Characterization of a dehydrogenase activity responsible for oxidation of 11-cis-retinol in the retinal pigment epithelium of mice with a disrupted RDH5 gene. A model for the human hereditary disease fundus albipunctatus.

In the vertebrate retina, the final step of visual chromophore production is the oxidation of 11-cis-retinol to 11-cis-retinal. This reaction is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs), prior to the chromophore rejoining with the visual pigment apo-proteins. The RDH5 gene encodes a dehydrogenase that is responsible for the majority of RDH activity. In humans, mutations in this gene are associated with fundus albipunctatus, a disease expressed by delayed dark adaptation of both cones and rods. In this report, an animal model for this disease, 11-cis-rdh-/- mice, was used to investigate the flow of retinoids after a bleach, and microsomal membranes from the retinal pigment epithelium of these mice were employed to characterize remaining enzymatic activities oxidizing 11-cis-retinol. Lack of 11-cis-RDH leads to an accumulation of cis-retinoids, particularly 13-cis-isomers. The analysis of 11-cis-rdh-/- mice showed that the RDH(s) responsible for the production of 11-cis-retinal displays NADP-dependent specificity toward 9-cis- and 11-cis-retinal but not 13-cis-retinal. The lack of 13-cis-RDH activity could be a reason why 13-cis-isomers accumulate in the retinal pigment epithelium of 11-cis-rdh-/- mice. Furthermore, our results provide detailed characterization of a mouse model for the human disease fundus albipunctatus and emphasize the importance of 11-cis-RDH in keeping the balance between different components of the retinoid cycle.     

Inhibition of ethanol metabolism by fructose in alcohol dehydrogenase-deficient deer mice in vivo

The purpose of this work was to compare the roles of a newly described mitochondrial dehydrogenase and catalase in ethanol elimination in deer mice deficient in alcohol dehydrogenase (ADH-). Fructose was used because of its well-known ability to stimulate dehydrogenase-dependent ethanol metabolism. Rates of ethanol metabolism in vivo were decreased significantly by about 60% in a dose-dependent manner by fructose in deer mice fed an ethanol-containing or a corn oil control diet. In addition, rates of metabolism of methanol, a selective substrate for catalase in rodents, were similar to rates of ethanol elimination and were decreased from 6.9 +/- 1.0 to 1.7 +/- 0.5 mmol/kg/h by fructose, supporting the hypothesis that catalase and not a mitochondrial dehydrogenase predominates in ethanol oxidation in ADH-deer mice. Glycolate, a substrate for peroxisomal H2O2 generation, reversed the inhibition of alcohol metabolism by fructose completely, indicating that fructose did not inhibit catalase directly. As expected, the ATP/ADP ratio was decreased by fructose significantly from 4.2 +/- 0.4 to 2.4 +/- 0.4 in deer mouse livers. These data are consistent with the hypothesis that fructose decreases catalase-dependent ethanol metabolism in vivo by inhibiting hepatic H2O2 generation.     

On the mechanism of isomerization of all-trans-retinol esters to 11-cis-retinol in retinal pigment epithelial cells: 11-fluoro-all-trans-retinol as substrate/inhibitor in the visual cycle

The synthesis of 11-fluoro-all-trans-retinol (11-F-tROL), which is shown to be an excellent substrate for processing by visual cycle enzymes, is described. It is isomerized to its 11-cis congener subsequent to its esterification by lecithin retinol acyl transferase (LRAT) approximately as well as is vitamin A itself. The enzymatic turnover of 11-F-tROL is unaccompanied by enzyme inhibition. The previously reported lack of isomerization of this substrate had been suggested as evidence for a carbonium mechanism in the critical enzymatic isomerization pathway in vision. The mechanism of this process remains unknown.     

Visual cycle and dark adaptation: a new approach in research

Visual cycle is the series of reactions that support regeneration of the visual pigmen after its photolysis in retinal rods and cones. Inherited or acquired deficiencies of the visual cycle impair dark adaptation and lead to a series of visual disorders. The paper describes a new approach to study of the visual cycle that uses fast dichroic microspectrophotometer. The method allows studying interconversion of bleaching products in single intact photoreceptors in condition approaching the situation in vivo. Using this approach, we established a complete scheme of transitions between metarhodopsins, retinal and retinol in amphibian rods. It appeared that the decay of metarhodopsins controls both the time course of rod dark adaptation following small bleaches and the production of retinol that is the substrate for rhodopsin regeneration. We also obtained novel data on kinetics of the decay of cone metapigments that was found to be by an order of magnitude faster than in rods. Possible application of the method for further study of the visual cycle in normal and pathological conditions is discussed.     

Multiple phosphorylation of rhodopsin and the in vivo chemistry underlying rod photoreceptor dark adaptation

Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all-trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.     

Role of noncovalent binding of 11-cis-retinal to opsin in dark adaptation of rod and cone photoreceptors

Regeneration of visual pigments of vertebrate rod and cone photoreceptors occurs by the initial noncovalent binding of 11-cis-retinal to opsin, followed by the formation of a covalent bond between the ligand and the protein. Here, we show that the noncovalent interaction between 11-cis-retinal and opsin affects the rate of dark adaptation. In rods, 11-cis-retinal produces a transient activation of the phototransduction cascade that precedes sensitivity recovery, thus slowing dark adaptation. In cones, 11-cis-retinal immediately deactivates phototransduction. Thus, the initial binding of the same ligand to two very similar G protein receptors, the rod and cone opsins, activates one and deactivates the other, contributing to the remarkable difference in the rates of rod and cone dark adaptation.     

Substrate specificities and mechanism in the enzymatic processing of vitamin A into 11-cis-retinol

The biosynthesis of 11-cis-retinol in the retinal pigment epithelium requires two consecutive enzymatic reactions. The first involves the esterification of all-trans-retinol by lecithin retinol acyltransferase (LRAT). The second reaction involves the direct conversion of an all-trans-retinyl ester into 11-cis-retinol by an isomerase-like enzyme. This latter reaction couples the free energy of hydrolysis of an ester to the thermodynamically uphill trans to cis conversion, thus providing the energy to drive the latter process. In this paper both enzymes are studied with respect to their substrate specificities to provide information on mechanism. The isomerase is shown to be highly specific with respect to the ionylidene ring system and substitution at C15, whereas sterically bulkier substituents at C9 and C11 are permitted. C5 and C13 demethyl retinoids are isomerized, removing from consideration isomerization mechanisms involving C-H abstraction at the C5 or C13 methyl groups of the retinoid. On the other hand, C9 demethyl retinoids are not isomerized. A C-H abstraction mechanism is unlikely at the C9 methyl group as well, because no kinetic deuterium isotope effect is found with all-trans-19,19,19-trideuterioretinoids and isomerization of unlabeled retinoids occurs without the incorporation of deuterium when the isomerization is performed in D2O. LRAT proved to be broadly specific for retinols but was relatively inert with other hydrophobic alcohols including cholesterol. The enzyme is also highly specific for phosphatidylcholine analogues versus other potential membranous acyl donors such as phosphatidylethanolamine and phosphatidylserine.     

Wavelength dependence of dark adaptation in Phycomyces phototropism

The wavelength dependence of phototropic dark adaptation in Phycomyces was studied between 347 and 545 nm. Dark adaptation kinetics were measured for wavelengths of 383, 409, 477, and 507 nm in the intensity range from 6.2 X 10(-2) to 2 X 10(-7) W X m-2. At these wavelengths, dark adaptation follows a biexponential decay as found previously with broadband blue light (Russo, V. E. A., and P. Galland, 1980, Struct. Bonding., 41:71; Lipson, E. D., and S. M. Block, 1983, J. Gen. Physiol., 81:845). We have found that the time constants of the fast and slow components depend critically on the wavelength. At 507 nm, dark adaptation kinetics were found to be monophasic. The phototropic latency after a step down by a factor of 500 was measured for 19 different wavelengths. Maximal latencies were found at 383, 477, and 530 nm; minimal latencies were found at 409 and 507 nm. With irradiation programs that employ different wavelengths before and after the step down, the dark adaptation kinetics depend critically on the sequence in which the two wavelengths are given. We have found too that not only do the adaptation kinetics vary with wavelength, but so also do the phototropic bending rate and the phototropic latencies in experiments without intensity change. The results imply that more than one photoreceptor is mediating phototropism in Phycomyces and that sensory adaptation is regulated by these photoreceptors.     

Social learning of predators in the dark: understanding the role of visual,chemical and mechanical information

The ability of prey to observe and learn to recognize potential predators from the behaviour of nearby individuals can dramatically increase survival and, not surprisingly, is widespread across animal taxa. A range of sensory modalities are available for this learning, with visual and chemical cues being well-established modes of transmission in aquatic systems. The use of other sensory cues in mediating social learning in fishes, including mechano-sensory cues, remains unexplored. Here, we examine the role of different sensory cues in social learning of predator recognition, using juvenile damselfish (Amphiprion percula). Specifically, we show that a predator-naive observer can socially learn to recognize a novel predator when paired with a predator-experienced conspecific in total darkness. Furthermore, this study demonstrates that when threatened, individuals release chemical cues (known as disturbance cues) into the water. These cues induce an anti-predator response in nearby individuals; however, they do not facilitate learnt recognition of the predator. As such, another sensory modality, probably mechano-sensory in origin, is responsible for information transfer in the dark. This study highlights the diversity of sensory cues used by coral reef fishes in a social learning context.     

Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol

Vertebrate retinas contain two types of light-detecting cells. Rods subserve vision in dim light, while cones provide color vision in bright light. Both contain light-sensitive proteins called opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde by absorption of a photon. Restoration of light sensitivity requires chemical re-isomerization of retinaldehyde by an enzymatic pathway called the visual cycle in the retinal pigment epithelium. The isomerase in this pathway uses all-trans-retinyl esters synthesized by lecithin retinol acyl transferase (LRAT) as the substrate. Several lines of evidence suggest that cone opsins regenerate by a different mechanism. Here we demonstrate the existence of two catalytic activities in chicken retinas. The first is an isomerase activity that effects interconversion of all-trans-retinol and 11-cis-retinol. The second is an ester synthase that effects palmitoyl coenzyme A-dependent synthesis of all-trans- and 11-cis-retinyl esters. Kinetic analysis of these two activities suggests that they act in concert to drive the formation of 11-cis-retinoids in chicken retinas. These activities may be part of a new visual cycle for the regeneration of chromophores in cones.     

Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.     

The time course of dark adaptation in the bee: a phototactic and electrophysiological investigation

ABSTRACT. The time course of dark adaptation in Apis melifera L. was investigated by analyses of phototactic behaviour and electroretinogram (ERG). The behavioural results give a function for dark adaptation showing that in the dark after strong light adaptation the sensitivity increases exponentially with a time constant of 3 min. The sensitivity changes c. 2.4 log units during the time span of 5–720 s. The electrophysiological results indicate a smaller change in sensitivity at the level of the photoreceptors. Within a time span between 20 and 720 s the sensitivity increases during dark adaptation by a factor of 4.3 on a linear scale.     

Light-sensitive swelling of isolated frog rod outer segments as an in vitro assay for visual transduction and dark adaptation

Frog rod outer segments swell slowly after being shaken from an excised retina into a modified Ringer's solution. The swelling has the following characteristics: (a) It is suppressed by illumination which bleaches only 500 rhodopsin molecules per outer segment per second. This is approximately the level required to saturate the in vivo receptor potential. (b) Light suppression is seen in NaCl but not in KCl solutions. (c) Dark swelling is labile and is enhanced by calf serum, low calcium concentrations, dithiothreitol, and cyclic nucleotide phosphodiesterase inhibitors. (d) Lowering the pH to 5.5 or removing magnesium reversibly reduces dark swelling to the same extent as illumination. (e) The amount of light required for maximal suppression of dark-swelling increases approximately 10-fold if the calcium concentrations is lowered by EGTA addition. (f) The effect of illumination is irreversibly abolished by antimycin and other inhibitors of mitochondrial electron transport. (g) A process analogous to dark adaptation in vivo can be observed: If 10-50% of the rhodopsin present is bleached and the outer segments are then kept dark, rapid dark swelling returns after a period of 15-45 min. This swelling is again sensitive to light. We tentatively ascribe the light suppression of swelling to the same decrease in sodium permeability which is observed on illuminating living receptor cells. The experiments suggest that outer segments retain their competence to perform both transduction and dark adaptation after their separation from the retina.     

In vivo two-photon imaging reveals a role of arc in enhancing orientation specificity in visual cortex

Cortical representations of visual information are modified by an animal's visual experience. To investigate the mechanisms in mice, we replaced the coding part of the neural activity-regulated immediate early gene Arc with a GFP gene and repeatedly monitored visual experience-induced GFP expression in adult primary visual cortex by in vivo two-photon microscopy. In Arc-positive GFP heterozygous mice, the pattern of GFP-positive cells exhibited orientation specificity. Daily presentations of the same stimulus led to the reactivation of a progressively smaller population with greater reactivation reliability. This adaptation process was not affected by the lack of Arc in GFP homozygous mice. However, the number of GFP-positive cells with low orientation specificity was greater, and the average spike tuning curve was broader in the adult homozygous compared to heterozygous or wild-type mice. These results suggest a physiological function of Arc in enhancing the overall orientation specificity of visual cortical neurons during the post-eye-opening life of an animal.     

Calcium: a role for neuroprotection and sustained adaptation

Sustained increases in intracellular calcium following prolonged seizures or other neurological insults have been thought to be responsible for neuronal cell death for well over two decades. For example, a seizure or a stroke can lead to excessive release of glutamate, an endogenous excitotoxin. Overactivation of receptors that interact with glutamate will raise calcium levels to stimulate a variety of signaling pathways that can impair neuronal respiration and eventually kill neurons. On the contrary, recent evidence shows that under numerous conditions calcium can prevent neurons from dying. Experimental epilepsy and ischemia models show that protection of neurons appears to depend upon the age of the animal, the amount and route of calcium elevation, timing of initial insults, and brain regions involved. This review will discuss novel findings on the protective signaling role of calcium under a wide range of pathological conditions.