Dark and light adaptation of pineal photoreceptors

Dark and light adaptation of pineal photoreceptors was studied in the isolated pineal organ of the rainbow trout, Salmo gairdneri. After intracellular recording, the photoreceptors were iontophoretically injected with Lucifer yellow CH or with horseradish peroxidase for morphological characterization. Pineal photoreceptor cells responded to light with a hyperpolarization whose amplitude was graded with intensity. Following a 30-60 s bleach, receptor responsiveness was greatly reduced with a gradual recovery in the dark. Recovery of membrane potential was complete within 2-4 min in the dark. In response to flashes the hyperpolarizing response increased in darkness in amplitude and duration over a period of more than 30 min and the voltage-intensity curves continuously shifted to lower intensities. After exposure to strong light the time-course of dark adaptation, determined with a threshold criterion, was monophasic and receptor sensitivity increased by at least 5-6 log units. The results show that pineal photoreceptors exhibited the full characteristics of dark adaptation processes previously ascribed to cells proximal to the receptors, i.e. to ganglion cells. Exposure to steady illumination of different intensities induced graded and sustained hyperpolarizations of the receptor membrane potential. The incremental voltage range of responses to test flashes superimposed on the backgrounds was reduced. Voltage-intensity curves were shifted to higher intensities with increasing background illumination indicating that adaptation occurred over a range of about 2.5 log units before the receptors saturated.     

Isolation and characterization of a retinal-binding protein from the squid retina

A retinal-binding protein (RALBP) was isolated from the squid retina, and purified by anion-exchange and size-exclusion chromatography. The molecular weight was determined to be 51,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration. The purified sample showed absorption maxima at about 330 and 400 nm in addition to a protein band, indicating the occurrence of retinol and retinal, respectively. The relative heights of these two peaks varied from preparation to preparation, depending on retinoid ligands. Irradiation of RALBP caused no marked change in absorption, but the amount of 11-cis-retinal decreased to form a photosteady state mixture with all-trans- and 13-cis-retinals. RALBP was fairly stable even in the presence of hydroxylamine (100 mM), but was affected by sodium borohydride (30 mM) or borane dimethylamine (400 mM), with the retinal reduced to retinol. When incubated with metaretinochrome-carrying membranes in the dark, RALBP specifically took up 11-cis-retinal and lost all-trans-retinol. Upon further incubation of this RALBP with opsin-containing membranes, rhodopsin was progressively formed in the dark. Squid RALBP may act as a shuttle in transferring the 11-cis-retinal from metaretinochrome to opsin in the visual cells.     

THE HUMAN PINEAL GLAND: A REVIEW OF THE "THIRD EYE"AND THE EFFECT OF LIGHT

The human pineal gland is an extremely active neuroendocrine transducer Environmental light acts through the retina and entrains the pineal gland's circadian rhythms by way of the hypothalamus and sympathetic nervous system Light depresses pinealocyte activity
It is possible to do without the pineal, but this tiny gland is considered to be the "regulator of regulators" and important In general homeostasis. A direct retino-hypothalamic pathway is probably involved, and a system of synchronising potentially independent oscillators is postulated     

The human pineal gland: a review of the "third eye" and the effect of light

The human pineal gland is an extremely active neuroendocrine transducer. Environmental light acts through the retina and entrains the pineal gland's circadian rhythms by way of the hypothalamus and sympathetic nervous system. Light depresses pinealocyte activity. It is possible to do without the pineal, but this tiny gland is considered to be the "regulator of regulators" and important in general homeostasis. A direct retino-hypothalamic pathway is probably involved; and a system of synchronizing potentially independent oscillators is postulated.     

Retinal melatonin production: role of proteasomal proteolysis in circadian and photic control of arylalkylamine N-acetyltransferase

PURPOSE: Dynamic day-night changes in melatonin synthesis are regulated by changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AA-NAT]). Similarly, a light-induced decrease in AA-NAT activity at night rapidly suppresses melatonin synthesis. The purpose of the current study was to test the hypothesis that in vivo changes of AA-NAT activity in chicken retina homogenates parallel changes in AA-NAT protein. This led to examination of the role of proteasomal proteolysis in the regulation of retinal AA-NAT activity and protein levels. METHODS: Chickens, entrained to a 12-hour light-12-hour dark cycle, were assessed under various lighting conditions, in some cases after in vivo intravitreal administration of the protein synthesis inhibitor cycloheximide or lactacystin, an inhibitor of the 20S proteasome. Tissue homogenates were prepared, AA-NAT enzyme activity was measured, and immunoreactive protein was estimated by Western blot using an anti-chicken AA-NAT(1-21) serum. RESULTS: The abundance of AA-NAT protein in both the retina and pineal gland exhibited a daily rhythm that was statistically indistinguishable from that of AA-NAT's activity measured in tissue homogenates. Acute exposure to light at night rapidly decreased AA-NAT protein and activity in a parallel fashion. Administration of cycloheximide at night decreased retinal AA-NAT activity in darkness and enhanced the effect of light. The light-evoked suppression of retinal AA-NAT protein and activity was blocked by intravitreal injection of lactacystin, which also was found to increase AA-NAT activity, either at night or during the daytime. CONCLUSIONS: AA-NAT activity measured in tissue homogenates reflects the steady state level of enzyme protein. AA-NAT protein in the retina turns over rapidly, reflecting a balance of de novo synthesis and proteasomal proteolysis. The suppressive effects of light at night are due primarily to enhanced AA-NAT proteolysis.     

Isolation by polyacrylamide gel electrophoresis of a light-sensitive vitamin A-protein complex from the retina of the honeybee drone

Polyacrylamide gel electrophoresis was performed on retinal homogenates from honeybee drones previously treated with tritiated vitamin A. After a 6-hr exposure to tritiated vitamin A in vivo or a 1-hr exposure in vitro, radioactivity appears as a single band in the gel of Davis and Ornstein for soluble proteins. No radioactivity is recovered in the SDS gel of Weber and Osborn for insoluble proteins. When labeled retinal homogenates are irradiated with white light, the radioactivity recovered in the gel of Davis and Ornstein diminishes proportionally with the time of illumination. In the presence of hydroxylamine, illumination removes all radioactivity from the protein band. Conversely, sodium borohydride added to the labeled homogenates prevents the radioactivity to be removed by illumination. These results indicate that the drone retina contains a soluble, photosensitive vitamin A-protein complex, which may represent a precursor of the visual pigment in the eye.     

Differential retinoid binding in chick pigment epithelium and choroid

A receptor for retinol but not for retinoic acid was demonstrated to be present in fresh chick embryo pigment epithelium and in pigment epithelial cells grown in tissue culture. In contrast, chick choroid was found to possess receptors for both retinoic acid and retinol.     

The eye as metronome of the body.

Vision is much more than just resolving small objects. In fact, the eye sends visual information to the brain that is not consciously perceived. One such pathway entails visual information to the hypothalamus. The retinohypothalamic tract (RHT) mediates light entrainment of circadian rhythms. Retinofugal fibers project to several nuclei of the hypothalamus. These and further projections to the pineal via the sympathetic system provide the anatomical substrate for the neuro-endocrine control of diurnal and longer rhythms. Without the influence of light and dark, many rhythms desynchronize and exhibit free-running periods of approximately 24.2-24.9 hours in humans. This review will demonstrate the mechanism by which the RHT synchronizes circadian rhythms and the importance of preserving light perception in those persons with impending visual loss.     

Effects of diamide on cyclic nucleotide levels in rat retina

The concentrations of guanosine 3',5' monophosphate (cyclic GMP) and adenosine 3',5' monophosphate (cyclic AMP) were measured in rat retinas incubated under control conditions and in retinas incubated with diamide, a relatively specific glutathione oxidant. Retinas incubated with either glucose or pyruvate as the substrate for 30 min in the dark contained about 50 picomoles cyclic GMP/mg protein and 7 picomoles cyclic AMP/mg protein. Light-exposed control retinas contained about 70% less cyclic GMP (14.4 picomoles/mg protein) and 20% less cyclic AMP (5.4 picomoles/mg protein). Addition of diamide to the incubation medium at concentrations between 0.1 and 1.0 mM produced a concentration-dependent decrease in the dark level of cyclic GMP, but did not affect its concentration in the light or the concentration of cyclic AMP in dark-maintained and light-exposed retinas. The major effect of diamide was to reduce the normal dark-light difference in the concentration of cyclic GMP. Oxidizing conditions thus appear to alter selectively the light-sensitive compartment of retinal cyclic GMP.     

Susceptibility of the bovine lens 115kDa beaded filament protein to degradation by calcium and calpain.

The 115kDa cytoskeletal beaded filament protein of bovine lens fibres is degraded during opacification induced by increased internal calcium. The monoclonal antibody R2D2 to this protein has been used in whole lenses and native homogenates to follow the process of degradation and the production of break-down products. In the opaque outer cortex of whole bovine lenses with an internal Ca2+ of 2.0mM, both the 115kDa parent protein and the main degradation product (57kDa) were reduced in amount by almost 60%. No additional products were detected by the antibody. When native homogenates were incubated overnight with 10mM Ca2+ the protein could no longer be detected in SDS gels, but faintly reactive bands were detected by the antibody. Since these changes were dependent on the presence of increased calcium they were compared with changes induced by incubating freshly isolated cytoskeletal proteins with Ca2+ and the Ca(2+)-activated protease, calpain. The 115kDa protein was shown to be susceptible to degradation by calpain, with the formation of a number of breakdown products. These results indicate that degradation of the beaded filament protein can be brought about by the activation of calpain. Since the enzyme is present in lens cortex it is likely to have a role in the protein degradation observed during Ca(2+)-induced opacification, and may also be involved in the changes occurring as the lens fibres mature.     

A receptor for retinol in the developing retina and pigment epithelium.

A specific receptor for retinol is present in the soluble fraction (cytosol) of the retina and pigment epithelium of the chick embryo. As demonstrated by sucrose density gradient centrifugation, the macromolecule is a protein of approximately 19 000–20 000 daltons and has different sedimentation properties from the serum retinol-binding macromolecule. Binding of retinol to the tissue receptor appears to be reversible and non-covalent; retinal and retinoic acid have little effect on the retinol-receptor interaction. Similar binding of retinol is also observed in the developing chick liver and brain.     

Dopaminergic agents affect the ability of brief periods of normal vision to prevent form-deprivation myopia

Placing a translucent diffuser over the eye of a chick causes the eye to grow excessively, resulting in form-deprivation myopia. For chickens kept on a 12:12 h light/dark cycle, removing the diffuser for 3 h during the light period protects against the excessive growth, but if the bird is kept in the dark for this 3-h period, the protective effect is abolished. Injecting dopamine agonists into the eye during this 3-h dark period restores the protective effect, which can be blocked by dopamine antagonists injected just prior to diffuser removal in the light. These responses are mediated by D2 receptors, suggesting that the protective effect of normal vision against form-deprivation is mediated through the stimulation of dopamine release and activation of D2-dopamine receptors.     

Changes in the expression of specific Müller cell proteins during long-term retinal detachment

Retinal detachments were produced in domestic cats by injecting fluid between the retinal pigment epithelium and neural retina. Retinas were allowed to remain detached for 30 or 60 days at which time the animals were killed. Tissue areas from detached and attached retinal regions from the same eye were processed for correlative biochemical and structural analysis, i.e. SDS-PAGE and Western blots of tissue homogenates were correlated with tissue processed for postembedding immunoelectron microscopy. Antibodies to six proteins were used as probes. Glial fibrillary acidic protein in Müller cells has previously been shown to increase after retinal detachment; here we show that vimentin, another intermediate filament protein present in Müller cells, also increases after detachment. In contrast, cellular retinaldehyde binding protein, cellular retinol binding protein, glutamine synthetase, and carbonic anhydrase C--all normally found in Müller cells--decrease after detachment. The results of this study indicate that retinal Müller cells dramatically alter their expression of proteins in response to retinal detachment.     

Ocular signs and symptoms and vitamin A status in patients with cystic fibrosis treated with daily vitamin A supplements

BACKGROUND/AIMS: Patients with cystic fibrosis (CF) may have low plasma vitamin A levels from malabsorption, zinc deficiency, liver disease, or poor compliance with prescribed supplements. In view of the increasing number of adults with CF, many of whom drive cars, it is important to assess vitamin A status. In our centre an attempt has been made to achieve normal levels of fat soluble vitamins by annual estimation of plasma levels and appropriate oral supplementation. This study aimed to determine if this approach prevents vitamin A deficiency and the consequent problems with dark adaptation. METHODS: The study was conducted at the regional adult and paediatric cystic fibrosis unit and the patients were recruited from there. Dark adaptation studies were conducted at the department of ophthalmology, St James's University Hospital. All patients are regularly seen in the outpatient department by a CF specialist dietitian and have a comprehensive annual dietary assessment. 28 patients had the following investigations: serum retinol, plasma zinc, serum retinol binding protein, liver function tests, dark adaptation, contrast sensitivity, and anterior ocular surface status. 25 age and sex matched controls without CF or ocular pathology were also recruited for the dark adaptation study. RESULTS: None of the patients had vitamin A deficiency, the median value of serum retinol being 48 microg/dl, range 31-80 microg/dl (normal range 30-80 microg/dl). Dark adaptation was normal in all cases compared with the control group where the mean value was 3.4 log units of threshold luminance (95% confidence interval 2.4-4.0). None of the test group had a value of threshold luminance 2 SD above the mean value for the control group. Eight patients had reduced contrast sensitivity. The median value for serum zinc was 14.2 micromol/ l, range 13-81 micromol/l (normal range 8-23 micromol/l) and the median value for retinol binding protein was 36 mg/l, range 13-81 mg/l (normal range 35-58 mg/l). There was no correlation between dark adaptation and serum retinol, zinc, or retinol binding protein. Two patients had clinical evidence of dry eye. CONCLUSION: Regular estimates of plasma vitamin A together with appropriate supplementation and expert dietetic review can maintain normal dark adaptation in patients with cystic fibrosis. The occurrence of reduced contrast sensitivity function is well documented but remains an unexplained phenomenon and deserves further study.     

Glycogen metabolism in an amphibian retina

The rate of incorporation of [3H]glucose into glycogen was determined in bullfrog retina incubated in vitro in dark and in the light. The rate of incorporation for glucose was found to be approximately two-fold greater in the dark than in the light, 0.108 vs. 0.061 nmol mg-1 protein min-1, respectively. The turnover rate for glycogen was found also to be approximately two-fold greater in the dark than in the light, 0.051 vs. 0.027% min-1, respectively.     

Hereditary retinal dystrophy in the rat: rhodopsin, retinol, vitamin A deficiency.

Measurements were performed on young hooded (B) and albino (Ba) rats with inherited visual cell degeneration of the Bourne-Campbell-Tansley type, in comparison to heterozygotes. The animals were reared and maintained in a dark environment. Rhodopsin of the dark-maintained B and Ba increased between 16 and 35 days to levels twice those of controls. These high levels were maintained up to the age of 100 days. Estimates indicate that no more than 50% of the rhodopsin is contained in surviving visual cells at the age of 25 days, only 25% at 35 days and virtually none at 80 days. The transfer of retinol from bleached rhodopsin to the pigment epithelium (Pe) is increasingly slowed from 17 days on as a function of the accumulation of debris. The conversion of retinal from bleached rhodopsin to retinol also occurs at a lower rate; about 45% of the retinal is converted to retinol at the end of a 10-min period of light exposure in 37-day-old Ba compared to 80% in the controls. The rate of rhodopsin regeneration after 1 hr exposure to strongly bleaching light is decreased after the age of 17 days; it is only about 30% normal for 6 hr in darkness at 37 days. Maintenance on a vitamin A deficient diet accelerates the rate of visual cell death and ERG decline as early as 12 days after withholding vitamin A from weaned B rats.     

Esterification of retinol in lacrimal gland. Evidence for acyl-CoA:retinol acyltransferase activity

Vitamin A is stored in cells as long-chain fatty acyl esters of retinol. Esterification in many tissues is catalyzed in part by acyl-CoA:retinol acyltransferase (ARAT). Since the lacrimal gland contains stores of retinyl esters, it was the goal of this study to determine whether the lacrimal gland contains ARAT activity. Rabbit lacrimal gland microsomes incubated with 3H-retinol synthesized retinyl esters. The reaction rate was stimulated 30-fold in the presence of a fatty acyl-CoA generating system, producing a mixture of esters including retinyl laurate, retinyl linoleate, retinyl palmitate, and retinyl stearate as determined by reverse-phase HPLC. Retinyl palmitate was synthesized at 1944 pmole/mg protein/30 min, representing 50% of total ester synthesis, and this activity was directly proportional to microsomal protein concentration. In the presence of 180 microM 3H-retinol and 100 microM palmitoyl-CoA, retinyl palmitate was synthesized at 175-220 pmole/mg/min, and the reaction fit Michaelis-Menten kinetics as a function of retinal concentration (theoretical Vmax = 329.4 pmole/mg/min). Lauroyl CoA and stearoyl CoA, but not linoleoyl CoA, were as effective as palmitoyl CoA as substrates for the reaction. The enzyme activity was inhibited by p-chloromercuriphenyl sulfonic acid and Na-taurocholate. The data show that the lacrimal gland synthesizes retinyl esters and that the characteristics of synthesis are consistent with the presence of acyl-CoA:retinol acyltransferase in lacrimal gland.     

Distribution of retinol isomerase in vertebrate eyes and its emergence during retinal development

Ocular tissue homogenates were incubated in darkness with [11,12-3H] all-trans retinol. Formation of radiolabeled 11-cis retinol was used as an index of isomerase activity and was determined by high-performance liquid chromatography. Isomerase was found in the eyes of cattle, human, rat, chicken, turtle, goldfish and frog, representing the mammals, birds, reptiles, bony fishes and amphibians. The enzyme was concentrated in the pigment epithelium (RPE). Variable activity was found in the retina, where the amount of radiolabeled 11-cis retinol formed under standard incubation conditions at protein concentrations of 0.03-1.08 mg/ml was 6.4 +/- 6.0% of that in the RPE-choroid. Using the same methodology, we could not detect isomerase in the retinas of three cephalopods (Octopus, Sepia and Loligo). In rats, isomerase was present at postnatal day 10 but not at postnatal days 0 and 4. Therefore, the expression in the RPE of retinol isomerase, which is essential for the formation of rhodopsin in the developing photoreceptors, is coordinated with the emergence of the rod outer segment in the retina. However, the continued expression of this enzyme in RCS rats does not depend on the presence of photoreceptors, because loss of photoreceptors was not associated with an absence of isomerase activity in RCS rats. Our findings suggest that a reciprocal flow of retinoids between the retina and the site of isomerase action in the RPE is a feature common to the visual cycle in all vertebrates.     

Effect of short-term, high-dose retinol on dark adaptation in aging and early age-related maculopathy

PURPOSE: To examine the effect of a short course of high-dose retinol (preformed vitamin A) on dark adaptation in older adults with normal retinal health or early age-related maculopathy (ARM). METHODS: The study design was a randomized, double-masked, placebo-controlled experiment. Adults > or = 50 years of age whose fundus photographs for the eye to be tested psychophysically fell within steps 1 to 9 of the Age-Related Eye Disease Study (AREDS) Grading System were randomly assigned to a 30-day course of 50,000 IU oral retinol or a placebo. At baseline and 30-day follow-up, dark adaptation was tested and the Low Luminance Questionnaire (LLQ), an instrument for assessing difficulty with vision in reduced lighting, was administered. Primary outcomes of interest were rod- and cone-mediated parameters of dark adaptation, with scores on the LLQ's six subscales as secondary outcomes. RESULTS: The sample consisted of 104 participants with 52 each in the intervention and placebo groups. There were no group differences in baseline variables. At 30-days, the dark-adaptation parameters of cone time-constant, cone threshold, rod-cone break, and rod threshold did not differ. The retinol intervention group had significantly larger (i.e., steeper) rod slopes, indicating faster sensitivity recovery, than did the placebo group (P = 0.0419). There were no group differences in scores on the LLQ subscales driving, extreme lighting, emotional distress, general lighting, or peripheral vision. The retinol group had a higher score by five points on the mobility subscale compared with the placebo group (P = 0.0141). Those who had the most self-reported change on the mobility subscale at day 30 were more likely to have greater change in the speed of dark adaptation, as indicated by the rod slope parameter (r = 0.24, P = 0.0141). CONCLUSIONS: A short-term, high-dose course of retinol increased the rate of rod-mediated dark adaptation in older adults who were in the early phases of ARM or were exhibiting normal retinal aging. These results are consistent with the hypothesis that depositions and other structural changes in the retinal pigment epithelium and Bruch's membrane in aging and early ARM cause a localized retinoid deficiency.