Axial gradients of rhodopsin in light-exposed retinal rods of the toad

Exposure of an intact vertebrate eye to light bleaches the rhodopsin in the photoreceptor outer segments in spatially nonuniform patterns. Some axial bleaching patterns produced in toad rods were determined using microspectrophotometric techniques. More rhodopsin was bleached at the base of the outer segment than at the distal tip. The shape of the bleaching gradient varied with the extent of bleach and with the spectral content of the illuminant. Monochromatic light at the lambda max of the rhodopsin gave rise to the steepest bleaching gradients and induced the greatest changes in the form of the gradient with increasing extent of bleach. These results were consistent with a mathematical model for pigment bleaching in an unstirred sample. The model did not fit bleaching patterns resulting from special lighting conditions that promoted the photoregeneration of rhodopsin from the intermediates of bleaching. Prolonged light adaptation of toads could also produce axial rhodopsin gradients that were not fit by the bleaching model. Under certain conditions the axial gradient of rhodopsin in a rod outer segment reversed with time in the light: the rhodopsin content became highest at the base. This result could be explained by an interaction between the pattern of bleaching and the intracellular topography of regeneration.     

Some properties of old and new rhodopsin in single Bufo rods

Rod photoreceptors renew the membranous disks of the outer segments (ROS). New disks are assembled at the proximal base and old disks are shed at the distal tip. Rhodopsin, the major protein of the disk, remains with the disk into which it was inserted. Thus, it is true that the oldest rhodopsin is at the tip and the newest at the base. A microspectrophotometer is used to examine the properties of rhodopsin in the two ends of the toad ROS. No differences between the two are found in absorption spectrum, concentration, dichroism, photoconversion rates, or lateral diffusion rates. Regeneration of rhodopsin from the bleached state is also studied but cannot be used to discriminate old from new rhodopsin because the point of entry of regeneration retinoids and/or their concentrations cannot be controlled. However, a new insight into pigment regeneration in the living toad eye is gained: regeneration is faster in the basal disks than in the distal.     

Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

Bovine rod rhodopsin. 1. Bleaching by luminescence in vitro by recombination of radicals from polyunsaturated fatty acids

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.     

Interaction of bovine rhodopsin with calcium ions. II: calcium release in bovine rod outer segments upon bleaching.

The calcium content of bovine rod outer segment (ROS) suspensions was determined by flame spectrophotometry to be about 0.2 Ca2+ per molecule rhodopsin. After bleaching of rhodopsin, a release of 0.01--0.1 Ca2+ per molecule rhodopsin from ROS into the solution was observed. These figures agree with some data in the literature (Appendix). A measured absorption increase of the Ca2+-indicator phthalein purple (10 degrees C, 562 nm, pH 9.3) occurs apparently simultaneously with the formation of metarhodopsin ii in ROS. This indicates that a light induced Ca2+-release of 12 calcium ions per photoactivated rhodopsin is coupled in time with the formation of metarhodopsin II.     

Differential Rhodopsin Regeneration in Photoreceptor Membranes is Correlated with Variations in Membrane Properties

Rhodopsin, the major transmembrane protein in both the plasma membrane and the disk membranes of photoreceptor rod outer segments (ROS) forms the apo-protein opsin upon the absorption of light. In vivo the regeneration of rhodopsin is necessary for subsequent receptor activation and for adaptation, in vitro this regeneration can be followed after the addition of 11-cis retinal. In this study we investigated the ability of bleached rhodopsin to regenerate in the compositionally different membrane environments found in photoreceptor rod cells. When 11-cis retinal was added to bleached ROS plasma membrane preparations, rhodopsin did not regenerate within the same time course or to the same extent as bleached rhodopsin in disk membranes. Over 80% of the rhodopsin in newly formed disks regenerated within 90 minutes while only 40% regenerated in older disks. Since disk membrane cholesterol content increases as disks are displaced from the base to the apical tip of the outer segment, we looked at the affect of membrane cholesterol content on the regeneration process. Enrichment or depletion of disk membrane cholesterol did not alter the % rhodopsin that regenerated. Bulk membrane properties measured with a sterol analog, cholestatrienol and a fatty acid analog, cis parinaric acid, showed a more ordered, less fluid, lipid environment within plasma membrane relative to the disks. Collectively these results show that the same membrane receptor, rhodopsin, functions differently as monitored by regeneration in the different lipid environments within photoreceptor rod cells. These differences may be due to the bulk properties of the various membranes.     

Adaptation of a deep-sea cephalopod to the photic environment. Evidence for three visual pigments

Watasenia scintillans, a bioluminescent deep-sea squid, has a specially developed eye with a large open pupil and three visual pigments. Photoreceptor cells (outer segment: 476 micron; inner segment: 99 micron) were long in the small area of the ventral retina receiving downwelling light, whereas they were short (outer segment: 207 micron; inner segment: 44 micron) in the other regions of the retina. The short photoreceptor cells contained the visual pigment with retinal (lambda max approximately 484 nm), probably for the purpose of adapting to their environmental light. The outer segment of the long photoreceptor cells consisted of two strata, a pinkish proximal area and a yellow distal area. The visual pigment with 3-dehydroretinal (lambda max approximately 500 nm) was located in the pinkish proximal area, giving high sensitivity at longer wavelengths. A newly found pigment (lambda max approximately 471 nm) was in the yellow distal area. The small area of the ventral retina containing two visual pigments is thought to have a high and broad spectral sensitivity, which is useful for distinguishing the bioluminescence of squids of the same species in their environmental downwelling light. These findings were obtained by partial bleaching of the extracted pigment from various areas of the retina and by high-performance liquid chromatographic analysis of the chromophore, complemented by microscopic observations.     

Control of light-activated phosphorylation in frog photoreceptor membranes.

In this paper, we examine some factors which regulate the efficiency of light in activating rhodopsin phosphorylation. We have measured phosphate incorporation after illumination in suspensions of bullfrog rod outer segments incubated with [gamma-32P]ATP. We observed that delaying ATP addition after illumination causes maximum phosphate incorporation to decrease 80% within 2 h. This decay occurs in urea-treated, extracted rod outer segment membranes. The decay of the light effect is not influenced by regeneration of opsin to rhodopsin or the presence of long-lived photoproducts. However, regeneration of opsin increases the amount of phosphorylation initiated by a second exposure to light. Further phosphorylation can also occur after phosphate groups have been removed from the membranes by dephosphorylation. Finally, we have confirmed our earlier observation that small amounts of light (bleaching less than 5% of the rhodopsin present) are more effective, by tenfold, in initiating phosphorylation than are larger amounts.     

Photobleaching and cyclic GMP dependences of rhodopsin phosphorylation in rod outer segment

The experimental data on the cGMP decrease under continuous illumination of rod outer segment have been theoretically analysed to study the bleaching and hence the cGMP dependence of the rhodopsin phosphorylation. From the agreement of the theoretical results with the experimental observations it has been found that the rate of phosphorylation depends on the rate of cGMP hydrolysis. If the rate of cGMP hydrolysis increases the rate of phosphorylation also increases. The results of the theoretical treatment predict that (i) the presence of cGMP in rod outer segment inhibits the rhodopsin phosphorylation and (ii) rhodopsin phosphorylation process is much faster than what has been reported in the literature.     

Dark adaptation processes in the amphibian rod

Rod dark adaptation in the amphibian retina appears to be due to three processes: 1. background adaptation, occurring immediately after the extinction of an adapting or bleaching light, 2. intermediate adaptation, that frequently lasts 30 min or more and 3. opsin adaptation, which in the isolated retina where regeneration of rhodopsin is insignificant, is observed as a permanent loss of sensitivity after the completion of intermediate adaptation. Intermediate adaptation is characterized by a linear relation between log threshold and the amount of retinal present, a similar relation is obtained between log threshold and the amount of rhodopsin bleached in opsin adaptation.These adaptation processes are discussed in terms of a model of the rod outer segment.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Aggregation of rhodopsin molecules during damaging exposure of photoreceptor membranes to light

Injuring light induced structural changes in rod outer segment (ROS) membranes are studied using "ST EST spectroscopy" for spin labelled rhodopsin, ESR of lipid spin label and SDS gel-electrophoresis. Free SH-group content of rhodopsin and lipid peroxidation level were simultaneously determined as well. A decrease of rotational mobility of rhodopsin in ROS induced by prolonged illumination is shown to result from irreversible protein aggregation caused by disulfide bond formation between "hydrophobic" SH-groups of rhodopsin. Some decrease of lipid microviscosity and degree of order are found, in contrast to considerable rise in microviscosity due to Fe2+-ascorbate induced lipid peroxidation of ROS membranes. Lipid oxidation is found to accelerate protein aggregation which in its turn influences the state of lipid bilayer.     

Biochemical aspects of the visual process. XXXVIII. Effects of lateral aggregation on rhodopsin in phospholipase C-treated photoreceptor membranes

Treatment of bovine rod outer segments with phospholipase C leads to largely lipid-depleted membranous structures. Under these conditions rhodopsin remains spectrally intact, but its thermal stability and regeneration capacity are decreased, whereas upon illumination the metarhodopsin I to II transition is blocked. These observations can be explained on the basis of the previously demonstrated lateral aggregation of rhodopsin molecules which, on the one hand leads to a (partial) shielding of these molecules and, on the other hand, might impose constraints on the flexibility of the molecule to undergo light-induced conformational changes.Upon reconstitution of these lipid-depleted preparations with amphipathic lipids by means of a detergent dialysis procedure, the aggregates are apparently rearranged to lipid bilayer structures with complete recovery of the original rhodopsin properties. Under our conditions the nature of the polar head groups and the fatty acids is not critical in this respect. Simple addition of amphipathic lipids, without the use of detergent, restores the rhodopsin properties only in the case of rod outer segment lipids and of didecanoylphosphatidylcholine, and even then only occasionally.These results are discussed in the light of the strong analogy in properties between phospholipase C-treated rod outer segment membranes and lipid- and detergent-free rhodopsin obtained by affinity chromatography. It is concluded that rhodopsin must be in a freely dispersed state in order to function properly. Apparently, a non-specific lipid bilayer fulfills this condition for the regeneration capacity, whereas normal photolytic behaviour requires, in addition, a minimal membrane fluidity according to the observations of other investigators. Presumably, the uniquely high phospholipid unsaturation of rod outer segment membranes is important for another, as yet unassessed, function of rhodopsin or the photoreceptor membrane.     

Kinetic study of transfer of 11-cis-retinal between rod outer segment membranes using regeneration of rhodopsin

The present study demonstrates some important facts on the regeneration of rhodopsin in rod outer segment membranes. 11-cis-Retinal added to a rod outer segment membrane suspension did not react directly with opsin but was rapidly solubilized into membranes and then recombined with opsin in the membrane. It was also revealed that the regeneration of rhodopsin was perturbed by the formation of retinylidene Schiff base with phosphatidylethanolamine in rod outer segment membranes, which decreased with increasing temperature. The activation energy of rhodopsin regeneration in rod outer segment membranes was 18.7 kcal/mol, being smaller than the value of 22 kcal/mol in 1% digitonin solution. 11-cis-Retinal could be found to transfer relatively fast (tau-1/k(1) R 10(3) s) between rod outer segment membranes by using the regeneration of rhodopsin. It was demonstrated that the kinetic measurement for the transport of membrane-soluble molecules such as retinal between membranes could be perform ed with ease and precisely by the method described in this paper.     

Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal.     

INHERITED RETINAL DYSTROPHY IN THE RAT

Retinal dystrophies, known in man, dog, mouse, and rat, involve progressive loss of photoreceptor cells with onset during or soon after the developmental period. Functional (electroretinogram), chemical (rhodopsin analyses) and morphological (light and electron microscopy) data obtained in the rat indicated two main processes: (a) overproduction of rhodopsin and an associated abnormal lamellar tissue component, (b) progressive loss of photoreceptor cells. The first abnormality recognized was the appearance of swirling sheets or bundles of extracellular lamellae between normally developing retinal rods and pigment epithelium; membrane thickness and spacing resembled that in normal outer segments. Rhodopsin content reached twice normal values, was present in both rods and extracellular lamellae, and was qualitatively normal, judged by absorption maximum and products of bleaching. Photoreceptors attained virtually adult form and ERG function. Then rod inner segments and nuclei began degenerating; the ERG lost sensitivity and showed selective depression of the a-wave at high luminances. Outer segments and lamellae gradually degenerated and rhodopsin content decreased. No phagocytosis was seen, though pigment cells partially dedifferentiated and many migrated through the outer segment-debris zone toward the retina. Eventually photoreceptor cells and the b-wave of the ERG entirely disappeared. Rats kept in darkness retained electrical activity, rhodopsin content, rod structure, and extracellular lamellae longer than litter mates in light.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.     

Orientation of acidic polysaccharides and rhodopsin-oligosaccharides in frog retinal rod outer segments

Summary Using topo-optical staining reactions, the presence and molecular order of three structural components of outer segments of frog retina were studied. These components included (1) an acidic polysaccharide texture, (2) free aldehyde groups which arise during formalin fixation and (3) the oligosaccharide chains of rhodopsin. Quantitative measurements of the dye binding and birefringence effects arising from the individual structural components in rod outer segments were made. Results indicated that all three structural components had a rather well-defined orientation within the ROS.The spherulites phagocytized from the apical ends of ROSs by the pigment epithelium also demonstrate preferred orientation of the three structural components investigated.     

Mechanism and specificity of rhodopsin phosphorylation.

Partial separation of protein kinase activity from rhodopsin in isolated bovine retinal photoreceptor outer segments was accomplished by mild ultrasonic treatment followed by ultracentrifugation. Residual kinase activity in the rhodopsin-rich sediment was destroyed by chemical denaturation which did not affect the spectral properties of the rhodopsin. The retinal outer segment kinase was found to be specific for rhodopsin, since in these preparations it alone of several bovine protein kinases was capable of phosphorylating rhodopsin in the light. The phosphorylation reaction apparently requires a specific conformation of the rhodopsin molecule since it is abolished by heat denaturation of rhodopsin, and it is greatly reduced or abolished by treatment of the visual pigment protein with potassium alum after the rhodopsin has been "bleached" by light. When kinase and rhodopsin or opsin fractions were prepared from dark-adapted and bleached outer segments and the resultant fractions were mixed in various combinations of bleached and unbleached preparations, the observed pattern of light-activated phosphorylation was consistent only with the interpretation that a conformational change in the rhodopsin molecule in the light exposes a site on the visual pigment protein to the kinase and ATP. These results rule out the possibility of a direct or indirect (rhodopsin-mediated) light activation of the kinase. Finally, phosphorylation of retinal outer segment protein in monochromatic lights of various wavelengths followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both rhodopsin and the higher molecular weight visual pigment protein reported by several laboratories have the same action spectrum for phosphorylation. This result is consistent with the suggestion that the higher molecular weight species is a rhodopsin dimer.