terms of phosphatidic acid formation from a) [3H]DAG and ATP in the presence of EGTA and R59022, a type I DAGK inhibitor, or b) [γ-32P]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 DAGKɛ content in the outer segments of these two species. Light-dependent DAGKɛ 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 [3H]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 DAGKɛ 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.

Regulation of Potassium‐induced [3H]D‐Aspartate Release from Isolated Bovine Retina by 5‐epi‐5‐F3t‐Isoprostane: Role of Prostanoid Receptors

Arachidonic acid‐derived F2‐isoprostanes (F2‐IsoPs) can regulate neurotransmitter release in bovine retina (Jamil J. et al, 2012). It is, however, unclear whether eicosapentanoic acid‐derived F3‐IsoPs can produce a similar effect in the retina.PurposeTo investigate the pharmacological actions of 5‐epi‐5‐F3t‐IsoP on K+‐induced [3H]D‐aspartate release from bovine retina. We also examined the role of prostanoid receptors on the F3‐IsoP‐induced response.MethodIsolated neural retina were incubated in oxygenated Krebs solution containing 200 nM of [3H]D‐aspartate and then prepared for studies of neurotransmitter release. Release of [3H]D‐aspartate was evoked by K+ (50 mM) stimuli applied at 90 mins (S1) and at 108 mins (S2) after the onset of superfusion.Results5‐epi‐5‐F3t‐IsoP (0.1 nM – 0.1 μM) elicited an inhibitory action on K+‐evoked [3H]D‐aspartate release in a concentration‐dependent manner, achieving a maximum inhibition of 46.9% at 0.1 μM (IC30 of 1 nM). The effect of 5‐epi‐5‐F3t‐IsoP (0.01 μM) was partially reversed by the prostanoid receptor antagonists, SC 19220 (1 μM; EP1), SC 51322 (10 μM; EP1) and AH 6809 (10 μM; EP1–3/DP1) while AH 23848 (1 μM; EP4/TP1) and SQ 29548 (10 μM; TP) had no effect on the IsoP.Conclusion5‐epi‐5‐F3t‐IsoP can inhibit K+‐evoked [3H]D‐aspartate release in isolated bovine retina, presumably via mechanisms that involve prostanoid receptors.